"nun”.h H. me A NIVE smr LIBRARIES I i I l Iiinn‘iii’iiii i ii 11m 1 us ‘I 3 1293 015 1 6529 This is to certify that the dissertation entitled EARLY AND LATE MECHANISMS FOR VOMITOXIN- INDUCED IgA NEPHROPATHY presented by DING YAN has been accepted towards fulfillment of the requirements for Ph.D. . Food Science degree in \ / 2940112") \/J Q v.7 /‘//’6 t, Major professor Date 7/ /7/ 9 7 MSUI'J an Affirmative Action/Equal Opportunity Institution 042771 LIBRARY Michigan State University PLACE IN RETURN BOX to romovo thio ohookout from your rooord. TO AVOID FINES roturn on or boioro ddo duo. WQUE DATE DUE DATE DUE MSU lo An N'flrmotivo Action/EM Opportunity Intuition EARLY AND LATE MECHANISMS FOR VOMITOXIN-INDUCED lgA NEPHROPATHY By Ding Yan A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1 997 ABSTRACT EARLY AND LATE MECHANISMS FOR VOMITOXlN-INDUCED lgA NEPHROPATHY By Ding Yan Prolonged oral vomitoxin (VT) exposure in mice results in increased cytokine gene expression, elevated production of lgA and lgA nephropathy (lgAN). In this study, both early and late mechanisms for VT-induced lgAN were examined. To assess the possible role of cytokines in this lgA dysregulation, the effects of a single oral VT exposure to mice on production of lgA and cytokines in Peyer's patch (PP) and spleen cell cultures were evaluated. The results indicated that PP cells exhibited an enhanced capacity for production of lgA, lL-5 and lL-6 as' early as 2 hr and as late as 24 hr after VT exposure. Both control and VT-induced lgA production were inhibited by lL-5 and lL-6 neutralizing antibodies. Subsequently, to determine the potential role of macrophages (Md) on regulation of lgA production, the effects of Md) on in vitro lgA and lL-6 production after oral VT exposure in mice were assessed. The results demonstrated that both lgA and IL-6 production by VT-treated and control PP and spleen cultures were diminished after Md) depletion. Enhanced secretion of lL-6 occurred concurrently with increased lgA production in Mtb-depleted PP cultures reconstituted with VT-treated peritoneal Md). PP B cells from control animals produced significantly more lgA when co-cultured with VT-treated Md) and, to a lesser extent, with VT-treated CD4‘ T cells. Both soluble mediators and cognate interactions between Md) and the lymphocyte populations appeared to be necessary for increased lgA production in PP cultures following VT exposure. The above two studies identify early mechanisms for VT- induced lgAN. Finally, to assess the role of lgA in inducing the experimental nephropathy, both 8603F1 and BALB/C mice were injected i.p. with VT-induced monoclonal lgA Abs and several immunopathologic markers were monitored. When treated with lgAs both strains of mice showed a marked elevation of serum IgA, lgA immune complexes (lC), lgG, lgM concentrations and mesangial lgA, lgG, 03 deposition and hematuria. Formation of lgA and lgG-casein complexes was detectable in BSC3F1 mice after injection suggesting that dietary casein might participate in lC-mediated pathogenesis of nephropathy. Except for lgG elevation, these same immunopathologic effects were observed in BALB/C mice after injection of lgA-secreting hybridoma cells. The final study identifies late mechanisms for VT- induced lgAN. Taken together, theses studies suggest that: (1) superinduction of lL-5 and lL-6 expression contribute to upregulation of lgA production in mice exposed orally to VT, (2) soluble mediators and cognate help from Md) are responsible, in part, for upregulation of lgA production in mice exposed orally to VT, and (S) nephropathy in mice could be induced by direct injection of lgAs encountered after VT feeding. Dedicated to my wife Wenqi Fan, my daughters Youfang Yan and Wendy Yan, my mother Zhongling Bao, my father Zhongshen Yan, and my sister Pan-Pan Yan for their love, encouragement, patience, understanding, and support. iv ACKNOWLEDGMENTS I would like to thank my major professor Dr. James J. Pestka for his advice, guidance, encouragement, assistance and understanding throughout my Ph.D. study at Michigan State University. I am also indebted to Dr. John E. Linz, Dr. William Helferich, Dr. Kathryn H. Brooks and Dr. Wilson K. Rumbeiha, the members of my guidance committee, for their invaluable suggestions and help. Special thanks are expressed to Dr. Hui-Ren Zhou and Dr. James Clarke for their advice and cooperation in research, and to Dr. Mohamed M. Abouzied, Dr. Juan l. Azcona-Olivera, Dr. Wumin Dong, Dr. Linda Rasooly, Dr. RoscoeL. Warner, Dr. Dana M. Greene, Dr. Sutikno, Dr. Yanli Ouyang and all other colleagues in the Food Microbiology Laboratory for their technical expertise. TABLE OF CONTENTS Page LIST OF TABLES ............................................... ix - LIST OF FIGURES ............................................... xi LIST OF ABBREVIATIONS ....................................... xiii INTRODUCTION ................................................ 1 CHAPTER 1. LITERATURE REVIEW ................................ 6 1.1 Deoxynivalenol (vomitoxin) and other trichothecenes ........... 7 1.1.1 History .......................................... 7 1.1.2 Chemical structure ................................. 7 1.1.3 Natural occurrence ................................ 9 1.1.4 Toxin production ................................. 12 1.1.5 lmmunotoxicity ................................... 17 1.2 IgA nephropathy ...................................... 21 1.2.1 History ......................................... 21 1.2.2 Geographic distribution ............................ 22 1.2.3 Clinical features .................................. 22 1.2.4 Laboratory features. .............................. 24 1.2.5 Animal models ................................... 27 1.3 Cytokines and lgA production ............................ 32 1.3.1 Characteristics of cytokines ......................... 32 1.3.2 Regulation of lgA production by T cell-secreted cytokines . 33 1.3.2.1 T lymphocytes ................................ 33 1.3.2.2 IL-2 ......................................... 34 1.3.2.3 lL-4 ........................................ 35 1.3.2.4 lL-5 ........................................ 36 1.3.2.5 lL-6 ........................................ 38 1.3.2.6 TGF-B ..................................... 40 1.3.2.7 IFN-y ....................................... 41 1.3.2.8 Th2 cells and mucosal lgA immune responses ....... 42 1.3.3 Macrophage-secreted cytokines. ..................... 44 vi CHAPTER 2. POTENTIAL ROLE FOR CYTOKINES IN ENHANCED lgA SECRETION BY PEYER'S PATCH CELLS ISOLATED FROM MICE ACUTELY EXPOSED TO VOMITOXIN ............................... 49 2.1 ABSTRACT ........................................... 50 2.2 INTRODUCTION ...................................... 52 2.3 MATERIALS AND METHODS ............................ 54 2.3.1 Chemicals and reagents ............................ 54 2.3.2 Animal and vomitoxin exposure regimen ................ 54 2.3.3 Cell cultures ...................................... 55 2.3.4 lgA quantitation ................................... 56 2.3.5 Cytokine quantitation ............................... 57 2.3.6 Statistics ........................................ 58 2.4 RESULTS ............................................ 59 2.5 DISCUSSION ......................................... 77 CHAPTER 3. ROLE OF MACROPHAGES IN ELEVATED lgA AND IL-6 PRODUCTION BY PEYER'S PATCH CULTURES FOLLOWING ACUTE ORAL VOMITOXIN EXPOSURE ............................. 84 3.1 ABSTRACT ........................................... 85 3.2 INTRODUCTION ...................................... 87 3.3 MATERIALS AND METHODS ............................ 90 3.3.1 Chemicals and reagents ............................ 90 3.3.2 Animal and vomitoxin exposure regimen ............... 90 3.3.3 Lymphocyte preparation ............................ 90 3.3.4 Peritoneal macrophages isolation ..................... 91 3.3.5 B cells isolation ................................... 92 3.3.6 CD4+ T cells isolation .............................. 92 3.3.7 Cell cultures ...................... . ............... 93 3.3.8 Reconstitution studies with fractionated cell populations . . . 93 3.3.9 Transwell culture studies ........................... 94 3.3.10 lgA quantitation .................................. 94 3.3.11 Cytokine quantitation .............................. 95 3.3.12 Statistics ....................................... 96 vii 3.4 RESULTS ............................................ 97 3.5 DISCUSSION ........................................ 120 CHAPTER 4. INDUCTION OF NEPHROPATHY BY INJECTION WITH DIETARY VOMITOXIN-INDUCED lgA MONOCLONAL ANTIBODIES INTO MICE ................................................... 130 4.1 ABSTRACT .......................................... 131 4.2 INTRODUCTION ..................................... 133 4.3 MATERIALS AND METHODS ........................... 136 4.3.1 Chemicals and reagents ........................... 136 4.3.2 Animal and lgA antibodies injection .................. 136 4.3.3 lgA-secreting hybridoma cell cultures ................. 137 4.3.4 lgA purification by TNP-BSA immuno-affinity gel ........ 137 4.3.5 Hematuria analysis ............................... 138 4.3.6 ELISAS ........................................ 139 4.3.7 Quantitation Of mesangial lgA, lgG, lgM and C3 ........ 140 4.3.8 Electron microscopy .............................. 141 4.3.9 Statistics ...................................... 142 4.4 RESULTS ........................................... 143 4.5 DISCUSSION ....................................... 162 CHAPTER 5. SUMMARY AND FUTURE STUDIES ................... 171 LIST OF REFERENCES ........................................ 174 viii LIST OF TABLES Table Page Table 1.1. Cytokines contributing to differentiation of lgA secretion cells . . . 47 Table 4.1. The effects of monoclonal lgA administration on serum lgA accumulation in B6C3F1 mice ............................... 144 Table 4.2. The effects of monoclonal lgA administration on serum IgA immune complexesformation in B6C3F1 mice .................. 145 Table 4.3. The effects Of monoclonal lgA administration on serum IgG accumulation in BGC3F1 mice ............................... 146 Table 4.4. The effects of monoclonal lgA administration on serum lgM accumulation in B6C3F1 mice ............................... 148 Table 4.5. The effects of monoclonal lgA administration on serum lgA-casein complexes formation in 86C3F1 mice ................ 149 Table 4.6. The effects Of monoclonal lgA administration on serum lgG-casein complexes formation in BGC3F1 mice ................ 150 Table 4.7. Erythrocyte counts in urine of B6C3F1 mice injected with monoclonal lgA .......................................... 151 Table 4.8. The effects Of monoclonal lgA administration on lgA, lgG, IgM and C3 deposition in the kidney Of the B6CBF1 mice .......... 152 Table 4.9. The effects of monoclonal lgA administration on serum Igs accumulation and lgA immune complexes formation in BALB/C mice . 155 Table 4.10. Erythrocyte counts in urine Of BALB/C mice injected with monoclonal lgA .......................................... 156 Table 4.11. The effects of monoclonal lgA administration on lgA, lgG, IgM and C3 deposition in the kidney Of the BALB/C mice ......... 156 Table 4.12. The effects of lgA-secreting hybridoma cell injection on serum IgA accumulation in BALB/C mice ............................ 158 Table 4.13. The effects Of lgA-secreting hybridoma cell injection on serum lgA immune complexes formation in BALB/C mice ............... 159 Table 4.14. The effects Of lgA-secreting hybridoma cell injection on serum IgG accumulation in BALB/C mice ............................ 160 Table 4.15. Erythrocyte counts in urine Of BALB/C mice injected with lgA-secreting hybridoma cell ................................ 160 Table 4.16. The effects Of lgA-secreting hybridoma cell injection on lgA, lgG, lgM and C3 deposition in the kidney of the BALB/C mice ...... 161 Table 4.17. ELISA reactivity Of representative monoclonal lgA supernatants with antigen panel (Rasooly et al,, 1994) ...................... 170 LIST OF FIGURES ELCIIILe m Figure 1.1. Structure Of trichothecenes .............................. 8 Figure 1.2. Classification Of trichothecenes based on their chemical structure ................................................. 10 Figure 1.3. Examples of some naturally identified trichothecenes ......... 11 Figure 2.1. Effect Of oral exposure Of mice to vomitoxin on lgA secretion in Peyer's patch cell cultures (2 hr after gavage) .................. 60 Figure 2.2. Effect Of oral exposure of mice tO vomitoxin on lgA secretion in Peyer's patch cell cultures (24 hr after gavage) ................. 62 Figure 2.3. Effect Of oral exposure of mice tO vomitoxin on lL-5 production in Peyer's patch cell cultures (2 or 24 hr after gavage) ............. 64 Figure 2.4. Effect Of oral exposure of mice to vomitoxin on lL-6 production in Peyer's patch cell cultures (2 hr after gavage) .................. 66 Figure 2.5. Effect Of oral exposure Of mice to vomitoxin on IL-6 production in Peyer's patch cell cultures (24 hr after gavage) ................. 68 Figure 2.6. Effect Of oral exposure Of mice tO vomitoxin on IL-6 production in spleen cell cultures (2 hr after gavage) ....................... 70 Figure 2.7. Effect Of-cytokine-Specific neutralizing antibodies on mitogen-driven lgA production in Peyer's patch cell cultures isolated from mice exposure to vomitoxin (2 hr after gavage) ........ 73 Figure 2.8. Effect Of cytokine-specific neutralizing antibodies on mitogen-driven lgA production in spleen cell cultures isolated from mice exposure to vomitoxin (2 hr after gavage) ............... 75 Figure 3.1. lgA production by complete or macrophages-depleted Peyer's patch cell cultures isolated from mice following exposure to vomitoxin .............................................. 98 Figure 3.2. lgA production by complete or macrophages-depleted spleen xi cell cultures isolated from mice following exposure to vomitoxin ..... 100 Figure 3.3. lL-6 production by complete or macrophages-depleted Peyer’s patch cell cultures isolated from mice following exposure to vomitoxin ............................................. 103 Figure 3.4. IL—6 production by complete or macrophages-depleted spleen cell cultures isolated from mice following exposure to vomitoxin ..... 105 Figure 3.5. lgA and IL-6 production in reconstituted cultures containing macrophages-depleted Peyer's patch cells and peritoneal macrophages (20:1 ratio) isolated from mice exposure to vomitoxin . . 108 Figure 3.6. lgA and IL-6 production in reconstituted cultures containing macrophages—depleted Peyer's patch cells and peritoneal macrophages (5:1 ratio) isolated from mice exposure to vomitoxin . . . 110 Figure 3.7. lgA and IL-6 production by purified Peyer's patch 8 cells reconstituted with purified peritoneal macrophages or splenic CD4“ T cells isolated from mice exposure to vomitoxin ........... 114 Figure 3.8. lgA production in reconstituted cultures (Transwell) containing macrophages-depleted Peyer's patch cells and peritoneal macrophages (20:1 ratio) isolated from mice exposure to vomitoxin . . 118 Figure 3.9. Costimulatory signals between T and B cells, and T cell and macrophage ......................................... 128 Figure 4.1a. Electron micrograph Of a glomerulus from a mouse injected with vomitoxin-induced monoclonal lgA after 6 weeks ............. 153 Figure 4.1b. Electron micrograph Of a glomerulus from a control mouse injected with PBS after 6 weeks .............................. 153 Figure 4.2. IgA and lgG-casein complexes formation ................. 165 xii LIST OF ABBREVIATIONS Ab antibody Ag antigen ANOVA analysis of variance BSA bovine serum albumin CHX cycloheximide C3 complement 3 CT cholera toxin DAS diacetoxyscirpenol DNP dinitrophenyl Con A concanavalin A DMEM Oulbecco's modified Eagle medium ELISA enzyme-linked immunosorbent assay FBS fetal bovine serum IC immune complexes lFN-y interferon-gamma lg immunoglobulin lgAN immunoglobulin A nephropathy lL interleukin ION ionomycin LPS lipopolysaccharide Md) macrophages xiii NIV nivalenol PBS phosphate-buffered saline PC PMA phosphorylcholin phorbol 12-myristate-1 3-acetate PP SRBC T-2 Peyer's patch sheep red blood cells T~2 toxin TGF—B TN F—Ot transforming growth factor-beta tumour necrosis factor-alpha TNP trinitrophenyl vomitoxin xiv INTRODUCTION Vomitoxin (VT) is a fungal secondary metabolite that belongs to a family Of mycotoxins referred to as trichothecenes (Tanaka et.al., 1988). This toxin is frequently found in cereal grains as well as other food and agricultural products (Abouzied et al., 1991; Rotter et al., 1996). The toxicological effects of VT as well as other trichothecene toxins include digestive disorders, skin inflammation, hemorrhagic syndrome, destruction of bone marrow, and nerve disorders (Ueno, 1983). One important toxicological effect of VT is its capacity to modulate immune function. VT can be both immunostimulatory and immunosuppressive in a variety Of animal and cell culture models depending on dose, experimental animal, and targeted immune function (Pestka and Bondy, 1994). In mice, dietary VT exposure induces extremely high levels of serum IgA (Forsell et al., 1986; Pestka et al., 1989), increases the circulating lgA immune complexes (lgA-IC), and causes glomerular lgA deposition and hematuria (Pestka et al., 1989; Pestka and Bondy, 1990; Dong et al., 1991; Dong and Pestka, 1993; Rasooly and Pestka, 1994; Greene et al., 1994a; 1994b). These symptoms are very similar, clinically, to human lgA nephropathy (Berger's Disease), which is the most common type of human glomerulonephritis world wide. Increases in percentages of membrane IgA“ cells and lgA-secreting cells in Peyer's patch (PP) and spleens of VT—fed mice occur concurrently with these effects (Pestka et al., 1990a; Bondy and Pestka, 1991), suggesting that VT can stimulate lgA secretion. Furthermore, polyspecificity and autoreactivity Of monoclonal lgA antibodies (Abs), typically have been identified in 2 hybridomas derived from PP Of VT-fed mice (Rasooly et al., 1994). Notably, elevation Of mesangial lgG and CS deposition and hematuria was observed in mice after injection of VT-induced monoclonal lgA Abs, thus suggesting that these polyreactive lgAs might be nephritogenic in the mouse (Rasooly et al., 1994). Cytokines are soluble protein or glycoprotein mediators that have marked regulatory effects in immune system. Cytokines influence 8 cell activation, class- switching, proliferation, and terminal differentiation to lgA-producing plasma cells (MCGhee et al., 1989). T cells are one major source Of helper cytokines for regulating lgA production (MCGhee et al., 1989; Beagley and Elson, 1992; McGhee and Kiyono, 1993; Kihira and Kawanishi, 1995). Previous studies have shown that exposure to VT i_n_vjtr_o superinduces interleukin (lL)-2, lL-4, IL-5 and lL-6 mRNA expression in murine splenic CD4+ T cells stimulated with concanavalin A (Con A) or phorbol myristate acetate (PMA) (Ouyang et al., 1995; 1996a; Azcona-Olivera et al., 19953; Warner et al., 1994) as well as PMA-stimulated EL-4 thymoma cultures (Dong et al., 1994). These cytokines have previously been shown to enhance differentiation of B cells to lgA secretion (Lebman et al., 19908; 1990b; Coffman et al., 1987; 1988; Beagley et al., 1988; 1989; POCkIey and Montgomery, 1991 a; Dieli et al., 1995). Recently, AzconaI-Olivera et al., (1995b) demonstrated that acute oral VT exposure to mice elevates cytokine mRNA levels with maximal effects occurring within 2 hr in the 25 mg/kg BW groups. VT has been previously shown to elevate the percentages of T cells, CD4+ T cells and CD4‘ICD8+ T cell ratios in PP and spleens of VT-fed mice (Pestka et al., 1990a). In vitro studies have 3 demonstrated that VT plus CD4” T cells can significantly increase lgA production in B cells (Warner et al., 1994). In these investigations, VT also increased lL-6 secretion in Con A-stimulated 004* T cells. Significantly elevated lgA production was also observed when PP T cells isolated from VT-fed mice were co-cultured with B cells (Bondy and Pestka, 1991). These findings suggest that T cells might play a role in regulating VT-induced lgA production. Macrophages (MD) are another important source of cytokines, some of which are capable of modulating T and B cell responses. Md) have been demonstrated to be major producers Of lL-1, IL-6 and TNF-a within the immune system (Bauer et al., 1988; Bauer, 1989). Recently, VanCott et al., (1996) suggested that lL-6 secreted by Md) may contribute to development of mucosal lgA responses. VT has been previously shown to stimulate IL-1 release from peritoneal Md) in vitro. Azcona- Olivera et al., (1995b) and Zhou et al., (1997) recently demonstrated that acute oral VT exposure in mice hyperelevates proinflammatory cytokines lL-6, lL-1 B, TNF-a and IFN-y mRNA expression in spleen and PP. Wong et al., (1997) have recently demonstrated that VT can superinduce lL-6 and TNF-a in a Cloned murine Md) cell line. Thus it is reasonable to suggest that VT might alter regulation of lgA. Based on the Observations of elevation cytokine gene expression, lgA production and lgA nephropathy after oral VT exposure in mice, we hypothesized that: (1) VT induces the specific stimulatory effect on lgA production via superinduction Of helper cytokines; (2) VT stimulation of lgA production is mediated via the Md); and (3) polyreactive lgA Abs induced by W contribute to experimental 4 nephropathy. The Objectives Of my research were as follows: 1. TO assess the effects of acute oral VT exposure on IgA and cytokine production in PP and spleen cell cultures in vitro. . To assess the effects Of cytokine-specific neutralizing monoclonal Abs on lgA production in PP and spleen cultures isolated from mice exposed orally to VT. . TO determine the effects Of Md) depletion on in vitro lgA and lL-6 production by PP and spleen cultures following acute oral VT exposure in mice. . TO compare the effects Of acute oral VT exposure on IL-6 secretion by Md) and CD4+ T cells and determine their capacity to increase lgA production. . TO assess whether cognate interactions and/or soluble factors contribute to Md)-mediated enhancement of IgA production following acute oral VT exposure in mice. . TO induce experimental nephropathy by injection of VT-induced lgA Abs into mice. The dissertation is composed Of five chapters. Chapter 1 is a review Of the literature on VT and other trichOthecenes, IgA nephropathy, and cytokines and IgA production. Chapter 2 describes the potential role for cytokines in enhanced lgA secretion by PP cells isolated from mice acutely exposed to VT. Chapter 3 describes the role of Md) in elevated lgA and IL-6 production by PP cultures 5 following acute oral VT exposure. The results from both chapter 2 and 3 also identify the early mechanisms for VT—induced IgA nephropathy. Chapter 4 describes the induction Of nephropathy by injection with dietary VT-induced lgA monoclonal Abs into mice and identifies the late mechanisms for VT-induced lgA nephropathy. Finally, chapter 5 summarizes these inter-related studies and makes suggestions for future studies. CHAPTER 1 LITERATURE REVIEW 7 1.1 Deoxynivalenol (vomitoxin) and other trichothecenes 1.1.1 History Deoxynivalenol (vomitoxin) is a fungal secondary metabolite that belongs tO a family Of mycotoxins referred to as trichothecenes (Tanaka et al., 1988). Deoxynivalenol (DON) was first identified in Japanese barley contaminated with Fusarium spp and given the trivial name Rd toxin (Morooka et al., 1972). Subsequently, it was named vomitoxin (VT) due to emesis (vomiting) in swine caused by Fusarium-infected corn (Vesonder et al., 1973). This toxin was also detected in culture of Fusarium graminearum by Yoshizawa and Morooka, (1973). VT was structurally identified as a trichothecene (Vesonder et al., 1973) and given the chemical name DON (Yoshizawa and Morooka, 1973). VT is important economically due to the prevalence Of grain and cereal products contamination by this toxin (Scott, 1989) and is a cause for a concern to human and animal health. 1.1.2 Chemical structure Trichothecenes consist Of oxygen, hydrogen. and carbon elements and they possess a tricyclic, epoxide ring system (Figure 1.1). All natural occurring trichothecenes contain a double bond at position C-9, 10 and an epoxy group at position C-12, 13 which are basic requirements for toxicity and biological activity (Ueno, 1980; Bamburg, 1983). The biological activity is completely lost after the epoxide group is reductively removed (Patterson, 1973). The trichothecenes 0:3 ‘° H O 2 H 1\ R 9 13¢03 1 6 R5 1‘5 i 12 4 H I ?"2 I14 R2 R4 R3 CH3 Figure 1.1. Structure of trichothecene 9 Dear oxygen-containing substituents located at one or more of positions 3, 4, 7, 8 and 15. These substituents may be hydroxyl, esterified hydroxyl, keto (position 8 only), or epoxide (position 7 and 8 only) groups or combinations thereof (Ueno, 1980). The trichothecenes have been classified into four groups according to their structural characteristics (Ueno, 1980) (Figure 1.2). VT belongs to group B of the trichothecenes and possesses a carbonyl group at position 08 (Ueno, 1980). VT has structural similarities to several other trichothecene toxins such as T-2, nivalenOI (NIV), and diacetoxyscirpenol (DAS) produced by strains of Fusarium, whose chemical structure is shown in Figure 1.3. The natural trichothecenes are colorless, mostly crystalline, optically active solids which are generally soluble in moderately polar organic solvents but only very slightly soluble in water (Ueno, 1980). Alcohol derivatives have a higher solubility in water than their esterified homologue (Betina, 1989a). These compounds are stable in the solid state but can undergo reactions in solution. For example, esters are saponified by treatment with alkali and the 12-13 epoxide is Opened by strong mineral acid (Ueno, 1977b). 1.1.3 Natural occurrence Each year, about 25% Of the world’s food crops are contaminated by . mycotoxins (Mannon and Johnson, 1985). Currently, over 148 trichothecenes have been isolated and identified (Scott, 1990; Buck and Cote, 1991), and the most Group D Figure 1.2. Classification of trichothecenes based on their chemical structure (from Ueno, 1980). 11 cu, it. o " 9"“ cu, ? 0 a 9H,, 0 ‘3 O a curéoo-Naé 02:20" . ONE cu, a-“ . 3 ’ cuzou Group A Group B Trichothecenes Group A R T-2 toxin A (CH3)ZCHCH2COO- Diacetoxyscirpenol A H Nivalenol 8 OH Deoxynivalenol (vomitoxin) B H Figure 1.3. Examples of some naturally identified trichothecenes (from Scott, 1990). 12 commonly encountered by animals and humans are VT and T-2 toxin (Council for Agriculture Science and Technology, 1989). In both US. and Canada, VT is the most important trichothecene in cereal grains (Scott, 1990). VT is a naturally occurring metabolite and produced predominantly by Fusarium graminearum which is a principal cause Of head blight in wheat (Sutton, 1982). They are frequently found in cereal grains as well as other food and agricultural products (Abouzied et al., 1991; Rotter et al., 1996). A number of reports have indicated that VT is the major toxicant in grains in Italy, Austria, South Africa, England, Canada, and United States (Vesonder and Ciegler, 1979; Bottalico et al., 1984; Ueno, 1984). Wide- spread occurrence Of VT in cereal products has been also reported in France, West Germany, Japan, China, Taiwan and Russia (Jemmali et al., 1978; Blaas et al., 1984; Tanaka et al., 1985; Ueno et al., 1986). Thus, VT has been widely detected throughout the world in wheat, barley, corn, rice, mixed feed, and other products (Mirocha et al., 1977; Jemmali et al., 1978; Yoshizawa et al., 1979). 1.1.4 Toxin production and toxicity The genus Fusarium contains important mycotoxin-producing species (Marasas et al., 1985). Under Optional conditions of temperature and humidity, preharvest cereal grains, especially barley, wheat and corn, can be invaded by Eusarium graminearum (Vesonder et al., 1973; Vesonder and Ciegler, 1979; Yoshizawa et al., 1979). Cool and wet conditions favor fungal growth and toxin production (Pathre and Mirocha, 1979; Bamburg, 1983). The production Of VT is l3 enhanced during cold and wet weather due to delayed harvest and extended growth of the fungus on the crop (Vesonder et al., 1978). VT can also be produced during warm, very humid weather (Richardson et al., 1985). Production of VT can continue during storage (Trenholm et al., 1981). These findings indicated that VT contamination is primarily confined tO temperate climate zones of the world and is predominantly a result of field infection rather than storage development. Once VT is produced by the fungus, it persists and cannot be decreased with feed additives, nor does it disappear with time (Wyllie and Morehouse, 1977). VT is heat stable and likely to survive during baking processes (Patey and Gilbert, 1989). It has been demonstrated that VT was not destroyed in the bread baked from naturally contaminated whole wheat flour (Abbas et al., 1985). This evidence suggests that VT is not inactivated during milling and processing of cereal grain products (Scott et al., 1983; Young et al., 1984) and detoxification may be difficult. A single report has indicated that soaking contaminated corn in 10% sodium bisulfite may detoxify VT completely (Hamilton, 1983). In general, detection and diversion Of VT contamination during early stages are important. In the past, the methods that may be particularly useful in detection and identification of VT were based on thin layer or gas chromatography and mass spectroscopy. However, an immunochemical assay which is also effective in determining this toxin has subsequently been developed and applied (Pestka, 1988; Pestka and Casale, 1990; Abouzied et al., 1991). The occurrence of VT in food and feeds creates a risk for humans and to .1 ”U 14 animals that ingest the contaminated products (Ueno, 1977a). It has been reported in one study that 60% of breakfast cereals contained VT (Trucksess et al., 1986). In another survey, the mean concentration Of VT was > 4.0 ug/g in all positive samples that come from various wheat and corn products including oat cereals, mixed grain cereals, and oat- and rice-based products (Abouzied et al., 1991). Due to the toxicity and wide occurrence of VT, some countries have set limits for this toxin in cereal grains. For example, in the US, an advisory level for VT (which has no legal force) is 2.0 pg/g in wheat and wheat products for milling process; 1.0 ug/g in finished wheat products for human consumption; and 4.0 ug/g for wheat and wheat milling by-products used in animal feed (Wood, 1992). Russia has an Official tolerance limit Of 0.5 -1.0 uglg for VT in wheat. In Canada, the guideline level for VT is 2.0 ug/g in uncleaned soft wheat (Van-Egmond, 1989). However, in Romania, a tolerance limit for VT is only 0.005 uglg in feeds (Van-Egmond, 1989). The toxicological effects of W as well as other trichothecene toxins (T-2, NIV and DAS) include digestive disorders, skin inflammation, hemorrhagic syndrome, destruction Of bone marrow, and nerve disorders (Ueno, 1983). A study on the acute toxicity of VT in mice revealed extensive necrosis in the gastrointestinal tract, bone marrow and lymphoid tissue, as well as focal lesions in kidney and cardiac tissue (Forsell et al., 1987). Since ingestion is the usual route of exposure to VT, it has been suggested that the gastrointestinal tract is one of the major target tissues for this toxin (Hunder et al., 1991). LD._.-,0 values Of VT in mice are 70 mg/kg i.p. (intraperitoneal) and 78 mg/kg orally (Forsell et al., 1987), which is less acutely IS toxic than other trichothecene toxins such as NIV, T-2 and DAS (Ueno, 1977b). The LDso Of NIV, T—2 and DAS in the mice (i.p.) is 4.1 mg/kg, 5.2 mg/kg and 23 mg/kg, respectively (Ueno, 1977b). Notably, farm animals have different sensitivities to VT. Swine are most sensitive to VT, refusing feed containing < 2 ppm VT (Trenholm et al., 1984). On the other hand, cattle and poultry may be relatively tolerant tO VT and have been shown to tolerate 2 20 ppm VT (Trenholm et al., 1984; Hamilton et al., 1985). A possible reason for cattle being less susceptible to VT is the ability Of rumen microorganisms that metabolize/detoxify this toxin (King et al., 1984). Furthermore, there is a sex-associated susceptibility to VT in that male animals appear to be more sensitive than female animals (lverson et al., 1995; Greene et al., 1994a; 1994b; Rotter et al., 1994). The half-life (t 1/2) of VT ranges from 2.08 to 3.65 hr in swine which receive VT intravenously (Coppock et al., 1985). Prelusky et al., (1984) observed that the half-life of VT is about 4 hr following oral exposure of VI" to dairy cows. Clearance of VT from turkey plasma was very rapid with the t 1/2 Of 44 min after VT exposure (Gauvreau, 1991). Recently, Azcona-Olivera et al., (1995b) reported that VT is rapidly absorbed from gut with peak levels being detected in plasma at s 30 min after mice received 25 mg/kg BW VT. Notably, at this dose, the initial and terminal elimination half-life were 0.56 and 88.9 hr, respectively. Since VT is Often found in corn and wheat infected by ngarium graminearum, these grains are high risk as an animal feeds. VT-contaminated 16 feeds has been associated with many cases of sublethal toxicosis in animals resulting in feed refusal, reduced weight gain, emesis and diarrhea (Yoshizawa and Morooka ,1974; Vesonder et al., 1976; Yoshizawa et al., 1978). There is also evidence to Show that an increase in hepatic neoplastic nodules occurs in rats fed Fusarium-contaminated corn (Wilson et al., 1985). However, some other results suggest that VT does not have carcinogenic potential and does not have mutagenic activity (Wehner et al., 1978; Rogers and Heroux-Metcalf, 1983; Lambert et al., 1995) Clinical symptoms such as emesis and diarrhea were also Observed in humans who ingested VT in India in 1987 (Bhat et al., 1989)_and similar‘reportes Of human illness were associated with VT in China (LUO, 1988). Several countries have reported outbreaks of wheat toxicoses in humans due to the consumption Of VT in the food infected by Fusarium graminearum (Ueno, 1983), but this toxicosis rarely causes death (LUO, 1988). Some reports indicated that human esophageal cancer may be associated with high levels of VT in Africa (Marasas et al., 1979) and China (LUO et al., 1990). These outbreaks Of disease indicate a need for caution, especially with regard to the potential health effects Of chronic exposure to VT. Taken together, the above studies support the hyp‘Othesis that trichothecene mycotoxins might be the causative toxicants in some food borne intoxications such as alimentary toxic aleukia (ATA) in Russia. mold corn toxicosis (most frequently associated with VT) in the United States, and red mold disease in Japan (Ueno et 17 al., 1972a; 1972b). Therefore, contamination Of VT in cereal grain products is a considerable food safety concern in many countries. 1.1 .5 lmmunotoxicity VT and other trichothecenes are potent protein synthesis inhibitors (Ueno, 1985; Betina, 1989b; Pestka and Casale, 1990) that can significantly alter cell- mediated immunity, humoral immunity and host resistance in animal models (Pestka et al., 1987; Pestka and Bondy, 1990). VT may inhibit elongation steps of protein synthesis by interaction with the 608 ribosomal subunit and suppression Of peptidyl transferase activity (Bamburg, 1983; Kiessling, 1986). Robbana-Barnat et al., (1985) reported that cardiac protein synthesis was decreased about 30% after intraperitoneal administration Of VT. Recently, Azcona-Olivera et al., (1995b) Observed that more than 70% Of protein synthesis is inhibited for as long as 9 hr in all tissue of mice receiving 25 mg/kg BW VT. Trichothecenes are acutely toxic to actively dividing cell populations in tissues such as bone marrow, thymus, lymph nodes, spleen and intestinal mucosa (Ueno, 1977b). Acute and chronic toxicities of trichothecenes cause depletion of lymphoid tissues (Ueno et al.. 1972a) which induces disorders of the immune system. One interesting finding is that VT can be both immunosuppressive and immunostimulatory (Pestka et al., 1987; Pestka and Bondy, 1994). Some studies demonstrated that VT was capable of producing immunosuppressive effects in experimental animals (Tryphonas et al., 1984; 1986), which may lead to increased l8 incidence of infection and disease (Vanyi and Sandor, 1988). VT can decrease murine thymus weight, humoral response, and inhibit murine splenocyte as well as rat and human lymphocyte cellular proliferation (Atkinson and Miller, 1984; Robbana-Barnat et al., 1988,). It is possible that impaired immunologic responsiveness may reduce resistance to infection and therefore cause animal disease (Pier et al., 1980). For example, it has been reported that oral VT exposure to mice results in reduced ability to resist Listeria monocytogenes and significantly increased splenic Listeria counts (Tryphonas et at, 1986; Pestka et al., 1987). In addition to the suppressive effects of VT, it also has the stimulatory effects on immune response. In mice, dietary VT exposure induces extremely high levels Of serum IgA (Forsell et al., 1986; Pestka, et al., 1989) and IgE (Pestka and Dong, 1994) as well as decreased lgG and lgM levels (Forsell et al., 1986; Pestka, et al., 1989). Subsequently, Pestka et al., (1989) and Greene et al., (1994a) found that the Optimal VT level for serum IgA elevation was at 25 ppm. In addition to increasing total amount of serum lgA, VT can also increase the polymeric/monomeric lgA ratio in serum (Pestka et al., 1989). A study on the effects of lgA reactivity to casein (a component of the diet), and cholera toxin (CT), indicated that VT can enhance antigen (Ag)-specific lgA and diminish Ag-specific lgG production (Pestka et al, 1990b). Casein-specific lgA was significantly increased in mice fed 25 ppm VT for 16 and 20 wks. Elevation of CT- specific lgA secretion was also found in CT-unimmunized mice fed 25 ppm VT for 16 and 20 wks but not in CT-immunized mice fed VT. Both casein and CT-specific IgG were inhibited in VT-fed mice. l_n_yijrg exposure to VT has been also shown to marginally stimulate lgA secretion in cloned B cell line CH12LX (Minervini et al., 1993). However, VT can not elevate lgA production in purified splenic B cell cultures (Warner et al., 1994). VT has been shown to increase the percentages Of membrane lgA+ cells, T cells and CD4+ T cells as well as alter CD4‘ICDS+ cell ratios in PP and spleens Of mice fed 25 ppm VT (Pestka et al., 1990a; Bondy and Pestka. 1991). The effects of VT increasing CD4+ T cells and CD4‘ICD8+ cell ratios implied that T helper cells may play a role in VT-induced lgA secretion (Pestka et al., 1990a). Bondy and Pestka, (1991) demonstrated that significantly increased lgA production occurs when control B cells are CO-cultured with PP T cells isolated from mice fed 25 ppm VT for 8 wks. This contribution from T cells is further supported by recent in vitro studies, where it was demonstrated that lgA production was significantly increased when murine splenic CD4+ T cells were exposed to 50 ng/ml Of VT and Con A for 24 or 48 hr and then CO-cultured with B cells for 7 days with lipopolysaccharide (LPS) (Warner et al., 1994). The T cell helper effects on elevation of lgA production may be mediated by cytokines such as lL-2, lL-4, lL-5 and IL-6. These cytokines have previously been shown tO enhance differentiation of B cells tO lgA secretion (Lebman et al., 1990a; 1990b; Coffman et al., 1987; 1988; Beagley et al., 1988; 1989; Pockley and Montgomery, 1991a; Dieli et al., 1995). Recent studies have shown that exposure to VT in vitro superinduces cytokine lL-2, lL-4, lL-5 and IL-6 mRNA expression in 20 murine splenic CD4+ T cells stimulated with Con A or PMA (Ouyang et al., 1995; 1996a; Warner et al. 1994; Azcona-Olivera et al., 1995a) as well as PMA-stimulated EL-_4 thymoma cultures (Dong et al., 1994). Azcona-Olivera et al., (1995b) demonstrated that acute oral VT exposure tO mice elevates cytokine mRNA levels with maximal effects occurring in the 25 mglkg BW group in as little as 2 hr. These Observations suggest that VT induced a specific stimulatory effect on lgA production, possibly mediated by elevating T helper cell responses and resulting in enhancement Of cytokine secretion. Beside VT disrupting normal regulation Of lgA production in the mouse, this toxin can alSo increase circulating lgA-IC in serum, and cause glomerular lgA deposition and hematuria in VT-fed mice (Pestka et al., 1989; Pestka andBondy, 1990; Dong et al., 1991; Dong and Pestka, 1993; Rasooly and Pestka, 1994; Green et al., 1994a; 1994b). These symptoms are very similar, clinically, to human IgA nephropathy which is the most common form Of glomerulonephritis world-wide with an unclear etiology (D’Amico, 1987). This information suggests that VT may be a possible etiological factor in IgA nephropathy. Taken together, all these findings are indicative of dysregulation of lgA production by VT. 1.2 IgA nephropathy 1.2.1 History immunoglobulin A nephropathy (lgAN) is a form of glomerulonephritis that has now been recognized to be the most common nephritis leading to end-stage renal failure in the world (D’Amico 1987). The etiology Of this disease is not well- understood. lgAN, first described by Berger and Hinglais (1968), is characterized by the presence of lgA and C3 (complement component) deposits in the mesangium. In this study, all 25 patients had a somewhat similar clinical history consisting Of macroscopic hematuria and moderate proteinuria. In most patients, episodes of recurrent macroscopic (gross) hematuria were Observed. lmmunofluorescence micrOSCOpy revealed diffuse and global glomerular mesangial staining with lgA, with less intense mesangial staining for lgG and C3. In 10 of these original patients, mesangial deposits were also Observed by electron microscopy. Thus, lgAN was also named Berger’ disease. Schena, (1990) indicated that an estimated 20% to 40% patients with lgAN develop end-stage renal failure 5 to 20 years after diagnosis because Of the progressive nature of the disease. Although the pathogenesis Of this disease is not fully understood, high serum levels of IgA and/or polymeric lgA and several kinds Of lgA-class Abs suggest that there is an aberration in the control Of the mucosal immune response resulting in a hyper-immune state Of lgA production in patients with lgAN (Czerkinsky et al., 1986). 22 1.2.2 Geographic distribution Although lgAN appears to be a ubiquitous disorder, its reported geographical distribution is irregular. Investigations from many countries have Shown that the apparent incidence of this disease has varied in studies from different countries. lgAN has been known to be very frequent in Asian-Pacific and South European countries (Schena, 1990). The disease is particularly prevalent and is one Of the most common forms of glomerular disease in Australia (Clarkson et al., 1979), Spain (Navas-PalaCios et al., 1981), Italy (Mandreoli et al., 1981), France (Levy et al., 1973), China (Zhou and Chen, 1986), Hong Kong (’Lai et al.. 1985), Singapore (Sinniah et al., 1981) and Japan (Shirai et al., 1978). In these countries, patients with lgAN may display more serve renal lesions and have a high rate Of progressive renal insufficiency leading to end-stage renal failure. The frequency noted on renal biopsy examination ranges from 12 to 40 percent. However, much lower rates of incidence have been observed in the United States (McCoy et al., 1974; Lee et al., 1982), England (Sissons et al., 1975), Germany (Michalk et al., 1980), Canada (Katz et al., 1976), India (Kher et al., 1983) and the Netherlands (van-der-Peet et al., 1977) with the frequency rate ranges from 2 to 8 percent in these countries. Thus, the high frequency of lgAN in certain countries in contrast to the lesser prevalence in others suggests that some geographical, genetic, or dietary factor may be relate to the development of this disease. 1.2.3 Clinical features 23 The Clinical presentation Of lgAN covers a wide range from isolated hematuria tO rapidly progressive renal failure (Nicholls et al., 1984). Most patients with this disease display mild to moderate mesangial proliferation associated with increase mesangial electron-dense deposits which are Often preceded and accompanied by hematuria (Emancipator et al., 1987; Hisano et al., 1991). Kidney damage is revealed as hematuria and proteinuria. Macroscopic hematuria was thought initially to be the presenting symptom in the majority Of patients with lgAN. The hematuria is painless, but is associated with systemic symptoms such as fever, malaise, fatigue, diffuse muscle aches, and abdominal pain (Walshe et al., 1984) as well as loin pain (MacDonald et al., 1975). Andreoli et al., (1986) reported that patients with heavy proteinuria have the most severe glomerular lesions and least favorable prognosis. Notably, many reports have indicated that the clinical features of lgAN in adults and children patients are different. These include elevated frequency Of proteinuria (Mina et al., 1985), serum levels of lgA (Clarkson et al., 1977), and systemic hypertension (Morel-Maroger et al., 1972; Droz et al., 1984) in adults as well as increased frequency of gross hematuria in children (Mina et al., 1985). In France, gross hematuria is the presenting symptom in 40% of adults and 80% of Children (Levy et al., 1973). Furthermore, Abuelo et al., (1984) Observed that 41 percent of patients with rapidly progressive form of lgAN were 16 years of age or younger, and suggested that younger patients (under 18 years of age) may be predominantly involved in this end-stage renal disease occurred. 24 There is a sex-related susceptibility to lgAN in that female patients appear to be associated with minor glomerular lesions as compared with males, either in adults or in children (Droz et al., 1984). This may account for the better prognosis found in females. lntereStingly, lgAN is rare in the black population and is more common in black female (Jennette et al., 1985). 1.2.4 Laboratory features Since the initial studies Of lgAN were characterized by staining of glomerular mesangial regions with antisera to lgA and IgG (Berger and Hinglais, 1968), positive mesangial immunofluorescence for lgA as the predominant lg is the diagnostic hallmark and requires renal biopsy. It has been reported that serum levels Of lgA are increased in about 50% of patients with lgAN (D’Amico, 1983), and there was a persistent elevation of serum lgA in many patients during the three year follow-up period. Such enhancement of serum lgA may be contribute to the persistent deposition Of lgA in the mesangial areas. The finding Of high serum levels Of polymeric lgA in patients with lgAN is possibly associated with these Observations (Trascasa et al., 1980). lgA is the primary lg associated with the mucosal membrane. There, lgA is dimerized and transformed into secretory lgA by a secretory component and J chain. About 70% of the, lgA eluted from renal biopsies from patients with lgAN is polymeric lgA (Monteiro et al., 1984). The importance Of the presence of dimeric or polymeric lgA in the Circulating lC in relation to the clinical activity Of the lgAN has 25 been further confirmed by Valentijn et al., (1984), where it was demonstrated that glomerular lgA deposits in lgAN is mostly lgA1 and polymeric. Mestecky et al., (1987) also verified that circulating lC isolated from patients with lgAN contain polymeric lgA1. Therefore, it is possible that ‘Overproduction of polyclonal or antigen-specific lgA1 and/or its defective Clearance may be associated with the pathogenesis of lgAN (Emancipator et al., 1989). lgA containing lC have been well demonstrated in the circulation of patients with lgAN (Woodroffe et al., 1980; Lesavre et al., 1982; Czerkinsky et al., 1986), and they suggest that lgA-IC may play a important role in the pathogenesis of lgAN. Woodroffe et al., (1980) indicated that dysregulation Of lgA production against dietary Ag or pathogens leads to form circulating lgA-lC and subsequent deposition in the kidney result in glomerular damage. Circulating IC were present intermittently and corresponded with episodes of gross hematuria. Woodroffe et al., (1980) had demonstrated that IC also contains the lgG class. The mixed lgA-lgG IC have been detected in more than 40% Of serum from lgAN patients (Czerkinsky et al., 1986). The Circulating IC are intermediate in size (9 to 17s) and contained lgA, lgG and, less commonly, lgM (Woodroffe et al., 1980; Lesavre et al., 1982). Hall et al., (1983) indicated that lgA-IC are more prevalent in the early stages Of the disease, and suggested that the initial immunologic insult to the glomerulus occurs most frequently in childhood and adolescence. Although the early reports from Berger and Hinglais (1968) indicated that characterization of lgAN is predominant deposition of lgA in the renal mesangium, 26 there other lgs are associated with this disease. According to the statistical results from different countries by D’Amico, (1983), I96 is found in combination with lgA in more than 50,percent Of the lgAN patients examined in most countries. About 25 to 30 percent Of biopsy specimens from patients with lgAN contain lgM class. The Southwest Pediatric Nephropathy Study Group, (1982) proposed that lgM deposits may occur as a secondary process deposits. C3 deposition parallels lgA in both distribution and intensity in most patients (more than 80%) with lgAN (D’Amico, 1983). However, the classical pathway complement components, C1 and C4, are usually absent or present in a lower percent (10% of patients) and exhibit low intensity staining. On the other hand, properdin is Often present in 50% to 100% Of patients with lgAN (Katz et al., 1976). C3 and properdin in the glomerular immune deposits have been assumed to indicate a predominant role for the alternative complement activation pathway in this disease (McCoy et al., 1974). A previous study has demonstrated that lgA-IC can activate the alternative complement pathway (Gotze and Muller-Eberhard, 1971 ). Julian et al., (1983) also Observed that certain types Of lgA aggregates or lgA myeloma complexes can activate complement in vitro. All these observations suggest that lC (including lgA-IC) may activate C3 through the alternative complement pathway, resulting in generation of activation fragments with the potential for mediating inflammatory injury in the glomeruli. Sakai (1988) suggested that cytokines may play an important role in the etiology and pathogenesis of lgAN. One interesting observation is that lgAN 27 patients have high urinary lL-6 activity (Dohi et al., 1991). Other studies have demonstrated that there is an increase in IL-5, lL-6 and TGF-B mRNA levels in CD4‘Tcells (de-Caestecker et al., 1993; Lai et al., 1994a; 1994b) as well as lL-4, lL-5 and IL-6 mRNA levels in peripheral blood mononuclear cells of lgAN patients (Ichinose et al., 1996). 1.2.5 Animal models Several animal models of lgAN can elicit clinical and morphologic features which closely resemble the human syndrome. The first experimental model Of lgAN employed IC Of the hapten-specific mouse myeloma lgA anti-dinitrophenyl derived from MOPC 315 plasmacytoma and dinitrophenyl hapten conjugated to bovine serum albumin (DNP-BSA) in BALB/C mice (Rifai et al., 1979). In this study, passive injection of lC of lgA and DNP-BSA, or separate injections of these two compounds, resulted in deposition of IgA and DNP-BSA in the mesangium. Similar depositions but more severe pathological changes were Observed upon injection Of DNP-BSA alone into MOPC 315 tumor-bearing mice. The degree Of deposition was associated with the size and dose Of complexes. Furthermore, Rifai and Millard, (1985) further demonstrated that lC induce C3 deposits and elicit hematuria. Complexes prepared with dimeric or polymeric lgA regularly generated mesangial deposits, whereas monomeric lgA at the same dose and degree of Ag excess failed to cause deposition. This may be related to the predominance Of polymeric lgA-IC occurring in glomerular deposits Of this disease. Thus, Rifai and 28 colleagues reproduced the clinical and pathologic Observations most frequently found in patients with lgAN. They proposed that the human disease is lC in nature, the Ab being predominantly of the lgA class. Induction of glomerular lgA deposits (lgA/lgA-IC) was also Observed in mice following administration of lgA anti-DNP (Ab) and DNP-conjugated lgA anti- phosphorylcholine (as an Ag) (Montinaro et al., 1991). This evidence demonstrated that the Ag plays a critical role in development of glomerulonephritis associated with lgA—IC. Some other studies have demonstrated that glomerular lgA, C3 and, with less amounts, lgM and lgG deposits were induced in Swiss Webster mice (lsaacs et al., 1981; lsaacs and Miller. 1982) or in Lewis rats (Fornasieri et al., 1993) by active injection of different sized (10, 70 and 500 kD) and different charged dextrans (neutral dextran, dextran sulfate and diethylaminoethyl-dextran). Both treatment mice and rats also developed hematuria. These Observations suggest that size and charge Of lgA-lC may be important factors in formation of glomerular deposits. Emancipator et al., (1983) developed another murine model Of lgAN by active oral immunization with proteins. In this experiment, after protracted oral immunization with three different protein Ags (bovine gamma globulin, ovalbumin and ferritin) for 14 wks. mice exhibited a heightened mucosal Ab response to these Ags with significant increases in serum lgA Ab and mesangial deposits of IgA. The immunofluorescence observations and ultrastructural appearance of glomeruli closely resemble those from patients with lgAN. These observations suggest that 29 prolonged mucosal immunization can induce a specific lgA response that leads to lgA-containing IC deposits in the mesangium. Emancipator et al., (1987) further demonstrated that serum lgA, lgG and lgM Abs were significantly elevated after oral immunization with protein Ags such as bovine gamma globulin to mice. In addition to mesangial deposition of lgA and oral immunogen, codeposition of lgG, lgM and C3 as well as microhematuria were also detected in these treatment mice. The investigators suggested that IgG and lgM codeposits in murine lgAN can induce the deposition Of complement, which is turn contributes to glomerular injury. Subsequently, a similar model was developed in Wistar and Lewis rats (Gesualdo et al., 1992). In this study, Lewis rats Showed predominantly glomerular lgA deposits with lesser lgG and C3 after 8 wks of continuous oral immunization with bovine gamma globulin. These immunized rats also developed microhematuria with negligible proteinuria. Other investigators also reported that oral immunization with gluten or ferritin resulted in elevation of serum IgA and mesangial lgA deposition as well as increase of serum anti-gluten or anti-ferritin Abs in BALB/C mice (Coppo et al., 1989) or in C3H/HeJ mice (Genin et al., 1986). Jessen et al., (1987) have induced an experimental model Of lgAN by injection Of Sendai virus into mice Since viral Ags have been implicated in the pathogenesis of human lgAN (Tomino et al., 1989). Immunization of mice with live viruses or viral protein extract resulted in a prevalent serum lgA (and lgG) anti-viral immune response, associated with mesangial deposition of lgA, C3 and viral Ag. Subsquently, Jessen et al.. (1992) further demonstrated that immunized mice 30 challenged with either live or dead virions showed a high incidence of hematuria. These results suggest that this animal model may be useful to probe infection- related lgAN. Interestingly, a spontaneous animal model for primary lgAN has been reported by lmai et at (1985) in ddY mice. ddY mice Older than 40 wks have a marked accumulation of lgA and C3 in their glomeruli and elevated serum lgA levels. In contrast to other animal models, the glomerulopathy and glomerular immune deposits developed without the deliberate admInistration Of exogenous Ag. The investigators suggest that mesangial lgA deposits might associate with the onset of retrovirus-induced tumors spontaneously occurring in ddY mice. This contention is supported by the Observation where the murine retroviral envelope glycoprotein, gp70, was deposited in the glomerular mesangial areas in ddY mice (Takeuchi et al., 1989). As mentioned earlier, Pestka and colleagues have Observed that dysregulation of lgA production and lgAN were induced by feeding the trichothecene VT in the murine model. Pestka et al., (1989) reported that increase serum lgA production after feeding 25 ppm VT into mice with beginning at 4 wks of exposure and peaking at 24 wks. Immunofluorescence staining showed marked accumulation of mesangial IgA and electron microscopy revealed electron-dense deposits in the glomeruli Of VT- fed mice. Subsequently, Dong et al., (1991), Dong‘ and Pestka, (1993), Rasooly and Pestka, (1994), and Greene et al., (1994a; 1994b) further demonstrated that dietary VT exposure induces elevation Of serum IgA and 31 Circulating lgA-IC as well as causes glomerular lgA deposition and hematuria in mice. All these Observations suggest that dietary exposure VT tO mice can dysregulate lgA production and induce glomerular lgA deposition in the experimental lgAN. 1.3 Cytokines and IgA production 1.3.1 Characteristics of cytokines Cytokines can be defined as the soluble protein or glycoprotein mediators that act as intercellular signals and mediate their effects via interaction with specific cell surface receptors on sensitive target cells (Callard, 1990). Cytokine production is transient . and the action radius is usually short. Cytokines can induce the synthesis and release of both positive and negative regulatory cytokines from their target cells. Generally, a cytokine may exhibit autocrine action, binding to the same cell that secreted it. An example is IL-2 secretion and utilization by an inflammatory T cells, or they may exhibit paracrine action, binding tO nearby cells. In a few cases, cytokines can also act systemically in an endocrine manner, binding to a distant cell (Arai et al., 1990). Cytokines have marked regulatory effects in immune system and stimulate and inhibit the growth, proliferation, differentiation of a wide variety of target cells or their secretion of Abs or other cytokines (Arai et al., 1990). Most cytokines have multiple biologic activities that overlap, and there is considerable redundancy, synergy, and antagonism effects during their actions (Paul and Ohara, 1987). Synergistic interactions are likely to occur between cytokines. For example, a combination Of lL-5 and lL-6 has been shown tO enhance lgA secretion in PP B cells cultures (Kunimoto et al.. 1989). There are also many examples of antagonistic interactions among cytokines. One example of this effect is the actions of lL-4 and lFN-y on the synthesis of lg subclasses in B cells (Snapper et al., 33 1988b). All these actions permit cytokines to regulate cellular activity in a coordinated interactive way. Cytokine-producing cells are Often physically located immediately adjacent to the responder cells (Metcalf, 1991) and generally secrete very small quantities Of cytokine and, in some cases, this is directed towards the responder cells (POO et al., 1988). Many cytokines bind tO elements Of the extracellular matrix around responder cells and increase their bioavailability to the responder cells (Gordon, 1991). An example Of this bound localization is the cell surface cytokines which presumably require cell-cell interaction for their action (Gordon, 1991). 1.3.2 Regulation of IgA production by T cell-secreted cytokines 1.3.2.1 T lymphocytes It is now clear that an appropriate lg response to Ag requires both 8 and T lymphocytes and Ag presenting cells. T lymphocytes have a "control role" in immune responses and have been divided into two groups Of cells expressing either CD4 or CD8 on their cell surfaces. "Helper" function is carried out by CD4*T cells, whereas CD8+ T cells are cytolytic to Ag-bearing target cells. CD4+ T cells have been subdivided further into two distinct categories based on their cytokine secretion profiles. T helper 1 (Th1) cells produce IF N-y, lL-2 and TNF-B but not IL- 4, lL-5, IL-6, or lL-10, and are responsible for cell-mediated immunity, and delayed type hypersensitivity (DTH) responses. In contrast, T helper 2 (Th2) cells produce lL-4, lL-5, IL-6 and iL-10 but not lFN-y or lL-2, and can provide "helper activity" for 34 humoral immune responses including lg isotype switching and secretion (Cherwinski et al., 1987; Mosmann and Coffman, 1989). Finally, T helper 0 (Th0) cells are characterized by lL-2, IFN-y and IL-4 production, and are thought to be precursors Of Th1 and Th2 cells (Street et al., 1990). Cytokines are critically important Since they influence 8 cell activation, proliferation, and terminal differentiation as well as class switching from other isotypes (McGhee et al., 1989). It has been reported that regulation of isotype switching during 8 cells development may be controlled by cytokines secreted during an immune response (Rizzo et al., 1995). For example, lL-4, TGF-B and lL-5, and IF N-y help switching Of lgM to IgE and lgG1, lgA, and lgG2a, respectively (Bond et al., 1987; Coffman et al., 1988; Snapper et al., 19888). 1.3.2.2 lL-2 lL-2 is derived from activated Th1 cells although it functions principally as a T cell growth factor, but it also exerts a range of effects on activated B lymphocytes (Callard, 1990). IL—2 was shown to act on previously activated 8 cells and support the growth and differentiation (Zubler et al.. 1984). B cells growth and lg production can be promoted by lL-2 (Jelinek and Lipsky, 1987). lL-2 has been observed to induce secretion Of lgA from tonsil B cells (Le-thi-Bich-Thuy and Fauci, 1985). Recent studies also show that induction of IL-2-secreting Th1 cells results in induction of high tetanus toxoid-specific lgA responses in the murine intestinal tract (VanCott et al., 1996). This result suggests that IL-2 may contribute a replacement 35 signal for the induction of IgA 8 cell responses. IL-2 was shown to increase lgA synthesis, but this ability was less efficient when compared with lL-5 or IL-6 (Coffman et al., 1991). Although TGF-B has been shown to be a switching factor for murine IgA class (Coffman et al., 1988; Lebman et al., 1990a), it has been reported that the addition of lL-2 to B cell cultures with TGF-B results in increased lgA secretion (Lebman et al., 1990b; lwasato et al., 1994). Nonoyama et al., (1994) also reported that lL-2 significantly elevated the secretion of IgA by anti-CD40-activated B cells cultured in the presence Of lL-10. These findings indicate that IL-2 is capable of inducing the synthesis of lgA when used combination with other cytokines. This contention is supported by recent studies that lL-2 greatly increases lgA production in B cells when in combination with lL-5 (Beagley et al., 1995) or with TGF-B (Min et al., 1996). 1.3.2.3 lL—4 lL-4 is a pleiotropic cytokine derived from Th2 cells with multiple biological effects on B cells (Ohara, 1989). In the mouse, 8 cell proliferation is augmented by lL-4 (Paul and Ohara, 1987). This cytokine acts by elevating the frequency of B cells responses following T cells help (Noelle et al., 1991). lL-4 is known to accelerate lg secretion from activated 8 cells (Jelinek and Lipsky, 1988). Although lL-4 is well knOWn as a switching factor for IgE and lgG1 (Bond et al., 198.7), it also enhances lgA class switching in splenic B cells and other two 8 cell lines, l.29u and CH12. LX (Shockett and Stavnezer. 1991; Whitmore et al., 1991; McIntyre et al., 36 1995). lL-4 itself plays a dominant role in developing the differentiation of native T cells into Th2-like cell type in vitro and in vivo (Swain etal.,_1990; KOpf et al., 1993). This role was also demonstrated by results that switching T cells to an lL-5 producing phenotype by lL-4 which is required for the induction of lung Th2 mucosal immunity (Coyle et al., 1995). These Th2 cells are important regulators of humoral immunity. 1.3.2.4 lL-5 lL-5 is produced primarily by stimulated but not resting T cells (Altman 1990) and plays a major role in T cell-dependent lgA production in murine system (Harriman et al., 1988). Under the absence Of other signals, a small number of apparently resting B cells can be activated to secrete lg by lL-5 (Lernhardt et al., 1987). Karasuyama et al., ( 1988) demonstrated that murine B cells directly mature into Ig-producing cells upon exposure to lL-5. A critical role for lL-5 is supported by fact that neutralizing anti-lL-5 Ab can inhibit the polyclonal Ab response induced by T cell clones on B cells (Rasmussen et al., 1988). The ability Of lL-5 tO elevate lgA production has been demonstrated by Bond et al., (1987) and Coffman et al., (1987). lL-5 also increases the release of lgA from activated human mucosal B cells (Schoenbeck et al., 1989) and stimulates lgA synthesis through promotion of maturation Of postswitch surface lgA‘ B cells into lgA-producing cells (Sonoda et al., 1992). This evidence is consistent with a recent report of increasing Ag-specific lgA secretion by in vitro addition or in vivo injection Of recombinant IL-5 (Dieli et al., 37 1995) The early finding that lL-5 increases lgA secretion suggested that this cytokine might be an important element of mucosal immunity (Harriman and Strober, 1987) and may ultimately represent a specific switching factor for lgA secretion (McGhee et al., 1989). In agreement with such a notion, an in vivo study by Ramsay and Kohonen-Corish, (1993) demonstrated that murine specific mucosal lgA responses are markedly elevated following intranasal immunization with recombinant vaccinia virus vector that expressed lL-5 in lung. This effect was inhibited by treating with neutralizing anti-IL-5 AD in mice. This further confirmed that IL-5 plays a critical role for Selectively increasing the development Of mucosal lgA responses. Interestingly, it has been reported that lL-5 alone, or together with lL-2 appears to augment lgM J chain mRNA synthesis (Matsui et al., 1989). In addition, B cells cultured with LPS plus lL-5 show increased levels of mRNA expression for the secreted form of lgA heavy chain (alpha) (Takatsu et al., 1988). These activities help to explain the potential for IL-5 to increase lgA secretion Early studies indicated that lL-5 could elevate lgA production in the presence of LPS and may act as a possible lgA switching factor (Bond et al., 1987; Coffman et al., 1987; Murray et al., 1987). However, several investigations have subsequently demonstrated that lL-5 enhanced the lgA secretion by acting on surface lgA” B cells that are presumably already committed to lgA synthesis. Thus, IL-5 appears to serve more as a terminal differentiation factor than as an isotype 38 switching factor (Beagley et al., 1988; 1989; Schoenbeck et al., 1989). Studies Of the effect of lL-5 on murine B cells revealed that lL—5 stimulates the secretion Of Abs when used combination with other cytokines such as lL-2 (McHeyzer-Williams, 1989), lL-4 (Murray et al., 1987), and TGF-B (Sonoda et al., 1989). Investigations by Beagley et al., (1995) indicated that lL-5 alone, or in combination with lL-2, greatly increased lgA secretion in murine B cells. lL-4 and IL-5 have been shown to be required for lg class switching and secretion (Hodgkin et al., 1991; Noelle et al., 1991). This was supported by experiments in which IL-4 induced the differentiation Of membrane lgM+ cells to membrane lgA“ types and IL-5 promoted the secretion Of lgA by membrane lgA+ cell types (Kunimoto et al., 1988). It was also found that interaction between lL-4 and lL-5 produced by PP T cells can increase lgA and IgG1 production (Coffman et al., 1987; Murray et al., 1987). Production of IgA can also be induced by the addition Of lL-5 tO TGF-B pretreated B cells (Sonoda et al., 1989). These results suggest that TGF-B is required for early activity while lL-5 appears only to act late in these 8 cell cultures (Sonoda et al., 1989). Recently, McIntyre et al., (1995) reported that cytokines, lL-4, IL-5 and TGF- (3 are required for high rates of IgA class switching to occur. This requirement involved T and B cell interaction. 1.3.2.5 lL-6 lL-6 is a pleiotropic mum-functional cytokine with regulative effects in immune response, inflammation, acute phase response and hematopoiesis (Van- 39 Snick, 1990). lL-6 is produced by T cells as well as a variety Of other cell types including Md), 8 cells, endothelial cells, fibroblasts, and epithelial cells under varied conditions (Van-Snick, 1990). lL-6, as a B cell stimulatory factor, induces terminal proliferation and differentiation Of mitogen- or Ag-activated B cells into lg secreting cells (Kishimoto and Hirano, 1988; Kishimoto, 1989). When lL-6 was added to PP B cell cultures, lgA secretion was increased (Beagley et al., 1989; 1991). In addition, IL-6.induced lgA production is abrogated by specific neutralizing anti-IL-6 AD in pokeweed mitogen (PWM)-activated B cells (Muraguchi et al., 1988). Of particular interest are recent Observations that reduction of mucosal lgA-secreting cells and lgA responses in lL-6-defIcient mice (Ramsay et al., 1994b), and that mucosal lgA responses were restored by intranasal inoculation with recombinant vaccinia viruses directed to express lL-6 in lung (Ramsay et al., 1994b). These studies demonstrate that IL-6 is important for the development of mucosal lgA responses. Purified lL-6 has been shown to elevate lg secretion without bias to particular isotypes (Muraguchi et al., 1988). lL-6 did not induce release Of lgA in membrane- bound lgA-negative B cells. This effect suggests that lL-6 is not an isotype switching factor (Beagley et al., 1989), but acts as a relatively late acting cytokine whose main function is directing terminal 8 cell differentiation (Kishimoto, 1989). Thus, it would appear that both IL-5 and lL-6 are integrally involved in the final differentiation Of lgA-committed B cells into lgA-secreting plasma cells in the mucosal tissue. This has been confirmed by elevation Of mucosal lgA responses 40 due tO lL-5 and IL6 (Ramsay et al., 1994a; Xu-Amano et al.,1994) and decrease number of mucosal lgA-producing cells Observed in lL-6 knock-out mice (Ramsay et al., 1994b). Several reports have suggested that lL-6 has strong activity on B cells in the presence of other cytokines. When PP B cells are stimulated by either cytokine alone, lgA secretion is only modestly increased, but is greatly enhanced by lL-5 and IL-6 in combination (Kunimoto et al., 1989). Some other in vivo studies revealed that lL-5 and IL-6 in combination with Ag can markedly elevate tear lgA response following ocular-topical route administration in the rat (Pockley and Montgomery, 1991a). TGF-B and lL-5, combined with lL-6, were demonstrated to upregulate lgA production in both rat lacrimal and salivary gland cultures (Pockley and Montgomery, 1991b; Rafferty and Montgomery, 1995). These findings offer the prospect Of using cytokines lL-5 and lL-6 as immune modulators in mucosal tissues. 1.3.2.6 TGF-B TGF-B is produced by a variety of cell types including activated T cells (Kehrl et al., 1986a), B cells (Kehrl et al., 1986b) and Md) (Assoian et al., 1987). TGF-B has been shown to specifically indUCe surface lgA’ cells to switch to lgA production (Coffman et al., 1988). CO-incubation of TGF-B with LPS-driven splenic B cells enhances the number of surface lgA+ cells (Lebman et al., 1990a). Thus, TGF-B appears to be an lgA switch factor as opposed to a terminal differentiation factor such as lL-5. Kunimoto et al., (1992) has demonstrated that TGF-B alone, or in combination with lL-4, can induce membrane lgA expression in CH12LX B cell 41 lines. Furthermore, increased production Of lgA by TGF-B is further enhanced by either IL-2 (Lebman et al., 1990b; lwasato et al., 1994; Rafferty and Montgomery, 1995; Min et al., 1996), lL-5 (Sonoda et al., 1989; Rafferty and Montgomery, 1995), lL-6 (Rafferty and Montgomery, 1995) or lL-4 and lL-5 (McIntyre et al., 1995), and lL-5 and IL—6 (Rafferty and Montgomery, 1995). 1.3.2.7 lFN-y lFN-y is a T cell derived cytokine with important immunomodulatory properties in addition tO its anti-viral and anti-proliferative activities (Farrar and Schreiber, 1993). lFN-y is one of the natural 8 cell differentiation factors and displays positive effects on murine B cells. Previous in vitro or in vivo studies have demonstrated that IFN-y could induce lg production (Snapper et al., 1988a; Finkelman et al., 1988). Prabhala and Wire, (1991) reported that in vivo treatment with lFN-y stimulates the mucosal immune system in the female rats reproductive tract by increasing secretory component and lgA levels in the uterine lumen. Recently, Snapper et al., (1995) demonstrated that IFN—y could increase lgA production in the presence Of lL-2 by TGF-B-induced membrane IgA‘ cells. Furthermore, a combination Of IFN—y and lL-4 has been shown to synergistically elevate polymeric lgA receptor levels in human intestinal epithelial cell line HT29 (Denning, 1996). 42 1.3.2.8 Th2 cells and mucosal lgA immune responses It is generally agreed that common muCosal humoral immune responses are predominantly of the lgA isotype (McNabb and Tomasi, 1981 ). lgA is considered to participate in the primary defense Of the host to exogenous pathogens (Kilian and Russell, 1994). The PP in the gut-associated lymphoid tissue are thought be a major source Of B cell precursors for lgA-secreting plasma cells (Craig and Cebra, 1971). PP are believed to be major inductive sites where B cells are activated and committed to lgA secretion (McGhee et al., 1989; Beagley and Elson, 1992). These activated B cells migrate to distant mucosal effector sites, such as lamina propria, where further stimulation and terminal differentiation into lgA producing cell takes place. The preferential switching Of mucosal B cells from lgM+ to lgA+ phenotypes and differentiation to lgA secretion are governed by Th cells which comprise a characteristic and dominant T cell subset in the PP (Kawanishi et el., 1982; 1983; Kawanishi and Mirabella, 1988; McGhee and Kiyono, 1993; Kihira and Kawanishi, 1995). These data suggest that PP contains T cells which specifically regulate the secretion of lgA by B cells (Kiyono et al., 1982). In support of this contention, in MELQ studies have demonstrated that PP T cells can elevate lgA synthesis in LPS- stimulated B cell cultures (Elson et al., 1979). Furthermore, CD4“ T cell involvement in regulation Of lgA production is likely based on the Observation that lgA plasma cells are significantly decreased after chronic treatment Of mice with anti-CD4+ T cell monoclonal Ab (Mega et al., 1992). Recent studies indicated that autoreactive gut PP CD4“ T cells can regulate lgA B cell heavy-chain switching and terminal 43 differentiation during gut mucosal B cell development (Kihira and Kawanishi, 1995). It seems reasonable that lgA responses are highly dependent on T cells (Clough et al., 1971) because Th1 type cytokines lL-2 and lFN-y as well as Th2 type cytokines lL-4, lL-5 and IL—6 have been Shown to enhance B cell responses and lgA synthesis. Although both Th1 and Th2 cells can regulate lgA synthesis, the Th2 cells may have the predominant effects (Coffman et al., 1988). Cytokine lL-4 and lL-5 secretion is high in lamina propria T cells of non-human primates and is important to the helper activity Of these cells (James at al., 1990). Xu-Amano et al., (1992b) reported that when mice were immunized orally with sheep red blood cells (SRBC), PP cells in mucosa-associated tissue exhibited a Th2-type response, whereas systemic administration Of SRBC elicited a TH1-type response in systemic Spleen tissue. Therefore, it was assumed that Th2 cells responses would predominate at the mucosal site (McGhee et al., 1989). This contention was supported by several studies demonstrating that Th2 cells are more abundant than Th1 cells at the mucosal site (Xu-Amano et al., 1992a; 1992b; 1993). In contrast, Th1-type responses occur mainly in systemic lymphoid tissue such as the spleen (Xu-AmanO et al., 1992a; 1992b; 1993). Furthermore, it was found that high numbers Of lgA plasma and lL-5-producing Th2 cells are present in lgA effector sites, especially in the intestinal lamina propria (Taguchi et al., 1990). Recent studies have also demonstrated that oral immunization with tetanus toxoid and CT results in enhanced Th2 cells and IgA responses in mucosal tissues (Xu-Amano et al., 1993; 1994; VanCott et al., 1996). 44 Many studies have shown that in_vm'_o incubation of IL-5 or lL-6 with PP B cells Or LPS-stimulated splenic B cells results in the induction of lgA synthesis (Coffman et al., 1987; Harriman et al., 1988; Beagley et al., 1988; 1989). In addition, both murine and human recombinant lL-6 have been reported to induce terminal differentiation of surface lgA+ (slgA’) B cells from gut-”associated lymphoid tissue to lgA-producing plasma cells (Fujihashi et al., 1991). The production of lL—6 from local intestinal mucosal has also been reported (Bao et al., 1993). Mucosal production Of lL-6 is important because this cytokine may regulate a number Of local and systemic immune responses, including lgA secretion, Md) differentiation and T cell proliferation (Akira et al., 1990). This conclusion is supported by a recent study demonstrating that the numbers Of mucosal lgA-producing cells were dramatically decreased in lL-6 knock—out mice and suggesting that mucosal immunity may be critically affected in the absence of lL-6 (Ramsay et al., 1994b). Thus, above observations suggest that Th2 type cytokines such as lL-5 and IL-6 are essential for inducing slgA+ B cells differentiation and increased rates Of lgA secretion in mucosal immunity. 1.3.3 Macrophage-secreted cytokines Although significant attention has been paid to. cytokine-mediated lgA production by Th cells, other cell types producing cytokines may also contribute to lg secretion. In addition to T lymphocytes, Md) are considered to be an important source Of cytokines and are capable of manipulating the T and B cell responses as 45 well as regulating the immune response. Md) are required accessory cells in the activation Of T lymphocytes (Rosenwasser and Rosenthal, 1978) and can be stimulated directly by endotoxin (LPS) (Oppenheim et al., 1980; Raetz et al., 1991; Verstovsek et al., 1994). Alternatively, Md) can be indirectly stimulated to secrete cytokine by contact with Ag (Farr et al., 1977) or mitogen activated lymphocytes (Mizel et al., 1978) and by cytokines such as lFN-y secreted from activated lymphocytes (Meltzer and Oppenheim, 1977; Young and Hardy, 1995)). It has been reported that Md) are required for the induction of Ag-specific helper T cells iLLiLl‘Q (Erb and Feldmann, 1975a). Pierce et al., (1977) also proposed that Ag-presentation by Md) is necessary to Th cells for optimal activity during induction Of Ab responses. A notable Observation is that transplantation Of peritoneal Md) from adult mice to neonatal mice resulted in an enhanced lg response to simultaneously injected SRBC (Argyris, 1968). This author suggested that a critical ratio Of Md) to immune competent cells may be important for stimulating lg synthesis. Erb and Feldmann, (1975b) demonstrated that Optimal Th cell induction required the admixture of about 3% Md) obtained from peritoneal exudate. Md), in response to LPS stimulation, are able to produce cytokines such as lL-1, IL—6, TNF-a and TGF-B which play important roles in immune activities (Cavaillon and Haeffner-Cavaillon, 1990: Libermann and Baltimore, 1990; Trinchieri, 1991; Ayala et al., 1993). Their roles include Ag-presentation, co- stimulation Of T and B cells, anti-tumor activity and anti-infection action. Some in 46 mg experiments have shown that a single stimulation by endotoxin results in a dose-dependent increase in the release Of lL-1, lL—6, and TNF by murine peritoneal Md) (West et al., 1992; 1993). TNF-or, as a important inflammatory cytokine, is produced mainly by Md) and has been shown to stimulate B cells increase lg secretion (Kehrl et al 1987). The most important sources Of lL-6 mm are believed to be Md) which, in fact, has been demonstrated to be major producer of lL-6 within the immune system (Bauer et al., 1988; Bauer, 1989). Recently, VanCott et al., (1996) reported that Md) secretion Of IL-6 was increased in murine PP and Spleen cultures after oral exposure to Salmonella expressing fragment C Of tetanus toxin, and that this IL—6 could increase plasma cell production of lgA at the mucosal effector site. Thus, this evidence suggests that lL—6 secreted by Md) may contribute the signals for development Of mucosal lgA responses in the lack Of classical Th2 cytokine IL-4, lL-5 and lL-6. Taken together, these findings indicate that lgA synthesis and responses are modulated by cytokines from certain immune cells. Mediators such as lL-4, lL-5 and lL-6 from Th2 cells or Md) provide "helper" function for B cells and may participate in the upregulation of lgA secretion in mucosal system. The effects of cytokines on B cells activation and lgA production are summarized in Table 1.1. 47 Table 1.1 Cytokines contributing tO differentiation Of lgA secreting cells CYTOKINE EFFECTS REFERENCE lL-2 Activate B cells to secrete lg Jelinek and Lipsky, 1987 Increase limited lgA synthesis Coffman et al., 1991 Increase lgA production (with lL-S) Beagley et al., 1995 Elevate lgA secretion (with lL-10) Nonoyama et al., 1994 Increase lgA secretion Lebman et al., 1990b; lwasato et al., (with TGF-B) 1994; Rafferty and Montgomery, 1995; Min et al., 1996 lL-4 Activate B cells to Secrete lg Jelinek and Lipsky, 1988 Induce mlgM’ cells to mlgA’ cells Kunimoto et al., 1988 Increase lgA production (with lL-5) Murray et al., 1987; Coffman et al., 1988 Increase rates of lgA class McIntyre et al., 1995 switching (with lL-5 and TGF-B) lL-5 Activate B cells to secrete lg Lemhardt et al., 1987 Increase lgA production Increase lgA production (with lL-2) Increase lgA production (with lL-4) Increase lgA production (with lL-6) Increase lgA production (with TGF-B) Increase rates of lgA class switching (with lL-4 and TGF-B) Elevate mucosal lgA responses Increase lgA plasma cell numbers A terminal differentiation factor for lgA secretion Bond et al., 1987; Coffman et al., 1987; Beagley et al., 1988; Harriman et al., 1988 ' Beagley et al., 1995 Murray et al., 1987; Coffman et al., 1987 Kunimoto et al., 1989 Sonoda et al., 1989; 1992; Rafferty and Montgomery, 1995 McIntyre et al., 1995 Ramsay et al., 1994a; Xu-Amano et al., 1994 Taguchi et al., 1990 Beagley et al., 1988; 1989; Schoenbeck et al.. 1989 48 Table 1.1 Continued CYTOKINE EFFECTS REFERENCE IL6 Activate B cells to secrete lg Kishimoto et al., 1988; Kishimoto, 1989 Increase lgA secretion Coffman et al., 1987; Beagley et al., 1989; 1991 Increase lgA production (with IL-5) Kunimoto et al., 1989 Increase lgA production Rafferty and Montgomery, 1995 (with TGF-B) Elevate mucosal lgA responses Ramsay et al., 1994a; Xu—Amano et al., 1994 Induce slgA‘ B cells to lgA- Fujihashi et al.. 1991 producing plasma cells Decrease lgA-producing cells Ramsay et al., 1994b following disruption of lL-6 gene TGF-B Switch factor for lgA class Coffman et al., 1988; Lebman et al.. 19903 Enhance sIgA+ cell numbers Lebman et al., 1990a Increase lgA secretion (with lL-2) Lebman et al., 1990b; lwasato et al., 1994; Rafferty and Montgomery, 1995; Min et al.. 1996 Induce mlgA expression Kunimoto et al., 1992 (with IL-4) Increase lgA production (with lL-5) Sonoda et al., 1989; 1992; Rafferty and Montgomery, 1995 Increase lgA production (with lL-6) Rafferty and Montgomery, 1995 Increase rates of lgA class McIntyre et al., 1995 switching (with IL-4 and lL-5) Increase lgA production Rafferty and Montgomery, 1995 (with lL-5 and lL-6) lFN-y Increase lgA production Prabhala and Wira, 1991 increase lgA production (with lL-2 and TGF-B) Elevate polymeric lgA receptor levels SnapperetaL,1995 Denning, 1996 CHAPTER 2 POTENTIAL ROLE FOR CYTOKINES IN ENHANCED lgA SECRETION BY PEYER'S PATCH CELLS ISOLATED FROM MICE ACUTELY EXPOSED TO VOMITOXIN 4c) 0 (J. 2.1 ABSTRACT Dietary exposure tO VT results in hyperelevated serum lgA and lgAN in mice. TO assess the possible role of cytokines in this lgA dysregulation, the effects of a single oral exposure in B6C3F1 male mice to 0, 5 or 25 mglkg BW VT on production of IgA and cytokines in PP and spleen cell cultures were evaluated. lgA levels were increased significantly in PP cell cultures prepared from mice at 2 or 24 hr after oral exposure to VT and subsequently stimulated with PMA and ionomycin (ION) or with LPS. Significant effects on lgA production were not Observed in spleen cell cultures. Since cytokines such as IL-2, lL-4, IL-5 and lL-6 have been shown to promote lgA production, the effect of the same VT exposure regimen on secretion of these mediators was determined in PP and spleen cultures. Supernatant IL-2 and lL-4 levels were unaffected by the prior treatment Of animals with VT. In contrast, lL-5 levels were increased significantly in 7 day PP cell cultures Obtained 2 hr after VT exposure both with and without PMA + ION exposure but not in other cultures. IL-6 levels were increased significantly in LPS-treated cultures prepared from PP at 2 and 24 hr following exposure to VT. lL-6 levels were also elevated significantly in both PMA + ION or LPS treated cultures from spleen isolated at 2 hr but not 24 hr post VT exposures. TO determine whether cytokines may play a role in IgA hyperelevation in vitro, PP and spleen cells from mice obtained 2 hr after'exposure to 25 mglkg VT were cultured in the presence Of neutralizing cytokine Abs and lgA production was monitored. Consistent with lL-5’s previously documented role in lgA production, anti-lL-S decreased lgA levels to background in cultures of both control 51 and VT-exposed PP or spleen cells in the presence Of either PMA + ION or LPS. Similar results were seen with addition of anti-lL—6. lgA levels were decreased to a lesser extent in PP cells cultured with LPS and in spleen cells cultured with PMA + ION from VT- exposed mice tO which anti-IL-2 Ab was added. The results indicated that the potential for enhanced lgA production exists in lymphocytes as early as 2 hr and as late as 24 hr after a single oral exposure to VT and that this may be related to the increased capacity to. secrete helper cytokines of T cell and Md) origin. Taken together, the results suggest that the superinduction Of cytokine expression may, in part, be responsible for upregulation of lgA secretion in mice exposed orally to VT. 52 2.2 INTRODUCTION VT is a fungal secondary metabolite that belongs to a family of mycotoxins referred to as trichothecenes (Tanaka et al., 1988). This toxin is frequently found in cereal grains as well as other food and agricultural products (Abouzied et al., 1991; Rotter et al., 1996). VT and other trichothecenes are potent protein synthesis inhibitors that can cause acute and chronic toxicity (Ueno, 1985; Betina, 1989b; Pestka and Casale, 1990). Trichothecenes can be both immunostimulatory and immunosuppressive in a variety Of animal and cell culture models (Pestka and Bondy, 1994). In mice, dietary VT exposure induces extremely high levels of serum IgA (Forsell et al., 1986; Pestka et al., 1989) and can increase the percentages Of membrane IgA" cells, T cells, and CD4+ T cells as well as CD4‘ICD8’ T cell ratios in PP and spleen (Pestka et al., 1990a; Bondy and Pestka, 1991). The toxin also causes glomerular lgA deposition and hematuria, which are very similar, Clinically, tO human lgAN (Pestka et al., 1989; Dong et al., 1991; Dong and Pestka, 1993; Rasooly and Pestka, 1994; Greene et al., 1994a; 1994b). All Of these findings are indicative of dysregulation of IgA production. Cytokines influence 8 cell activation, class-switching, proliferation, and terminal differentiation to lgA-producing plasma cells (McGhee et al., 1989). Previous studies in our laboratory have shown that exposure to VT Mm superinduces lL-2, lL-4, IL-5 and lL-6 mRNA expression in murine splenic CD4“ T cells stimulated with Con A or PMA (Ouyang et al., 1995; 1996a; Azcona-Olivera et al., 1995a; Warner et al., 1994). as well as PMA-stimulated EL-4 thymoma 53 cultures (Dong et al., 1994). These cytokines have previously been shown to enhance differentiation Of B cells to lgA secretion (Lebman et al., 1990a; 1990b; Coffman et al., 1987; 1988; Beagley et al., 1988; 1989; Pockley and Montgomery, 1991 a; Dieli et al., 1995). Recently, Azcona-Olivera et al., (1995b) demonstrated that acute oral VT exposure to mice elevates cytokine mRNA levels with maximal effects occurring in the 25 mglkg BW group in as little as 2 hr. We hypothesize that VT stimulates lgA production via superinduction Of T helper cytokines. To test the hypothesis, l: (_1_) related the effects Of acute oral VT exposure on lgA tO cytokine production in mucosal and systemic lymphocytes cultured in vitro and (2) assessed the effects Of cytokine-specific neutralizing monoclonal Abs on lgA production in these cultures. The results indicate that PP lymphocytes exhibited enhanced capacity for elevated production of lgA, IL-5 and lL-6 when isolated as early as 2 hr and as late as 24 hr after VT exposure i_n_yi_v_o. Both control and VT-induced lgA secretion could be inhibited by neutralizing Abs for lL-5 and lL-6 and, to a lesser extent, IL-2. Taken together, these results suggest that superinduction of in vivo cytokine expression by VT may be responsible, in part, for upregulation of IgA secretion. 54 2.3 MATERIALS AND METHODS 2.3.1 Chemical and reagents All chemicals were Of reagent grade quality or better and Obtained from Sigma Chemical (St Louis, MO) except where otherwise noted. 2.3.2 Animal and VT exposure regiment Male B6C3F1 mice (8-9 weeks) were used because: (1) male mice are more sensitive than female to VT-induced IgA nephropathy (Greene et al., 1994a) and chronic effects (lverson et al., 1995), and (2) males have been previously shown to be more sensitive to VT superinduced cytokine mRNAs expression (Azcona-Olivera et al., 1995b). Mice were obtained from Charles River Laboratories (Wilmington, MA) and kept in the university animal care facility room with a humidity- and temperature-controlled and a 12 hr light and dark cycle. Mice were housed in cages equipped with filter bonnets (Nalgene, Rochester, NY) and fed powdered semi-purified AIN-76A diet (ICN Nutritional Biochemical, Cleveland, OH) upon arrival. Animals were acclimated for at least one wk prior to usage. Food and water were withdrawn from cages 2 hr before toxin administration. VT was purchased from Romer Labs (Washington, MO). Mice (4 per group) were orally gavaged with 5 or 25 mglkg BW VT in 500 ul of 0.01M carbonate- bicarbonate buffer (pH 9.6) and control mice received 500 pl vehicle only (Azcona- Olivera et al., 1995b). Food and water were restored after gavaging. Mice were euthanized at 2 or 24 hr after gavage, and their spleens and PP were removed for isolation and culture. 55 2.3.3 Cell cultures Spleens were teased apart with sterile tissue forceps in harvest buffer consisting Of 0.01M phosphate buffered saline (PBS, pH 7.4), containing 2% (vlv) heat inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY), 100 U/ml penicillin, and 100 rig/ml streptomycin. Cell suspensions were held on ice for 10 min to allow settling of tissue particles. Supernatant was removed following centrifugation at 450 x g for 10 min. Erythrocytes were Iysed for 3 min at room temperature in 0.02 M Tris buffer (pH 7.65) containing 0.14M ammonium chloride. Cells were centrifuged, resuspended in RPMI-1640 medium supplemented with 10% (vlv) F BS, 1mM sodium pyruvate, 100 U/ml penicillin, 100 ug/ml streptomycin, 0.1 mM nonessential amino acid and 5 x 10‘5 M 2-mercaptoethanol, and then counted using a hemacytometer (American Optical, Buffalo, NY) (Strober, 1991). PP were teased apart in harvest buffer, passed through a sterile 85 -mesh stainless steel screen and resuspended in the same buffer. Cells were centrifuged at 450 x g for 10 min, resuspended in supplemented RPMI-1640 medium and counted. Cells (1 x 105) were cultured in 1 ml Of supplemented RPMI-1640 medium in flat-bottomed 24-well tissue culture plates (Fisher Scientific 00., Corning, NY) at 37° C under a 7% CO2 in a humidified incubator. Cultures were unstimulated or stimulated with the T cell inducing agents, PMA (10 ng/ml) plus ION (500 ng/ml) (PMA + ION) or the Md) inducing agent, Salmonella typhimurium LPS (20 ug/ml). Supernatant was collected at 1, 4 and 7 days and stored in aliquots at -20° C until 56 analysis. For cytokine-specific neutralizing Ab blocking studies, neutralizing rat anti- mouse cytokine Abs, (anti-lL-2 , lL—4, lL-5 and lL-6 from PharMingen, San Diego, CA) were included at a concentration of 10 ug/ml in the cultures (5 x 105/ml cells). lsotype matched rat-lgG Ab (PharMingen) was used as a control. Supernatant was collected at 5 days and stored in aliquots at -20° C until analysis. 2.3.4 lgA quantitation lgA was measured in culture supernatants by enzyme-linked immunosorbent assay (ELISA) (Bondy and Pestka, 1991). Immunolon 4 Removawell microtiter strip wells (Dynatech Laboratories Inc., Chantilly, VA) were coated overnight at 4°C with 50 pllwell of heavy-Chain specific goat anti-mouse lgA (Cappel Worthington, Malvern, PA) at a concentration Of 10 ug/ml in 0.1M bicarbonate buffer (pH 9.6). Coated plates were washed 3 times with 0.01M PBS (pH 7.2) containing 0.2% Tween 20 (PBST) to remove excess capture Abs. plates were incubated with 300 pl of 1% (w/v) BSA in PBST (BSA-PEST) at 37° C for 30 min to block nonspecific protein binding, and then washed 4 times with PBST. For lgA determination, standard mouse reference serum (Bethyl Laboratories, Inc, Montgomery, TX) or samples were diluted in 10% (vlv) FBS RPMI-1640 medium and 50 III was added to appropriate wells. Plates were incubated at 37° C for 60 min, washed 4 times with PBST, and then 50 pl Of goat anti-mouse lgA horseradish peroxidase (or-chain specific, Cappel Worthington, Malvern, PA), diluted 1:1000 in 1% (w/v) BSA in PBS, was added to each well. Plates were incubated at 370 C for 30 min and washed 6 57 times with PBST. Bound peroxidase was determined with 2,2-azinO-bis (3- ethylbenzthiazolin-6-sulfonate) (ABTS) substrate [0.4mM ABTS, 50mM Citrate buffer (pH 4.0), and 1.2mM hydrogen peroxide] as described previously by Pestka et al., (1980). Absorbance was measured at 405 nm on a Vmax Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA) and IgA was quantitated by using Vmax Software (Molecular Devices). 2.3.5 Cytokine quantitation Commercial mouse recombinant cytokine IL-2 (Collaborative Research Inc. Bedford, MA), IL-4 (Cellular Products Inc. Buffalo, NY), lL-5 (Genzyme, Cambridge, MA) and lL-6 ( PharMingen, San Diego, CA) were used as standards for cytokine quantitation. Cytokine production was monitored by ELISA using modification of the procedure of Dong et al., (1994). Briefly, Immunolon 4 Removawell microtiter strip wells (Dynatech Laboratories Inc., Chantilly, VA) were coated overnight at 4°C with 50 III/well of 1.0 ng/ITII purified rat anti-mouse cytokine capture Abs (PharMingen) in 0.1M sodium bicarbonate buffer (pH 8.2). Plates were washed 3 times with PBST, blocked with 300 pl Of 3% (w/v) BSA-PEST at 37° C for 30 min and washed 4 more times with PBST. Standard murine cytokines or samples were diluted in RPMI-1640 medium containing 10% (vlv) FBS and 50 ul aliquots were added to appropriate wells. Plates were incubated at 37° C for 60 min, washed 4 times with PBST, and 50 ul Of biotinylated rat anti-mouse cytokine detection monoclonal Abs (1.5 jig/well; PharMingen) diluted in BSA-PBST were added to each well. After incubation at room temperature for 60 min, plates were washed 6 times with PBST, 50 III of 58 streptavidin-horseradish peroxidase conjugate (1.5 jig/well in BSA-PBST) were added to each well and plates were incubated at room temperature for 60 min. The plates were then washed 10 times with PBST, and 100 pl Of substrate [1 OmM Citric- phosphate buffer (pH 5.5), containing 0.4mM tetramethylbenzidine (TMB; Fluka Chemical Corp, RonkOnkoma, NY) and 1.2mM H202] were added to each well. The reaction was stopped by adding an equal volume of 6 N H2804. Absorbance was read at 450 nm on a Vmax Kinetic Microplate Reader (Molecular Devices) and cytokine concentrations were quantitated by using Vmax Software (Molecular Devices). 2.3.6 Statistics The data were analyzed by Dunnett’s test following one way analysis of variance (ANOVA) using SigmaStat Statistical Analysis System (Jandel Scientific, San Rafael, CA). A p value Of less than 0.05 was considered statistically significant. 59 2.4 RESULTS The effects of single oral VT exposures Of 5 or 25 mglkg BW on lgA production MILI’Q were determined in cultures prepared from PP and spleens as representative Of mucosal and systemic lymphoid tissues, respectively. lgA levels were increased significantly in PP cell cultures from VT-exposed mice with or without T cell activators, PMA + ION or the Md) activators, LPS and obtained at 2 (Figure 2.1) or 24 hr (Figure 2.2) after toxin exposure as compared to controls with the greatest effects being Observed in the 25 mglkg group. Significant effects on lgA were not Observed in spleen cells (data not shown). These results suggested that oral VT administration to mice can increase the potential of PP cells to secrete lgA. The effects of oral W on cytokines were assessed in cultures prepared from PP and spleens. lL-2 and lL-4 supernatant levels were not affected in PP and spleen cell cultures by VT exposure. In contrast, lL-5 levels were Significantly higher in 4 and 7 day PP cell cultures obtained from mice 2 hr after VT exposure but not in other cultures. lL-5 was not appreciably affected in 1, 4 and 7 day _PP cultures prepared 24 hr after VT exposure (Figure 2.3) or in 1, 4 and 7 day spleen cultures prepared 2 and 24 hr after VT exposure (data not shown). lL—6 levels were significantly greater in LPS-stimulated cultures prepared from PP at 2 hr (Figure 2.4) and 24 hr (Figure 2.5) following exposure to VT. lL-6 levels were also elevated significantly in both PMA + ION- or LPS-stimulated cultures from spleen isolated at 2 hr (Figure 2.6) but not 24 hr (data not shown) post VT exposure. These results 60 Figure 2.1. Effect of oral exposure of mice to O. 5 and 25 mglkg BW VT on lgA secretion in PP cell cultures. Mice were sacrificed at 2 hr after gavage. PP cells (1x105/ml) were cultured with or without mitogens in 24-well plates for 1, 4 and 7 days, respectively. Supernatant lgA levels were analyzed by ELISA. Data are mean .t SEM (n=4). Bars marked with letter (a) are significantly different (p < 0.05) from corresponding control (0 mglkg) group. 61 éiifoE‘4“;‘i3¥sf~. W v 800 z 9 : _ 75 E > N " 13 V d) ' .E I- ; . l‘ . <- °§ 8;; ill I l l I O c O O 8 s a (lefiuwa. 62 Figure 2.2. Effect of oral exposure of mice to O, 5 and 25 mglkg BW VT on lgA secretion in PP cell cultures. Mice were sacrificed at 24 hr after gavage. PP cells (1x105/ml) were cultured with or without mitogens in 24-well plates for 1, 4 and 7 days, respectively. Supematant lgA levels were analyzed by ELISA. Data are mean 1 SEM (n=4). Bars marked with letter (a) are significantly different (p < 0.05) from corresponding control (0 mg/kg) group. LPS I N‘mpm w \~.\\\\‘\ :\\;‘\t\\§x\x\\:\\\k\x\x\\‘? I PMA + ION :~~;\‘ ;<\\x\-\\‘x .\\ ; :..\\.. Time (days) I \Vy \\‘: NARVx Unstlmulatod : s;\\‘3:_\‘:\\\‘ C kak [:3 Control //// VT 5 mglkg - VT 25 molko l l O C O O O 600 (151/Bu) c{rm 64 Figure 2.3. Effect of oral exposure of mice to O, 5 and 25 mglkg BW VT on lL-5 production in PP cell cultures. Mice were sacrificed at 2 or 24 hr after gavage. PP cells (1x105/ml) were cultured with or without mitogens in 24-well plates for 1, 4 and 7 days, respectively. Supernatant lL-5 levels were analyzed by ELISA. Data are mean i SEM (n=4). ND indicates non-detectable. Bars marked with letter (a) are significantly different (p < 0.05) from corresponding control (0 mglkg) group. 65 (24 hr) \\\\ \\ PMA + ION PMA + ION I 1 (2 hr) (24 hr) Time (days) ' \ ‘ \‘ ‘\‘ g\..\.\.\;\ [x ;\\<\\\ ;\i .x I O Unstimulated Unstimulatcd I \\ \ I I _I N on ‘ (IN/91W") 9-1i (lwlsuun) 9"" O 66 Figure 2.4. Effect of oral exposure of mice to O, 5 and 25 mglkg BW VT on lL-6 production in PP cell cultures prepared 2 hr after gavage. PP cells (1x105/ml) were cultured with or without mitogens in 24-well plates for 1, 4 and 7 days, respectively. Supernatant lL-6 levels were analyzed by ELISA. Data are mean i SEM (n=4). Bars marked with letter (a) are significantly different (p < 0.05) from corresponding control (0 mglkg) group. 67 PMA + [on '- - Time (days) Unstlmmated [:3 Control T I | 4 68 Figure 2.5. Effect of oral exposure of mice to O, 5 and 25 mglkg BW VT on lL-6 production in PP cell cultures prepared 24 hr after gavage. PP cells (1x105/ml) were cultured with or without mitogens in 24-well plates for 1, 4 and 7 days, respectively. Supernatant lL-6 levels were analyzed by ELISA Data are mean :t: SEM (n=4). Bars marked with letter (a) are significantly different (p < 0.05) from corresponding control (0 mglkg) group. 69 — §\\\\\\\\\\\\\\\\\\\\\\\\\\ PMA + ION '~ \V' — -N F Unstimuiated C] COMro| 0.8 I I "- N o o 6 — — Wzstlkg 0.0 70 Figure 2.6. Effect of oral exposure of mice to O, 5 and 25 mg/kg BW VT on lL—6 production in spleen cell cultures prepared 2 hr after gavage. Spleen cells (1 x1 05/ml) were cultured with or without mitogens in 24-well plates for 1, 4 and 7 days, respectively. Supernatant IL-6 levels were analyzed by ELISA. Data are mean 2*: SEM (n=4). Bars marked with letter (a) are significantly different (p < 0.05) from corresponding control (0 mglkg) group. 71 PMA + ION Time (days) Unstimulatod :3 Control 7% VT 5 mglkg - VT 25 mglkg T T (nu/Bu) 9-1I 3 72 indicated that VT exposure increased the potential for secretion of lL-5 and lL-6 by PP cells as well as lL-6 by spleen cells. To determine the potential role of cytokines on VT-induced lgA hyperelevation, PP and spleen cells from control and treatment mice (2 hr after exposure) were cultured in the presence of cytokine neutralizing Abs and supernatant lgA monitored after 5 days. Cells were stimulated with PMA + ION or with LPS and co-cultured with anti-lL-2, lL-4. lL-5 or IL-6 singly. lgA levels were significantly lower in VT-treated PP (Figure 2.7) and spleen (Figure 2.8) cell cultures stimulated with PMA + ION or with LPS and containing anti-IL-5 or lL-6. The effects were also observed in the control animal groups (Figure 2.7 and 2.8). lgA levels were partially decreased in LPS-stimulated PP (Figure 2.7) and PMA + ION-stimulated spleen (Figure 2.8) cells from VT-exposed animals to which anti-IL- 2 Ab was added . These results suggested that increased levels of both lL-5 and IL—6 and to a lesser extent, lL-2, might enhance IgA production in cultures from VT- treated mice. Figure 2.7. Effect of cytokine-specific neutralizing Abs on mitogen-driven lgA production in PP cell cultures isolated from mice exposed to 0 and 25 mg/kg BW VT 2 hr after gavage. PP cells (5x105/ml) were cultured with PMA + ION or with LPS in 24-well plates for 5 days in the presence of anti-lL-2, lL-4, lL-5 or lL-6 singly. As a control, isotype matched rat-lgG Ab was used. Supernatant lgA levels were analyzed by ELISA. Open and solid bars indicate 0 and 25 mglkg BW VT, respectively. Data are mean .+_ SEM (n=4). Bars marked with letter (a) are significantly different (p < 0.05) from no anti-IL Ab group and letter (b) are significantly different (p < 0.05) from isotype matched Ab group. 74 ooow _ 2555 <9 ooow o _ =55... <2 88 ooov coon o coon _ _ . E I . .228 D h. 20. + (2.. n< .3 Smog... oz a< on..._uc< n< m..=.::< n< v4.59: w. n< 41.5 oz Figure 2.8. Effect of cytokine-specnfic neutralizing Abs on mitogen—driven lgA production in spleen cell cultures isolated from mice exposed to 0 and 25 mg/kg BW VT 2 hr after gavage. Spleen cells (5x105/ml) were cultured with PMA + ION or with LPS in 24-well plates for 5 days in the presence of anti-lL-2. lL-4, lL-5 or IL- 6 singly. As a control, isotype matched rat-lgG Ab was used. Supernatant lgA levels were analyzed by ELISA. Open and solid bars indicate 0 and 25 mglkg BW VT, respectively. Data are mean i SEM (n=4). Bars marked with letter (a) are significantly different (p < 0.05) from no anti-IL Ab group and letter (b) are significantly different (p < 0.05) from isotype matched Ab group. 76 2535 <2 ooc cow com _ L ; I .223 mu =55... <2 on? r on: _ 20. + (En. n< ecu comogs oz n< al.-55‘ a< “3222 it 2 32.2 ...f[ .2 3.-...2 [3 85.2: 838. IE .=.=§ oz 77 2.5 DISCUSSION The aberrant elevation of serum lgA and development of lgAN (Pestka and Bondy, 1994) following chronic oral exposure to VT in mice represents a novel and isotype-specific dysregulation of the humoral immune response by a toxin. lgA has a critical role in the humoral immune response of the intestinal mucosa and is considered to participate in the primary defense of the host to exogenous pathogens (Kilian and Russell, 1994). The PP in the gut-associated lymphoid tissue is a major source of B cell precursors for lgA-secreting plasma cells (Craig and Cebra, 1971). The observations that production of lgA, IL-5 and IL—6 were affected more by VT in PP cultures than in spleen cultures suggests that VT exerts a greater effect in the gut mucosal compartment as compared to the systemic compartment. PP are believed to be major inductive sites where B cells are activated and committed to lgA secretion (McGhee et al., 1989; Beagley and Elson, 1992;). These activated B cells migrate to distant mucosal effector sites, such as lamina propria, where further stimulation and terminal differentiation into lgA-producing cells takes place. T cells and accessory cells as well as their secreted cytokines are involved in regulating the differentiation of B cells into lgA-secreting plasma cells in the PP (McGhee et al., 1989; Beagley and Elson, 1992; McGhee and Kiyono, 1993). Several major results were observed in this study suggest that PP function is a primary target for VT. First, oral VT administration to mice increased the capacity of PP cell cultures to secrete IgA and the cytokines IL-5 and lL-B. Secondly, the capacity for elevated lgA and lL-6 production was observed in lymphocytes as early 78 as 2 hr and as late as 24 hr after VT exposure. Third, both control and VT-induced lgA production could be inhibited by lL-5 and lL-6 neutralizing Abs. These data suggest that VT-induced lgA production may be directly related to increased levels of helper cytokines. Many cytokines have been shown to elevate lgA production including the Th1 type cytokine lL-2 (Coffman et al., 1991) and the Th2 type cytokines lL-4 (Kunimoto et al., 1988) , lL-5 and lL-6 (Beagley et al.,1988; 1989; Beagley and Elson, 1992). Although both Th1 and Th2 cells can regulate lgA synthesis, the Th2 cells may have the predominant effects (Coffman et al., 1988). Oral immunization with tetanus toxoid and CT results in enhanced Th2 cells and lgA responses in mucosal associated tissues (Xu-Amano et al., 1993; VanCott et al., 1996). The Th2 cytokines lL-5 and lL-6 have been associated with B cell differentiation and promotion of lgA secretion (Beagley et al., 1988; 1989). Sonoda et al., (1992) determined that lL-5 can stimulate lgA synthesis through promotion of maturation of postswitch slgA" B cells into lgA-producing cells. lL-B, as a B cell stimulatory factor, is capable of enhancing lgA production from mlgA“ PP B cell cultures (Beagley et al., 1989). Synergy between lL-5 and lL-6 has been suggested based on the observations when PP B cells are stimulated by either cytokine alone, lgA secretion is only modestly increased, but is greatly enhanced by lL-5 and lL-6 in combination (Kunimoto et al., 1989). Thus, it would appear thatboth lL-5 and lL-6 are integrally involved in the final differentiation of lgA-committed B cells into lgA- secreting plasma cells in the mucosal tissue. This has been further confirmed by 79 elevation of mucosal lgA responses due to lL—5 and lL-6 (Ramsay et al., 1994a) and the decreased numbers of mucosal lgA-producing cells observed in lL-6 knock-out mice (Ramsay et al., 1994b). My findings that lgA levels were significantly decreased in both control and VT-treated PP and spleen cell cultures upon addition of anti-lL-5 or lL-6 Abs further suggest that these cytokines are critical to elevated differentiation of B cells to lgA production. This is consistent with early studies demonstrated that induction of lgA production by lL-5 and lL—6 is inhibited by treating with specific neutralizing Abs (Muraguchi et al., 1988; Ramsay and Kohonen-Corish, 1993). The preferential switching of mucosal B cells from lgM+ to lgA+ phenotypes and differentiation to lgA secretion is governed by T helper (CD4‘) cells which comprise a characteristic and dominant T cell subset in the PP (Kawanishi et al., 1983; Kawanishi and Mirabella, 1988; McGhee and Kiyono, 1993; Kihira and Kawanishi, 1995). In support of this contention, mum studies have demonstrated that PP T cells can elevate lgA synthesis in LPS-stimulated B cell cultures (Elson et al., 1979). Furthermore, CD4+ T cell involvement in regulation of lgA production is likely based on the observation that lgA plasma cells are significantly decreased after chronic treatment of mice with anti-CD4” T cell monoclonal Ab (Mega et al., 1992). The results provided herein support earlier findings that VT induces hyperelevated lgA production and that CD4+ cells play a critical role in this effect (Forsell et al., 1986; Pestka et al., 1989; Bondy and Pestka, 1991; Dong et al., 1991; Dong and Pestka, 1993; Greene et al., 1994a; Rasooly and Pestka, 1994; 80 Warner et al., 1994). They are also consistent with previous observations that percentages of membrane lgA+ cells, T cells and CD4‘ T cells as well as CD4‘ICDB” cell ratios are increased in PP and spleens of mice fed 25 ppm VT (Pestka et al., 1990; Bondy and Pestka, 1991). Bondy and Pestka, (1991) reported that significantly increased lgA production occurs when control B cells are co-cultured with PP T cells isolated from mice fed 25 ppm VT for 8 wks. ]n_vthQ exposure of CD4“ T cells to VT can significantly increase lgA production by B cells (Warner et al., 1994). Thus, one possible interpretation of these data is that T cell dysregulation in the PP caused by VT may contribute to aberrantly increased lgA production. It should be recognized that in addition to Th2 cells, Md) are major producers of IL-6 within the immune system (Bauer et al., 1988, Bauer, 1989). Recently, VanCott et al., (1996) reported that Md) secretion of lL-6 was increased in murine PP and spleen after oral exposure to Salmonella expressing fragment C of tetanus toxin, and that this lL-6 could elevate plasma cells production of lgA at mucosal effector sites. In this study, it is critical to note that elevation of lL-6 secretion was observed in LPS-driven PP and spleen cell cultures after oral exposure to VT. Since M¢ are responsive to this mitogen (Raetz et al., 1991; Verstovsek et al., 1994) whereas T cells are not, it is likely that Md) are specifically associated with the VT- induced upregulation of lgA production via secretion of cytokine lL-6. Further investigation of the effects of VT on Md) in mice is thus warranted. lL-2 has also been shown to' increase lgA production by B cells when in 81 combination with TGF-B (lwasato et al., 1994). Nonoyama et al., (1994) also reported that lL-2 significantly elevated the secretion of IgA by anti-CD40-activated B cells cultured in the presence of lL-10..These findings indicated that lL-2 is capable of inducing the synthesis of IgA when in Combination with other cytokines. The observation that lgA secretion is reduced in the presence of anti-lL-2 suggests that lL-2 may contribute synergistically in the presence of lL-5 and lL-B to the induction of lgA production. This contention is supported by recent studies that lL-2 greatly increases lgA production in B cells when in combination with lL-5 (Beagley et al., 1995) or with TGF-B (Min et al., 1996). Although I did not detect increases in supernatant lL-2 in my cultures, this may have resulted from binding to membrane or soluble receptors (Mohler and Butler, 1991). In this study, the observed enhancement of lL-5 and lL-6 secretion in PP cell cultures after oral VT exposure supports previous observations that VT superinduces cytokine gene expression and secretion following exposure in_VLtLQ or m (Warner et al., 1994; Dong et al., 1994; Azcona-Olivera et al., 1995a; 1995b). Superinduction of cytokine gene expression by protein synthesis inhibitors such as VT have been observed previously. For example, VT has been shown to stimulate lL—1 secretion by peritoneal macrophages i_r_t_v_it_m (Miller and Atkinson, 1986). Furthermore, other protein synthesis inhibitors such as T-2 toxin and cycloheximide (CHX) superinduce cytokine lL-2 mRNA expression and secretion (Efrat et al., 1984; Holt et al., 1988; Zubiaga et al., 1991). One explanation for the superinduction by trichothecenes or CHX relates to the ability of these compounds 82 to inhibit synthesis of a labile protein repressor of cytokine mRNA expression thus leading to increased cytokine mRNA expression (Efrat et al., 1984). Recently, Ouyang et al., (1996b) have demonstrated that VT induces NF—KB/Rel binding activity particularly through inhibiting resynthesis of lKB-a in murine EL-4 and primary CD4’ T cells. A second possible mechanism for VT-induced superinduction could involve to alteration of cytokine mRNA half-life. Studies of lL-2 mRNA stabilization suggest that labile RNases may be responsible for CHX-mediated stabilization of IL-2 mRNA since CHX may directly inhibit synthesis of labile RNases (Shaw et al., 1988). Based on the ability of VT to inhibit protein synthesis, it is possible that VT could inhibit such labile RNases synthesis and consequently enhance the half-life of cytokine mRNA. It is notable that the effects of VT exposure on cytokines and IgA production were observed following tissue harvest as early as 2 hr with greatest effect being observed in the 25 mglkg group. Consistent with this observation, Azcona-Olivera et al., (1995b) and Zhou et al., (1997) reported that VT-induced maximal effects in elevation of lL-6 as well as lL-1B, lL-2, TNF-a and lFN-y mRNA expression in PP and spleen occurred in 2 hr after mice received 25 mglkg VT. Interestingly, at this dose, VT is rapidly absorbed from gut with peak levels being detected in plasma at s 30 min after exposure, but more than 70% protein synthesis is inhibited as long as 9 hr in all tissues of mice receiving 25 mglkg VT (Azcona-Olivera et al., 1995b). Thus it is appears that VT superinduces IL-6 and other cytokines concurrently with impairment of protein synthesis. The observation that lL-6 effects were still 83 observable 24 hr after VT exposure suggests that the effects on this cytokine were longJasfing. Sakai (1988) has indicated that cytokines may play an important role in the etiology and pathogenesis of lgAN. One interesting observation is that lgAN patients have high urinary lL-6 activity (Dohi et al., 1991). Recently, other studies have demonstrated that there is an increase in lL-5, IL-6 and TGF-B mRNA levels in CD4+ T cells (de-Caestecker et al., 1993; Lai et al., 1994a; 1994b) as well as IL- 4, lL—5 and lL-6 mRNA levels in peripheral blood mononuclear cells of lgAN patients (Ichinose et al., 1996). Thus, induction of cytokines may be intimately associated with immunopathologic sequelae associated with both human lgAN and experimental VT-induced lgAN. Taken together, the results presented herein indicated that a single oral VT administration to mice can increase the capacity of PP cells to secrete lgA, IL-5 and lL-6 and that Ab neutralization of these cytokines prevented lgA secretion m. The observation that neutralization of these cytokines also depressed lgA production in control cultures, suggest that VT may elevate lgA secretion via regulatory mechanisms already existing in the PP. Thus, VT’s isotype-specific effects may not be primarily a direct effect on lgA production but rather an indirect effect of altered cytokine levels which regulate lgA production. Further clarification of both the leukocyte phenotypes involved in enhanced cytokine production and the mechanisms by which this occurs are warranted. CHAPTER 3 ROLE OF MACROPHAGES IN ELEVATED lgA AND lL-6 PRODUCTION BY PEYER'S PATCH CULTURES FOLLOWING ACUTE ORAL VOMITOXIN EXPOSURE 84 85 3.1 ABSTRACT Oral VT exposure in mice results in elevated cytokine gene expression, increased production of lgA and lgAN. To determine the potential role of Md) in these effects, PP and spleen cell cultures, prepared from mice 2 hr after oral exposure to O or 25 mglkg BW VT, were evaluated for lgA and cytokine lL-6 production. Both PP and spleen cells from treatment mice produced more lgA over a 7 day period than did corresponding control cells when cultured without a co- stimulus or in the presence of either the T cell activators PMA + ION or the Md) activator LPS. The VT effect was completely ablated in PP cultures depleted of Md) but not in spleen cells. In LPS-treated cultures, supernatant IL-6 Was higher in the VT treatment groups as compared to controls at 7 days for PP cells and 1, 4 and 7 days for spleen cells whereas these effects were not observed in unstimulated or PMA + ION cultures. VT-induced elevation of IL-6 secretion in LPS-treated PP and spleen cells was also ablated by Md) depletion. A potential co-stimulatory role for Md) was further suggested because both lgA and lL-6 production increased when Md)-depleted PP cells from VT-treated animals were co-cultured with peritoneal Md) from VT-treated animals as compared to both: (1) control Md)-depleted PP cells plus control peritoneal Md) and (2) treatment Md)-depleted PP cells plus control peritoneal Md). Higher lgA and lL-6 concentrations were also observed in supernatants from cultures containing VT-treated Md)-depleted PP cells and control peritoneal Md) as compared to control Mcp-depleted PP cells plus control peritoneal Md). Furthermore, PP B cells from control animals secreted elevated levels of lgA 86 and IL-6 when co-cultured with peritoneal Md) from VT-treated animals. Direct contact with the VT-treated Md) appeared to be necessary for an optimal stimulatory signal because the degree of IgA increase was lower in the reconstituted cell cultures where VT-treated Md) were separated by a semi-permeable membrane from PP cells as compared to co-cultures without a membrane. Taken together, these results suggest that Md) were primarily responsible for upregulation of lgA production in mice exposed orally to VT and that this was likely to involve both secretion of soluble mediators such as lL-6 and cognate cell-cell interactions. 87 3.2 INTRODUCTION VT is a common fungal toxin that occurs in wheat and corn-based foods and belongs to a family of mycotoxins referred to as trichothecenes (Tanaka et al., 1988). Prolonged dietary VT exposure can induce extremely high levels of serum IgA (Forsell et al., 1986) as well as lgAN (Pestka et al., 1989; Pestka and Bondy, .1990; Dong et al., 1991; Dong and Pestka, 1993; Rasooly and Pestka, 1994; Greene et al., 1994a; 1994b). Increases in percentages of membrane lgA+ cells and lgA-secreting cells in PP and spleens of mice occur concurrently with these effects (Pestka et al., 1990a; Bondy and Pestka, 1991), suggesting that VT stimulates lgA secretion. Cytokines influence B cell activation, class-switching, proliferation, and terminal differentiation to lgA-producing plasma cells (McGhee et al., 1989). Previous studies in our laboratory have shown that exposure to VT JILLiLIQ superinduces lL-2, lL-4, lL-5 and IL-6 mRNA expression in murine splenic CD4” T cells stimulated with Con A or PMA (Ouyang et al., 1995; 1996a; Azcona-Olivera et al., 1995a; Warner et al., 1994) as well as PMA-stimulated EL-4 thymoma cultures (Dong et al., 1994). These cytokines enhance differentiation of B cells to lgA secretion (Lebman et al., 1990a; 1990b; Coffman et al., 1987; 1988; Beagley et al., 1988; 1989; Pockley and Montgomery, 1991a; Dieli et al., 1995). Azcona- OIivera et al (1995b) further demonstrated that acute oral VT exposure to mice superinduces mRNA expression of the proinflammatory cytokines IL-6, lL-1B, TNF- or and IFN-y. These effects are maximal in mice exposed to 25 mglkg BW VT for 88 as little as 2 hr (Zhou et al., 1997). Md) are important sources of cytokines, some of which are capable of modulating T and B cell responses. Md) have been demonstrated to be major producers of lL-6 within the immune system (Bauer et al., 1988; Bauer, 1989). Recently, VanCott et al., (1996) suggested that lL-6 secreted by Md) may contribute to development of mucosal lgA responses. T cells are another major source of helper cytokines for regulating lgA production (McGhee et al., 1989; Beagley and Elson, 1992; McGhee and Kiyono, 1993; Kihira and Kawanishi, 1995). In vitro studies have demonstrated that VT-treated CD4+ T cells can significantly increase lgA production in B cells (Warner et al., 1994). Significantly elevated lgA production was also observed when PP T cells isolated from VT-fed mice were co-cultured with B cells (Bondy and Pestka, 1991). The purpose of this study was to test the hypothesis that VT stimulation of lgA production is mediated via the Md). Specific goals were to assess the potential role of Md) by: (1 ) determining the effects of Md) depletion on iJLflILQ lgA and lL-6 production by PP and spleen cultures following acute oral VT exposure in mice; (2) comparing the effects of acute oral VT exposure on IL-6 secretion by Md) and CD4‘ T cells and determining their capacity to increase lgA production; and (3) assessing whether cognate interactions and/or soluble factors contribute to Md)-mediated enhancement of lgA production following acute oral VT exposure in mice. The results indicate that prior exposure to VT can enhance the capacity of both Md) and, to a lesser extent, CD4“ T cells to help lgA secretion in vitro. Both superinduction 89 of lL-6 secretion and increased capacity for cognate help by Md) appeared to facilitate VT-induced upregulation of lgA production in cultures from mucosal lymphoid tissues. 90 3.3 MATERIALS AND METHODS 3.3.1 Chemical and reagents All chemicals were of reagent grade quality or better and obtained from Sigma Chemical (St Louis, MO) except where otherwise noted. 3.3.2 Animals and VT exposure regimen Male B6C3F1 mice (89 weeks) were obtained from Charles River Laboratories (Wilmington, MA) and kept in a humidity and temperature controlled university animal care facility room with a 12 hr light and dark cycle. Mice were housed in cages equipped with filter bonnets (Nalgene, Rochester, NY) and fed powdered semi-purified AIN-76A diet (ICN Nutritional Biochemical, Cleveland, OH) on arrival. Animals were acclimated for at least one wk prior to usage. Food and water were withdrawn from cages 2 hr before toxin administration. VT was purchased from Romer Labs (Washington, MO). Mice (4 per group) were gavaged orally with 25 mglkg BW VT in 500 pl of 0.01M carbonate- bicarbonate buffer (pH 9.6) and control mice received 500 pl vehicle only (Azcona- Olivera et al., 1995b). Food and water were restored after gavaging. Mice were humanely euthanized 2 hr after gavage, and their spleens, PP and/or peritoneal Md) were removed for isolation and culture. 3.3.3 Lymphocyte preparation Spleens were teased apart with sterile tissue forceps in harvest buffer consisting of 0.01M PBS (pH 7.4), containing 2% (vlv) heat inactivated FBS (Gibco, Grand Island, NY), 100 U/ml penicillin, and 100 ug/ml streptomycin. Cell 91 suspensions were held on ice for 10 min to allow settling of tissue particles. Supernatant was removed following centrifugation at 450 x g for 10 min. Erythrocytes were lysed for 3 min at room temperature in 0.02 M Tris buffer (pH 7.65) containing 0.14M ammonium chloride. Cells were centrifuged, resuspended in RPMI-1640 medium supplemented with 10% (vlv) FBS, 1mM sodium pyruvate, 100 U/ml penicillin, 100 ug/ml streptomycin, 0.1 mM nonessential amino acid and 5 x 10’5 M 2-mercaptoethanol, and then counted using a hemacytometer (American Optical, Buffalo, NY) (Strober, 1991). PP were teased apart in harvest buffer, passed through a sterile 85 -mesh stainless steel screen and resuspended in the same buffer. Cells were centrifuged at 450 x g for 10 min, resuspended in supplemented RPMI-1640 medium and counted. For some cultures, Md) were depleted as described by Warner et al., (1994) by gently mixing and incubating the whole PP or. spleen cells with Myloclear cell Reagent (Biotex Labs. Inc., Edmonton, Alberta, Canada) for 1 hr. The mixture was layered onto Histopaque-1.119, centrifuged for 10 min at 200 x g and the buffy layer containing lymphocytes was collected. Effectiveness of Md) depletion was verified by esterase staining (Yarn et al., 1971). 3.3.4 Peritoneal Md) isolation Md) were collected from the peritoneal cavity of euthanized mice without activation as described by Kruisbeek (1994). Briefly, Md) were obtained by injecting 10 ml of RPMI medium into the peritoneal cavity and then withdrawing the peritoneal fluids slowly. Pooled peritoneal lavage fluids were centrifuged at 450 x 92 g for 10 min. The cell pellet was washed once in RPMI medium by centrifugation at 450 x g for 10 min and then resuspended in RPMI with 10% F BS for culture. The purity of collected Md) was verified by esterase staining (Yam et al., 1971). 3.3.5 B cell isolation B cells were prepared by depletion of T cells as described by Warner et al., (1994). Briefly, M¢~depleted PP lymphocytes were incubated with a mixture of 100 pl each of monoclonal anti-Thy 1.2, anti-Lyt 2, and anti-L3T4 Abs (PharMingen, San Diego, CA) suspended in 2 ml of10% FBS-RPMI medium for 45 min on ice. The ‘ mixture was washed with cold PBS and centrifuged for 10 min at 450 x g. The pellet was resuspended in 3 ml solution composed of 2.5 ml of 10% FBS-RPMI medium and 0.5 ml of baby rabbit complement (Accurate Chemical & Scientific Corporation, Westbury, NY) and incubated for 45 min at 37° C in a CO2 incubator. The mixture was then washed with FBS-RPMI and centrifuged for 10 min at 450 x g. The cell pellet was resuspended in RPMI with 10% FBS for culture. 3.3.6 CD4‘ T cell isolation CD4+ T cells were purified from splenic lymphocyte preparations after Md) depletion by using the mouse CD4+ cell Kit (Biotex) as described by Warner et al., (1994). Briefly, B cells were removed by absorption on a column coated with goat anti-mouse and goat anti-rat Abs. Lymphocytes were incubated on ice for 45 min with Ab specific for mouse CD8+ cells (rat anti-mouse) prior to passage through the column. Thus both B and C08“ cells should adhere to the column. After cells were collected, erythrocytes were lysed for 2 min at room temperature in a buffer ()3 containing 0.14 M ammonium chloride in 0.02 M Tris buffer (pH 7.65). Cells were centrifuged and resuspended in RPMI with 10% F BS for culture. 3.3.7 Cell cultures Complete (undepleted) and Md)-depleted PP and spleen cells (5 x 105) were cultured in 1 ml of supplemented RPMI-1640 medium in flat-bottomed 24-well tissue culture plates (Fisher Scientific Co., Corning, NY) at 37° under 7% CO2 in a humidified incubator. Cultures were unstimulated or stimulated with the T cell inducing agents, PMA (10 ng/ml) plus ION (500 ng/ml) (PMA + ION) or the Md)- inducing agent LPS (20 ug/ml) from Salmonella typhimurium. Supernatant was collected at 1, 4 and 7 days and stored in aliquots at -20° C until analysis. 3.3.8 Reconstitution studies with fractionated cell populations Mdi-depleted PP cells (5 x 105) and peritoneal Md) from treatment and control animals were reconstituted in 1 ml RPMI-1640 medium with 10% FBS in 24-well tissue culture plates. Previous studies have suggested that a critical ratio of Md) to immune competent cells was important for stimulating lg synthesis (Argyris, 1968). The ratio of Min-depleted PP cells vs peritoneal Md) was chosen at 20:1 because the physiological concentration of Md) is 2 to 5% of the whole cell population in spleen (Verstovsek et al., 1994). A (5:1) ratio was also used for comparative purposes. Mixed cultures were unstimulated or stimulated with PMA + ION or with LPS as described above. Supernatant from reconstituted cell cultures was collected at 1, 4 and 7 days and stored in aliquots at -20° C until analysis. Fractionated splenic CD4‘ T cells and peritoneal Md) from treatment or 94 control animals were reconstituted with purified PP B cells (1 x 10‘) from treatment or control animals in 1 ml RPMI-1640 medium with 10% FBS in 24- well tissue culture plates. Supernatant from reconstituted cell cultures was collected at 7 days and stored in aliquots at -20° C until analysis. 3.3.9 Transwell culture studies Fractionated Md)—depleted PP cells and peritoneal Md) from treatment and control animals were reconstituted in Transwell 24-well cell culture plates (Costar, Cambridge, MA) with a permeable membrane to separate peritoneal Md) and Md)- depleted PP cells. As a control, mixed cells were cultured together in identical plates without a membrane insert. The ratio of Md)-depleted PP cells vs peritoneal Md) was 20:1. Mixed cultures were unstimulated or stimulated with PMA + ION or with LPS. Supernatant from reconstituted cell cultures was collected at 1 and 7 days and stored in aliquots at -20° C until analysis. 3.3.10 lgA quantitation lgA was measured in culture supernatants by ELISA (Bondy and Pestka, 1991). Immunolon 4 Removawell microtiter strip wells (Dynatech Laboratories Inc, Chantilly, VA) were coated overnight at 4° C with 50 ullwell of heavy-chain specific goat anti-mouse lgA (Cappel Worthington, Malvern, PA) at a concentration of 10 itng in 0.1M bicarbonate buffer (pH 9.6). Coated plates were washed 3 times with 0.01M PBS (pH 7.2) containing 0.2% Tween 20 (PBST) to remove excess capture Abs. Plates were incubated with 300 pl of 1% (w/v) BSA-PBST at 37° C for 30 min to block nonspecific protein binding and then washed 4 times with PBST. For lgA 95 determination, standard mouse reference serum (Bethyl Laboratories, Inc, Montgomery, TX) or samples were diluted in 10% (v/v) FBS RPMI-1640 medium and 50 pl was added to appropriate wells. Plates were incubated at 37° C for 60 min, washed 4 times with PBST, and then 50 pl of goat anti-mouse lgA horseradish peroxidase (or-chain specific, Cappel Worthington, Malvern, PA), diluted 1:1000 in 1% (wlv) BSA in PBS, was added to each well. Plates were incubated at 37° C for 30 min and washed 6 times with PBST. Bound peroxidase was determined with 2,2- azino-bis (3-ethylbenzthiazolin-6-sulfonate) (ABTS) substrate [0.4mM ABTS, 50mM citrate buffer (pH 4.0), and 1.2mM hydrogen peroxide] as described previously by Pestka et al., (1980). Absorbance was measured at 405 nm and lgA was quantitated by using Vmax Software (Molecular Devices). 3.3.11 Cytokine quantitation Commercial mouse recombinant cytokine lL-6 (PharMingen, San Diego, CA) was used as standard for IL-6 quantitation. IL-6 production was monitored by ELISA using modification of the procedure of Dong et al. (1994). Briefly, Immunolon 4 Removawell microtiter strip wells (Dynatech Laboratories Inc., Chantilly, VA) were coated overnight at 4° C with 50 pl/well of 1.0 pg/ml purified rat anti-mouse lL-6 capture Ab (PharMingen) in 0.1M sodium bicarbonate buffer (pH 8.2). Plates were washed 3 times with PBST, blocked with 300 pl of 3% (wlv) BSA-PBST at 37° C for 30 min and washed 4 more times with PBST. Standard murine lL-6 or samples were diluted in RPMI-1640 medium containing 10% (v/v) FBS and 50 pl aliquots were added to appropriate wells. Plates were incubated at 37° C for 60 min, washed 4 96 times with PBST, and 50 pl of biotinylated rat anti—mouse IL-6 detection monoclonal Ab (1.5 pglwell; PharMingen) diluted in BSA-PBST were added to each well. After incubation at room temperature for 60 min, plates were washed 6 times with PBST, 50 pl of streptavidin-horseradish peroxidase conjugate (1.5 pglwell in BSA-PBST) were added to each well and plates were incubated at room temperature for 60 min. The plates were then washed 10 times with PBST, and 100 pl of substrate [1 OmM citric-phosphate buffer (pH 5.5), containing 0.4mM tetramethylbenzidine (TMB; Fluka Chemical Corp, Ronkonkoma, NY) and 1.2mM H202] were added to each well. The reaction was stopped by adding an equal volume of 6 N H2804. Absorbance was read at 450 nm and IL-6 concentration was quantitated by using Vmax Software (Molecular Devices). 3.3.12 Statistics The data were analyzed by Dunnett’s test or Student-Newman-Keuls (SNK) test following one way ANOVA using SigmaStat Statistical Analysis System (Jandel Scientific, San Rafael, CA). A p value of less than 0.05 was considered statistically significant. 97 3.4 RESULTS Effects of prior oral VT exposure on in vitro lgA and IL-6 production in PP and spleen cell cultures. To assess the role of Md) on VT-induced elevation of lgA and lL-6 secretion, l : (1) exposed mice orally to 0 and 25 mglkg BW VT, (2) isolated PP and spleen cells 2 hr later, (3) cultured the cells with and without Md) depletion, and (4) monitored lgA and lL-6 concentrations in supernatants over a 7 day period. Both complete PP and spleen cultures isolated from VT-exposed mice produced as much as 2- to 6-fold more lgA at 1, 4 and 7 days than corresponding control cells when cultured in the absence or presence of PMA + ION or LPS (Figure 3.1 and 3.2). The greatest elevation in lgA responses (2- to 10-fold over control) were found in the LPS-stimulated PP and spleen cell cultures. Since both Md) and B cells are responsive to LPS (Raetz et al., 1991; Liu and Janeway, 1991; Berberich and Schimpl, 1992; Verstovsek et al., 1994), it seemed likely that VT had its greatest effects on one or both of these populations. It was further notable that overall lgA production was higher in VT-treated PP cultures (about 10—40 fold) (Figure 3.1) than in VT-treated spleen cultures (Figure 3.2). This evidence suggests that, relative to lgA, VT'may have a greater effect in the gut mucosal compartment as compared to the systemic compartment, and that PP Md) or B cells are primary targets for VT-induced lgA production in this model. Md) depletion markedly reduced lgA production by PP cells (about 85 to 95%) from VT-treated and control animals cultured without stimulation or with stimulation by PMA + ION or with LPS as compared to undepleted whole cultures 98 Figure 3.1. lgA production by PP cell cultures isolated from mice following exposure to 0 and 25 mglkg BW VT. Mice were sacrificed at 2 hr after gavage. Both complete and Md)-depleted PP cells (5x105/ml) were cultured with or without mitogens in 24-well plates for 1, 4 and 7 days. Supernatant lgA levels were analyzed by ELISA. Data are mean i SEM (n=4). Bars marked with letter indicate significantly different (p < 0.05) as follows: a, different from corresponding complete PP cells; and b, different from corresponding control PP cells. 99 i o i can i coo i com 93 OONV 20:5... 682%.. es: E E I 683%.. es. .380 was as: oov 33:52: I O O (‘0 .880 D coo (mi/fin) V5| I 00 Figure 3.2. lgA production by spleen cell cultures isolated from mice following exposure to 0 and 25 mglkg BW VT. Mice were sacrificed at 2 hr after gavage. Both complete and Md)-depleted spleen cells (5x105/ml) were cultured with or without mitogens in 24-well plates for 1, 4 and 7 days. Supernatant lgA levels were analyzed by ELISA. Data are mean i SEM (n=4). Bars marked with letter indicate significantly different (p < 0.05) as follows: a, different from corresponding complete spleen cells; and b, different from corresponding control spleen cells. 101 ma... 882%.. :5 ._.> Ens 2:: 20. + <2.— 83:53:: .5' l O ‘- on 683% 3:. .350 .380 U O ('0 (iwifiu) V5I 102 (Figure 3.1). In contrast to PP, VT-enhanced lgA production in spleen cultures was much less dependent on Md) (Figure 3.2). These results suggest that Md) was a key target of VT and was intimately involved with lgA hyperproduction. Since lL-6 has been previously shown to promote B cell differentiation to lgA secretion in PP cells (Beagley et al., 1991; Beagley and Elson, 1992), the effect of acute VT exposure on production of this cytokine also was evaluated. As with lgA, lL-6 secretion was enhanced by VT pretreatment (Figure 3.3 and 3.4). IL-6 in LPS- stimulated complete cultures was 2- to 3-fold higher in the VT treatment groups as compared to controls at 7 days for PP cells (Figure 3.3) and 1, 4 and 7 days for spleen cells (Figure 3.4) whereas IL-6 was unaffected in unstimulated or PMA + ION-stimulated cultures. Since LPS is a potent stimulator of Md) (Raeta et al., 1991; Verstovsek et al., 1994) whereas PMA and ION have been used primarily for stimulating primary T cells (Gonzalez et al., 1994; Jeannin et al., 1995), the results suggested that Md) were the primary source for IL~6 in both PP and spleen. This possibility was supported by the observation that Md) depletion markedly depressed lL-6 levels in LPS-stimulated PP (GO-95%) (Figure 3.3) and spleen (40- 75%) (Figure 3.4) cell cultures from VT-treated and control animals as compared to undepleted whole cell cultures. In view of the key role of lL-6 in differentiation to lgA secretion and my previous finding that anti-IL-6 neutralizes VT’s effects on lgA (see chapter 2), this cytokine is likely to play a mediatorial role in VT-enhanced lgA production. Figure 3.3. lL-6 production by PP cell cultures isolated from mice following exposure to 0 and 25 mglkg BW VT. Mice were sacrificed at 2 hr after gavage. Both complete and Md)-depleted PP cells (5x105/ml) were cultured with or without mitogens in 24-well plates for 1, 4 and 7 days. Supernatant IL-6 levels were analyzed by ELISA. Data are mean 3: SEM (n=4). ND indicates non-detectable. Bars marked with letter indicate significantly different (p < 0.05) as follows: a, different from corresponding complete PP cells; and b, different from corresponding control PP cells. 104 an... 622%.. 22. ._.> cow oov com com .263 2:: \\\\\\\\\\\\\\\\\\\\§\‘ solo-Dololoto-I'ouuonho onoon-on-non-co-on-oausno. CIIOIIDIIIIIIOCOOC 20. + (in. low 19‘ low 35:53.... .5' O I O N I on .2858 2... .228 \\ .35 Du (iwifid) 9-1i O Q 105 Figure 3.4. lL-6 production by spleen cell cultures isolated from mice following exposure to O and 25 mglkg BW VT. Mice were sacrificed at 2 hr after gavage. Both complete Md)-depleted spleen cells (5x105/ml) were cultured with or without mitogens in 24-well plates for 1, 4 and 7 days. Supernatant lL—6 levels were analyzed by ELISA. Data are mean -.t SEM (n=4). Bars marked with letter indicate significantly different (p < 0.05) as follows: a, different from corresponding complete spleen cells; and b, different from corresponding control spleen cells. VT (in depleted) -VT Control (m depleted) 3 C O U 106 LPS 7 4 1 Time (days) I I I I I O O O O O O C O O O O o 0 (D V N ‘_ 2 Q + 0- =2\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\‘ r.\\\\\\\\\\\\\\\\\\\\\\\\\s =W I I O O C) '0 N O) ‘- ‘- E .9 g r: 3 I I I I I D C O O O D IO N O) (O (0 ‘- ‘- (iwifid) 9-1i 107 Effects of prior oral VT exposure on 111mg IgA and IL-6 production in Imp-depleted PP cell cultures reconstituted with peritoneal Md). The potential role of Md) in VT-induced lgA and IL-6 production was further assessed on Md)- depleted PP cultures reconstituted with Md). Peritoneal exudate was used as an enriched source of Md). Comparisons were made using cultures that contained: (1) control peritoneal Md) + control Md)-depleted PP cells; (2)~control peritoneal Md) + VT-treated Mdi-depleted PP cells; (3) VT-treated peritoneal Md) + control Md)- depleted PP cells; and (4) VT-treated peritoneal Md) + VT-treated Mdi-depleted PP cells. lgA levels were consistently the highest in cultures where Mo—depleted PP cells from VT-treated animals were co-cultured with peritoneal Md) from VT-treated animals using a ratio of 20:1 as compared to the combinations of control Md)- depleted PP cells with control peritoneal Md) (Figure 3.5). These effects were most prominent when LPS was used as the mitogen. Notably, lgA production further increased when the ratio of VT-treated Md)-depleted PP cells to VT-treated peritoneal Md) was decreased to 5:1 (Figure 3.6). When compared to control Md)- depleted PP cells plus control peritoneal Md), lgA levels were also increased to a lesser extent when VT-treated Md)-depleted PP cells were co-cultured with control peritoneal Md) or control Md)-depleted PP cells were co-cultured with VT-treated peritoneal Md) (Figure 3.5 and 3.6). IL-6 levels were markedly increased in cultures where Md)-depleted PP cells from VT-treated animals were co-cultured with peritoneal Md) from VT-treated animals as compared to the combination of control Md)-depleted PP cells with 108 Figure 3.5. IgA and IL-6 production in reconstituted cultures containing Md)- depleted PP cells and peritoneal Md) at a 20:1 ratio. Mice were exposed to 0 and 25 mglkg BW VT and sacrificed at 2 hr after gavage. Md)-depleted PP cells (5x105lml) and peritoneal Md) from animals were reconstituted at ratio of 20:1 in 24- well tissue culture plates as follows: (1) control peritoneal Md)+ control Md)-depleted PP cells (CON Md) + CON Md)‘ PP); (2) control peritoneal Md) + VT-treated Md)- depleted PP cells (CON Md) + VT Md)‘ PP); (3) VT-treated peritoneal Md) + control Md)—depleted PP cells (VT Md) + CON Md)‘ PP); (4) VT-treated peritoneal Md) + VT- treated Md)-depleted PP cells (VT Md) + VT Md)’ PP) . Mixed cultures were unstimulated or stimulated with the mitogen for 1, 4 and 7 days, respectively. Supernatant lgA and IL-6 levels were analyzed by ELISA. Data are mean :I: SEM (n=4). Bars marked with letter indicate significantly different (p < 0.05) as follows: a, different from CON Md) + CON Md)‘ PP; and b, different from CON Md) + VT Md)‘ PP. 109 [:1 1081! W 4 Days Unstimulatod I CON I“ + CON Mi PP Wu I CON "6+ VT Mii' PP W. I VT MQ + CON my pp %////// .- VTM¢+vrmr PP W/ , PMA + ION I CON Md + CON 316' PP //// - 7" . ,, CON MH- VT Mt pp ////%/////,. . VT M¢ + CON If pp g////%///////’,. a. -.&b VT I“ + VT Md" pp /////////////////////// of). LPS I CON I“ + CON Mo. PP 7/////////. I CON M¢+ VT Hf pp %////////////_ . iiiri 012 lgA (Mg/ml) 3450510 - 7 Days Unstlmulated I — I-h 'u PMA + ION 37%//%'% — I — . 8.3 ////////%///////////%//%//o%/~//// ' u LPS — . I i4/////%////%/////%/%/;%,. . _ , on I I I I IL-6 (nglml) 110 Figure 3.6. IgA and lL-6 production in reconstituted cultures containing Md)- depleted PP cells and peritoneal Md) at a 5:1 ratio. Mice were exposed to 0 and 25 mglkg BW VT and sacrificed at 2 hr after gavage. Md)-depleted PP cells (5x105/ml) and peritoneal Md) from animals were reconstituted at ratio of 5:1 in 24-well tissue culture plates as follows: (1) control peritoneal Md)+ control Mcb-depleted PP cells (CON Md) + CON Md)‘ PP); (2) control peritoneal Md) + VT-treated Md)-depleted PP cells (CON Md) + VT Md)‘ PP); (3) VT-treated peritoneal Md) + control Mcb—depleted PP cells (VT Md) + CON Md)‘ PP); (4) VT-treated peritoneal Md) + VT-treated Md)- depleted PP cells (VT Md) + VT Md)‘ PP) . Mixed cultures were unstimulated or stimulated with the mitogens for 1, 4 and 7 days, respectively. Supernatant IgA and lL-6 levels were analyzed by ELISA. Data are mean i SEM (n=4). Bars marked with letter indicate significantly different (p < 0.05) as follows: a, different from CON Md) + CON Md)’ PP; and b, different from CON Md) + VT Md)’ PP. 111 E: 1 Day "Hi 4 Days - 7 Days U u I” Unstimulated CON M5 + can I; pp 5:3; CON Mi} + VT If pp VT Md) + CON If PP ‘ VT ”5 + VT M" PP 52...,“ P“ * '°" PMA + ION con m + can If PP CON M¢ + VT u.’ pp VT M. ‘I’ GO" M.’ PP :52; l.;.‘./ ,;/-H ,//. U ',-/", a,” .r 2 /////////////////. ///’///'// :2) //J . ////// I . VT Md) + VT Hf pp - . .CON "9 + CON M.“ pp . CON M. + VT M‘I' PP $412377. VT "9 "' CON ”V pp VT "9 "' W “I. PP 31:. Illj o 2 4 6 8010203040 lgA (pg/ml) IL-6 (nglml) 112 control peritoneal Md) (Figure 3.5 and 3.6). The effects were most pronounced in LPS-stimulated cell cultures thus suggesting that Md) were a primary source of IL-6. In further support of this contention, 2- to 4-fold more IL—6 was prodUced in cultures where VT-treated Md)-depleted PP cells were co-cultured with VT-treated peritoneal Md) at ratio of 5:1 (Figure 3.6) as compared to cultures employing a 20:1 ratio (Figure 3.5). Higher IL-6 concentrations were observed in cultures where VT-treated Md)- depleted PP cells were co-cultured with VT-treated peritoneal Md) as compared to the combination of VT-treated Md)-depleted PP cells with control peritoneal Md) (Figure 3.5 and 3.6). However, lL-6 levels were also elevated in cultures when VT- treated Md)-depleted PP cells were combined with control peritoneal Md) as compared to the combination of control Mdi-depleted PP cells with control peritoneal Md) (Figure 3.5 and 3.6). These results suggest that increased lgA production might be mediated by VT-induced enhancement of IL-6 production both in part by Md) and in part by a non-Md) populations such as Th cells. Effects of prior oral VT exposure or) MEL!) IgA and IL-6 production by B cell cultures reconstituted with peritoneal Md) or splenic CD4+ T cells. Previous results suggested the potential of both Md) and Th cells to promote lgA and IL-6 production. To further assess these possibilities, purified PP B cells were reconstituted with peritoneal Md) or splenic CD4*T cells. All three cell types were isolated from mice 2 hr after exposure to VT or vehicle and then cultured as 113 follows: (1) control B cells only; (2) VT-treated B cells only; (3) control B cells + control CD4” T cells; (4) VT-treated B cells'+ control CD4+ T cells; (5) control B cells + VT-treated CD4+ T cells; (6) VT-treated B cells + VT-treated CD4+ T cells; (7) control B cells + control peritoneal Md); (8) VT-treated B cells + control peritoneal Md); (9) control B cells + VT-treated peritoneal Md); and (10) VT-treated B cells + VT-treated peritoneal Md). A ratio of 3:2 for PP B cells vs CD4+ T cells was chosen based on the study of Bondy and Pestka (1991‘). A ratio of PP B cells vs peritoneal Md) was chosen at 20:1 for the reasons described above. As in previous experiments, cultures were unstimulated or stimulated with PMA + ION or with LPS. PP B cell cultures from control animals produced significantly more lgA when co-cultured with peritoneal Md) or CD4+ T cells from VT-treated animals as compared to cultures containing control PP B cells and control peritoneal Md) or CD4‘ T cells (Figure 3.7). Similarly, lgA was markedly amplified in cultures when VT-treated PP B cells were co-cultured with VT-treated peritoneal Md) (about 14- fold) or CD4* T cells (about 5-fold) as compared to cultures containing VT-treated PP B cells and control Md) or CD4“ T cells (Figure 3.7). As shown in previous experiments, the greatest lgA responses in Md)-containing cultures were observed in the LPS-stimulated cultures where VT-treated or control PP B cells were co- cultured with VT-treated peritoneal Md). In general, these observations suggest that lgA production can be elevated by VT-treated Md) or, to a lesser extent, VT-treated CD4” T cells. lL-6 levels were also markedly elevated in cultures where PP B cells from 114 Figure 3.7. IgA and IL-6 production by purified PP B cells reconstituted with purified peritoneal Md) or splenic CD4+ T cells. Mice were exposed to 0 and 25 mglkg BW VT and sacrificed at 2 hr after gavage. PP B cells (1x10‘lml) and splenic CD4+ T cells or peritoneal Md from animals were reconstituted at ratios of 3:2 and 20:1, respectively, in 24-well tissue culture plates as follows: (1) control or VT- treated B cells only (B cells); (2) control or VT-treated B cells + control CD4‘T cells (B cells + CON CD4” cells); (3) control or VT-treated B cells + VT-treated CD‘ T cells (B cells + VT CD4” cells); (4) control or VT-treated B cells + control peritoneal Md ( B cells + CON Md); (5) control or VT-treated B cells + VT-treated peritoneal Md (B cells + VT Md). Mixed cultures were unstimulated or stimulated with the mitogens for 7 days. Supematant IgA and IL6 levels were analyzed by ELISA. Data are mean :1: SEM (n=4). ND indicates noncetectable. Bars marked with letter indicate significantly different (p < 0.05) as follows: a, control B cells vs VT-treated B cells; b, control T cells vs VT-treated T cells in control B cell groups; c, control T cells vs VT-treated T cells in VT-treated B cell groups; d, control Md vs VT- treated Md in control B cell groups; and e, control Md vs VT-treated Md in VT- treated B cell groups. 115 I: Control B cell - VT B cell Unstlmulatod Unstimulatod B cells a coils + con coil‘oslls a coils + VT coil‘coiis B coils + co» m "2 a coils + VT Md PMA+ ION PMA-l» ION B cells '3 cells + con corosils B cells + VT census 3 cells 4» CON Md 3 coils + VT in 0 1O 20 30 400 2 4 6 IgA (nglml) lL-6 (nglml) 116 control animals were co-cultured with peritoneal Md from VT-treated animals as compared to control PP B cells plus control peritoneal Md (Figure 3.7). This effect was markedly amplified in cultures when VT-treated PP B cells were co-cultured with VT-treated peritoneal Md as compared to cultures containing VT-treated PP B cells and control peritoneal Md (Figure 3.7). The greatest response was observed in the LPS-stimulated cultures (35-60 fold increase) whereas PMA + ION appeared to have no effect when compared to unstimulated cultures. IL-6 levels were only significantly increased in PMA + ION-stimulated cultures where control PP B cells were co-cultured with VT-treated CD4“ T cells as compared to the combinations of control PP B cells with control CD4+ T cells (Figure 3.7). Notably, approximately 2-fold more IL-6 secretion was observed in cultures where VT-treated or control PP B cells were co-cultured with VT-treated peritoneal Md as compared to co-cultures containing control peritoneal Md. These data suggest that increased lgA production in mucosal tissues could be mediated by both Md and to a lesser extent 004* T cells but that the elevation of IL-6 levels was mediated mainly by Md. Interestingly, prior exposure of B cells to VT in vivo consistently enhanced the capacity of Md to increase lgA and lL-6 production in combination cultures. Role of soluble mediators and cell-cell interactions on the capacity of Md to stimulate mm lgA production. It was of further interest to verify whether Md enhanced lgA production by providing cognate cell-cell interactions and/or soluble mediators (e.g. IL-6). Peritoneal Md and Md-depleted PP cells were obtained from 117 mice 2 hr after exposure to 0 and 25 mglkg BW VT and were co-cultured in plates containing a permeable membrane (Transwell) to separate peritoneal Md and Md- depleted PP cells. As controls, mixed cells were cultured together in identical plates without a membrane. The cells were reconstituted as follows: (1) control peritoneal Md + control Md-depleted PP; (2) control peritoneal Md + VT-treated Md-depleted PP cells; (3) VT-treated peritoneal Md + control Md—depleted PP cells; and (4) VT-treated peritoneal Md + VT-treated Md—depleted PP cells. As expected, lgA levels were increased in cultures where VT-treated Md—depleted PP cells were co-cultured with VT-treated peritoneal Md in the absence of a membrane (Figure 3.8). lgA levels were significantly decreased (about 50%) in the reconstituted cell cultures with or without PMA + ION or LPS and employing a membrane as compared to those cell cultures without a membrane (Figure 3.8). These findings suggest that in addition to Md-secreted mediators such as the cytokine IL-6, cell- cell interactions might be important for mediating VT-enhanced lgA production. 118 Figure 3.8. lgA production in reconstituted cultures containing Md-depleted PP cells and peritoneal Md at a 20:1 ratio. Mice were exposed to 0 and 25 mglkg BW VT and sacrificed at 2 hr after gavage. Md-depleted PP cells (1x105/ml) and peritoneal Md from animals were reconstituted at ratio of 20:1 in 24 well Transwell cell culture plates which contain a permeable membrane to separate Md and Md- depleted PP cells. As controls, mixed cells were cultured together in identical plates without a membrane. The cells were reconstituted as follows: (1) control peritoneal Md+ control Md-depleted PP cells (CON Md + CON Md' PP); (2) control peritoneal Md + VT-treated Md-depleted PP cells (CON Md + VT Md' PP); (3) VT-treated peritoneal Md + control Md-depleted PP cells (VT Md + CON Md‘ PP); (4) VT- treated peritoneal Md + VT-treated Md-depleted PP cells (VT Md + VT Md' PP) . Mixed cultures were unstimulated or stimulated with the mitogens for 1 and 7 days, respectively. Supernatant lgA levels were analyzed by sandwich ELISA. Data are mean :1: SEM (n=3). Bars marked with letter indicate significantly different (p < 0.05) as follows: a, different from cell cultures without a membrane; b, different from CON Md + CON Md‘ PP; and c, different from CON Md + VT Md' PP. 119 [:3 Without membrane - With membrane 1 Day 7 Days Unstimulated Unstinulated cou Md +con iilli' PP E a ' con m +VT lu' PP F” a?" VT m +CON m’ PP El :3“ VTm +VT Md' PP Ff Il—IJ PMA+ ION PMA + ION con Md +co~ m‘ PP i7," CON m +VT will" PP E" VT m +cou Md' PP :1 a VT Md +V'r llli' PP F4” LPs LPS * con lu +co~ m’ PP ; con mi +V'l' Md’ PP ‘ IjIIIJ 1M VT Md +cou lu’ PP I VTMd +VT Md' PP E} "" —i:i..——’_""°' i T I I T I I I 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 1.0 19A (“91"“) '9!" (pg/ml) 120 3.5 DISCUSSION VT has been shown to be both immunostimulatory and immunosuppressive in a variety of animal and cell culture models (Pestka and Bondy, 1994). The stimulatory effects of prolonged dietary VT exposure previously observed in our laboratory include: (1 ) elevation of serum lgA production (Forsell et al., 1986; Pestka et al., 1989), (2) induction of lgAN (Pestka et al., 1989; Pestka and Bondy, 1990; Dong et al., 1991; Dong and Pestka, 1993; Rasooly and Pestka, 1994; Greene et al., 1994a; 1994b); and (3) increase in the percentages of membrane lgA+ cells, T cells, and CD4“ T cells as well as CD4*ICD8* T cell ratios in PP and spleen (Pestka et al., 1990a; Bondy and Pestka, 1991). Collectively, all these findings are indicative of dysregulation of lgA production by VT. Several lines of evidence suggest that VT can specifically dysregulate cytokine production. For example, mum superinduction of lL-2, IL-4, IL-5 and lL-6 mRNA expression by VT has been observed in murine splenic CD4+ T cells (Ouyang et al., 1995; 1996a; Azcona-Olivera et al., 1995a; Warner et al., 1994) as well as EL-4 thymoma cultures (Dong et al., 1994). Additionally, mm: enhancement of mRNA levels of the proinflammatory cytokines (IL-6, lL-1B, TNF-a and lFN-y) that are suggestive of Md activation is observed in PP and spleens of mice exposed acutely to VT (Azcona-Olivera et al., 1995b; Zhou et al., 1997). Thus, the potential exists for VT to alter lg secretion via cytokines. The results presented herein revealed that both lgA and lL-6 secretion were markedly decreased in Md—depleted PP and spleen cell cultures isolated from mice 2 hr after exposure to 25 mglkg VT. This evidence 121 suggests that Md produce lL-6 and that this cytokine may be related to VT-induced lgA production. In this study, I used a short term VT exposure (2 hr) model to identify potential leukocyte populations that mediate this novel isotype specific dysregulation. Several key findings were made. First, PP was a primary target for VT-induced lgA production as compared to spleen. Second, both IgA and lL-6 secretion by VT-treated and control PP cultures were diminished after depletion of Md. Third, enhanced production of murine lL-6 occurred concurrently with increased lgA production in Md-depleted PP cell cultures reconstituted with peritoneal Md. Fourth, increased lgA production was evident in PP B cell cultures reconstituted with both VT-exposed Md and CD4+ T cells with Md having the greatest effect. Finally, both soluble factors and cognate interactions between Md and the lymphocyte populations appeared necessary for increased lgA production in PP cultures following VT exposure. PP in the gut-associated lymphoid tissue is a major source of B cell precursors for lgA-secreting plasma cells (Craig and Cebra, 1971). In contrast to PP, spleen is not a major source of B cells for IgA production. Both Md and B cells are responsive to LPS (Raetz et al., 1991; Liu and Janeway, 1991; Berberich and Schimpl, 1992; Verstovsek et al., 1994). Elevation of IL-6 secretion in LPS- stimulated spleen cultures coupled with a small relative increase in IgA production suggests that Md were the primary source for IL-6 in spleen but that spleen was not a primary target for VT-induced lgA production in this model. This contention was supported by the observation that production of IgA was affected more by VT in PP 122 cultures than in spleen cultures. Thus, VT potentially has a greater effect in the gut mucosal compartment as compared to the systemic compartment. It well-established that certain cytokines are involved in regulating lgA response in PP via enhancement of activation, Switching and differentiation of B cells into lgA-secreting plasma cells (McGhee et al., 1989; Beagley and Elson, 1992; McGhee and Kiyono, 1993). Notably, lL-6 has been demonstrated to elevate lgA production when it is added to PP B cell cultures (Beagley et al., 1989; 1991). One of the primary sources of IL-6 is the Md (Bauer et al. 1988; Bauer, 1989). In previous studies, I found that acute oral VT exposure in mice can increase the capacity of PP and spleen cell cultures to secrete lL-6 and, furthermore, specific neutralizing anti-lL—6 Ab inhibits in_\_/_i1r_o lgA production by PP cultures obtained from mice exposed to single oral dose of VT (see chapter 2). Thus, there is a strong possibility that, by secreting cytokines such as IL—6, Md may contribute co- stimulatory function for B cell production of IgA. Recently, VanCott et al., (1996) demonstrated that oral exposure to Salmonella expressing fragment C of tetanus toxin induces high levels of Md-secreting IL-6 in murine PP and spleen cultures, and that this lL—6 elevated plasma cell production of lgA at mucosal effector sites. The production of IL-6 from local intestinal mucosa has also been reported (Bao et al., 1993). Mucosal production of lL-6 is important because this cytokine could regulate a number of local immune responses including lgA production (Akira et al., 1990). This conclusion was supported by recent observations that the numbers of mucosal lgA-producing cells were dramatically decreased in IL6 knock-out mice 123 (Ramsay et al., 1994b). Thus, it is reasonable to suggest that lL-6 produced by Md plays a central role in regulation of VT-induced lgA responses. Recently, Okahashi et al., (1996) demonstrated that oral administration of lL-4 knock-out mice with tetanus toxin and CT induced CT-specific CD4“ T cell- producing IL-6 and mucosal lgA anti-CT responses. This suggested that IL-6 from Th2-type cells plays an important compensatory role in the induction and regulation of mucosal lgA response. Thus, T cells may be another source of IL—6 that arises oral VT exposure in mice. This is supported by several lines of evidence. First, increased lL-6 and lgA production were also observed in cultures where VT-treated Md—depleted PP cells were co—cultured with control peritoneal Md. After depleting Md from PP cells, T cells are still presumed to be present and therefore could provide an alternative signal for lL-6 secretion. Second, lgA production in control PP B cell cultures was enhanced when co-cultured with VT-treated CD4“ T cells as compared to cultures containing control PP B cells and control CD4+ T cells. lL-6 secretion was significantly increased only in PMA + ION-stimulated cultures where control PP B cells were co-cultured with VT-treated CD4+ T cells as compared to .co-cultured with control CD4+ T cells. Third, previous studies have observed that wild exposure of murine splenic CD4+ T cells to VT can significantly elevate lgA production by B cells (Warner et al., 1994). In these studies, lL-6 levels were enhanced significantly in Con A-stimulated CD4‘ T cells after exposure to VT when compared to control. Fourth, previous studies have demonstrated that significant increases in IgA production occur when control B cells are co-cultured with PP T 124 cells isolated from mice fed 25 ppm VT for 8 wks (Bondy and Pestka, 1991). Finally, elevated percentages of CD4“ T cells and CD4‘ICD8‘ T cell ratios were also found in PP and spleens of VT-fed mice (Pestka et al., 1990a). Collectively, these results suggest that T cells might also play a role in regulating VT-induced lgA production. Since the results indicated the potential of both Md and T cells from VT- treated animals to promote IgA and lL-6 secretion, a key question relates to which cell type (Md or T cells) is the major contributor. It is critical to note that lgA and IL- 6 production were highest in cultures where VT-treated or control PP B cells were co-cultured with VT-treated Md than co—cultured with VT-treated CD4” T cells. Furthermore, the greatest effects on lgA and lL-6 production were found in the LPS- stimulated cultures consisting of VT-treated or control PP B cells and VT-treated peritoneal Md. These observations suggest in the model described herein that Md may have a much greater effect on lgA and IL-6 production than CD4” T cells. There were several reasons for selecting peritoneal Md and splenic CD4" T cells in reconstitution studies. First, results of preliminary experiments indicated that lL-6 secretion was increased in peritoneal Md cultures obtained from mice 2 hr after oral exposure to VT (data not shown). This evidence suggests that VT was capable of stimulating peritoneal Md to release IL-6. Second, peritoneal exudates are afar richer source of Md than PP and thus minimized the need for experimental animals. Third, murine spleen was used as an enriched source of CD4” T cells because previous studies have shown that exposure to VT in vitro superinduces IL- I25 2, IL-4 IL-5 and lL-6 mRNA expression in murine splenic CD4’ T cells (Ouyang et al., 1995; 1996a; Azcona-Olivera et al., 1995a; Warner et al., 1994). Finally, in vitro exposure of murine splenic CD4+ T cells to VT can significantly enhance lgA production by B cells (Warner et al., 1994). These observations suggest that splenic CD4+ T cells may play a role in regulating lgA production by secretion of cytokines such as lL-6. In addition to Md and Th cells, it is worth noting that B cells can also produce IL-6 (Van-Snick, 1990). Yee et al., (1989) has proposed that B cells synthesize, secrete and utilize IL-6, and this autocrine pathway could promote both growth and lg secretion. Clearly, the autocrine or paracrine actions of B cell-secreted lL-6 could lead to augmented differentiation of lgA-committed B cells and result in increasing lgA production. Thus, it was notable that control and VT-treated B cells cultured alone did not show a major difference of IgA and lL—6 production following oral exposure to VT whereas there was a marked increase in lgA and lL-6 production when VT-treated B cells were combined with untreated and treated Md or treated CD4’ T cells. A number of studies have demonstrated that the regulation of the Ig response not only needs soluble cytokines but also requires cell-cell interactions. Vercelli et al., (1989) have observed that a physiological concentration of lL-4 induces purified B cells to synthesize IgE only after a cognate interaction with T cells. Cytokine combinations alone were unable to replace T cells and monocytes in inducing IgE synthesis in peripheral blood mononuclear cells, suggesting that 126 cell-cell contact is an essential requirement. It was notable that lgA levels were markedly less in the reconstituted cell cultures where VT-treated Md were separated with a semi-permeable membrane as compared to those cell cultures without a membrane. One explanation for these findings is that although the soluble mediators were allowed to pass through the membrane, cell-cell interactions between Md and the lymphocyte populations were prevented. Md are required as accessory cells in the activation of T lymphocytes (Rosenwasser and Rosenthal, 1978). Md can be stimulated to secrete cytokines by contact with Ag (Farr et al., 1977), mitogen activated lymphocytes (Mizel et al., 1978) and by cytokines secreted from activated lymphocytes (Meltzer and Oppenheim, 1977). This indicates that Md—Iymphocyte interactions are bidirectional and can be mediated either by cell-cell contact or by cytokines. Erb and Feldmann, (1975a) reported that Md and T cell interactions are required for the induction of Th cell in the reaction to Ags i_n_vjt_rg. Some previous studies have demonstrated that activation of Md effector function is required the cognate (cellzcell) interactions between Md and Th cells (Sypek and Wyler, 1991; Sypek et al., 1991; Stout and Suttles, 1993). Subsequently, many recent studies also demonstrated that interactions between activated T cells and monocytes are necessary for the induction of IL-1 8 mRNA expression (McAllister and Ellis, 1996) as well as the production of lL-1 (3 (See et al., 1992; Dayer et al., 1993; Wagner et al., 1994), TNF-a (See et al., 1992) and lL-12 (Shu et al., 1995) by monocytes. Therefore, I believe that in addition to soluble mediators, interactions between Md and T cells are required in VT-induced enhancement of IgA 127 production. These interactions are likely to play an integral role in regulation of lgA response in mucosal tissues and susceptibility to VT-induced lgAN. It is now also clear that induction of T-cell activation for regulating immune responses requires the costimulatory signals which involve the attachement of CD40 ligand (CD40L) on activated T cells to CD40 on B cells (Noellet et al., 1992; Van-den-Eertwegh et al., 1993) and the interactions of CD28 on the T cells with B7 on the Ag presenting cells (Mondino and Jenkins, 1994). The costimulatory signals involved in interactions between T and B cells, and T cell and Md are shown in Figure 3.9. B7 is a cell surface molecule and is expressed by multiple cell types including Md and B cells (Hathcock et al., 1994). Previous studies have demonstrated that interactions of CD28 with its natural ligand B7 expressed on the surface of activated Md or B cells will induce proliferation and cytokines secretion in activated T cells (Freedman et al., 1991; Razi-Wolf et al., 1992; Linsley et al., 1991). Based on the observation that VT could induce secretion of IL-6 by Md for regulating lgA production, it seems possible that induction of expression of cell surface molecule B7 also occurred on VT-treated Md and resulted in providing a costimulatory signal for regulating lgA response. Furthermore, cognate interactions between Md and T cells appeared necessary for VT-induced enhancement of IgA production. Further investigation of the potential of VT on inducing expression of B7 molecule on Md is thus warranted. Taken together, these results suggest that superinduction of IL-6 secretion 128 68:3me new =8 ._. oca i=8 m new ._. 5053 29.67. bo.m_:E_.moo ad 239“. owonqooooz :00 m =00 mu. :00 an. 129 by both Md and to a lesser extent CD4+ T cells may be responsible for upregulation of lgA production in mice exposed orally to VT. For Md, this is likely to involve both secretion of soluble mediators such as IL-6 and cognate interactions. CHAPTER 4 INDUCTION OF NEPHROPATHY BY INJECTION WITH DIETARY VOMITOXIN-INDUCED IgA MONOCLONAL ANTIBODIES INTO MICE 131 4.1 ABSTRACT Oral exposure to VT induces elevated levels of serum lgA, circulating IgA—lC and causes mesangial lgA deposition and hematuria in mice. These manifestations mimic the hallmark clinical signs of human lgAN. To further assess the role of VT- induced lgA in inducing this disease in the mouse, two strains of mice, B603F1 and BALBIC, were injected i.p. with various monoclonal lgA Abs previously obtained from hybridomas derived from PP of VT-exposed mice and 1 several immunopathologic markers were subsequently monitored. In B6C3F1 mice, 2- to 5-fold serum IgA and lgA-IC levels were increased in treatment groups after 4 and 6 wks as compared to controls. Serum lgG and IgM levels as well as urinary erythrocyte counts were elevated in treatment groups after 2, 4 and 6 wks as compared to controls. Concurrent increases in IgA and IgG complexes containing casein, the dietary protein source, were found in treatment mice. Mesangial lgA, lgG, lgM and C3 deposition were significantly increased in all treatment mice after 6 wks. Furthermore, electron microscopy revealed that there was an elevation of electron-dense deposits in the glomeruli of lgA-injected mice after 6 wks. The above parameters were similarly affected in BALBIC mice. Injection of lgA-secreting hybridoma cells into BALBIC mice also increased serum lgA, lgA-IC and IgG levels as well as elevated mesangial lgA, lgG and C3 deposition and hematuria in treatment mice after 2 to 3 wks as compared to controls. These results indicated that injection of VT-induced monoclonal lgA Abs or lgA-secreting hybridoma cells into mice can induce elevation of serum lgA, lgA-IC, lgG and cause mesangial lgA, 132 lgG, C3 deposition and hematuria. Casein, an Ag found in the diet used for these mice, appeared to form IC with lgA or lgG and these lC may participate in pathogenesis leading to nephropathy. 133 4.2 INTRODUCTION VT is a fungal secondary metabolite that belongs to a family of mycotoxins referred to as trichothecenes (Tanaka et al., 1988). This toxin is frequently found in cereal grains as well as other food and agricultural products (Abouzied et al., 1991; Rotter et al., 1996). VT and other trichothecenes are potent protein synthesis inhibitors (Ueno, 1985; Betina, 1989; Pestka and Casale, 1990) that can significantly alter cell-mediated immunity, humoral immunity and host resistance in animal models (Pestka et al., 1987; Pestka and Bondy, 1990). Trichothecenes can be both immunostimulatory and immunosuppressive in a variety of animal and cell culture models (Pestka and Bondy, 1994). In mice, dietary VT exposure induces extremely high levels of serum IgA (Forsell et al., 1986; Pestka et al., 1989), and increases circulating lgA-IC formation, glomerular lgA deposition and hematuria (Pestka et al., 1989; Pestka and Bondy, 1990; Dong and Pestka, 1993; Rasooly and Pestka, 1994; Greene et al., 1994a; 1994b). These symptoms are clinically similar to human lgAN (Berger's Disease) which is the most common glomerulonephritis worldwide (D’Amico, 1987). These observations suggest that VT may be used as a probe to understand mechanism of lgAN and that it might be a possible etiological factor in lgAN. Although the pathogenesis and etiology of lgAN are not fully understood, lgAN is likely to be caused by an aberrant mucosal immune response resulting in large increases in polyspecific serum lgA (especially polymeric lgA) (Monteiro et al., 1984). This apparently leads to a dramatic increase in glomerular IgA and lgA-IC 134 deposition within the kidney (Czerkinsky et al., 1986). lgG, lgM and C3 complement component deposition occurs concurrently and are also likely to contribute to glomerular dysfunction. It has been suggested that the mucosal stimulation following gastrointestinal [respiratory infection, genetic background or diet factors may contribute to the development of this disease (D’Amico, 1987). There are several animal models for lgAN that mimic the clinical and morphologic features of the human syndrome (Rifai et al., 1979; Emancipator et al., 1983; Rifai and Millard, 1985; Emancipator et al., 1987). Notably, Rifai et al., (1979) and Rifai and Millard, (1985) have demonstrated that passive injection of lgA-IC results in deposition of IgA and C3 as well as elicit hematuria. These symptoms are frequently observed in human lgAN. It has also been noted that oral immunization with different proteins (bovine gamma globulin, ovalbumin and ferritin) can induce elevation of serum lgA and mesangial lgA deposits in mice (Emancipator et al., 1983). Recent studies in our laboratory have demonstrated that exposure to VT can dysregulate lgA production and induce glomerular lgA deposition and .hematuria in both B6C3F1 and BALBIC mice (Pestka et al., 1989; Dong et al., 1991; Dong and Pestka, 1993; Rasooly and Pestka, 1994; Greene et al., 1994a; 1994b). Furthermore, polyspecific and autoreactive monoclonal lgA Abs have been produced by hybridomas derived from PP of VT-fed mice and these resemble lgAs encountered in sera of VT-exposed mice (Rasooly et al., 1994). In that study, elevation of mesangial lgG and C3 deposition and hematuria was also observed in BALBIC mice after injection of VT-induced monoclonal lgAs, thus suggesting that 135 autoreactive polyspecific lgAs might be pathogenic in this murine model (Rasooly et al., 1994). Further investigation of potential of VT-induced monoclonal lgAs in inducing nephropathy on both B6C3F1 and BALBIC mice is of considerable interest because it might provide an experimental model for understanding immunopathologic mechanism of lgA-induced nephropathy. We hypothesize that the polyreactive lgA Abs induced by VT contribute to experimental lgAN induced by this trichothecene. The objective of this study was to induce experimental nephropathy by injection of VT-induced lgA Abs into mice. I specifically assessed serum IgA, circulating lgA-IC formation, mesangial'lgA and CS deposition and hematuria as indicators of lgAN. The results indicated that injection of lgA Abs into two strains of mice can induce the elevation of serum lgA, lgA-IC, lgG, lgM as well as cause accumulation of mesangial lgA, IgG and CS and hematuria. One potential dietary Ag, casein, appeared to form IC'with lgA or IgG and these IC may participate in pathogenesis of this disease. 136 4.3 MATERIALS AND METHODS 4.3.1 Chemical and reagents All chemicals were of reagent grade quality or better and obtained from Sigma Chemical (St Louis, MO) except where othenivise noted. 4.3.2 Animal and IgA Abs injection Two strains of mice were used because: (1 ) induction of experimental lgAN was previously observed in VT-fed B6C3F1 mice (Pestka et al., 1989; Pestka and Bondy, 1990; Dong et al., 1991; Dong and Pestka, 1993) and BALBIC mice (Rasooly and Pestka, 1994; Greene et al., 1994a), and (2) BALBIC mice have been previously used to produce lgA-secreting hybridomas derived from PP of VT- exposed mice (Rasooly et al, 1994). Female mice (B6C3F1 and BALBIC) were obtained from Charles River Laboratories (Wilmington, MA) and kept in a university animal care facility room with a humidity- and temperature-controlled and a 12 hr light and dark cycle. They were housed in cages equipped with filter bonnets (Nalgene, Rochester, NY) and fed powdered semi-purified AlN-76A diet (ICN Nutritional Biochemical, Cleveland, OH).upon arrival. Animals were acclimated for I at least one wk prior to‘usage. For Ab studies, mice (3 per group) were injected i.p. with purified lgA Abs (1 mg/mouse) in PBS twice per wk for 4 wks and 6 wks respectively. Control mice were injected with an equal volume of PBS. BALBIC mice (3 per group) were injected i.p. with lgA-secreting hybridoma cells (1 x 107/mouse) once for 3 wks. Control mice were injected with NS-1 cells (1 x 107/mouse) or with an equal volume of DMEM 137 medium. 4.3.3 lgA-secreting hybridoma cell cultures Seven different monoclonal lgA-secreting hybridoma cell lines derived from PP of VT-fed mice, 3-1-GS, 7-1-E4, 8-2-F6, 11-1 B5, 12-1-B5, 25-1-D5, and 42-2-85 were kindly provided by Rasooly et al., (1994) and .MOPC 315 is a murine lgA standard (American Type Culture Collection, Rockville, MD). These cells were cultured in DMEM medium supplemented with 20% (v/v) F BS ( Gibco, Grand Island, NY), 1% NCTC (vlv), 10 mM sodium pyruvate, 100 U/ml penicillin, 100 pglml streptomycin and 20% (vlv) MCM in tissue culture plates (Fisher Scientific 00., Corning, NY) at 37° C under a 7% CO2 in a humidified incubator. Monoclonal lgA supernatants were precipitated three times with 50% saturated ammonium sulfate (Harlow and Lane, 1988). Samples were dialyzed against 3 changes of 0.01 M PBS (pH 7.4). 4.3.4 lgA purification by TNP-BSA immuno-affinity gel The above described polyreactive monoclonal lgAs reacted strongly with TNP. This facilitated purification of these monoclonal lgAs using TNP-BSA immuno- affinity gel columns. TNP-BSA (trinitrophenylated BSA) was prepared by trinitrophenylation using picric acid (Good et al., 1980). Briefly, 20 mg of BSA was mixed with 20 mg potassium carbonate in 1 ml distilled water and then 20 mg of picrylsulfonic acid was added. The mixture was covered with aluminum foil and stirred at room temperature overnight. The conjugate was dialyzed in the dark at 4° C for 3 days against sodium bicarbonate (0.1 M, pH 9.0) with several buffer 138 changes. Following dialysis, the ratio of TNP bound to BSA was calculated by spectrophotometric analysis at 278 nm for protein and 348 nm for TNP (Good et al., 1980). TNP-BSA conjugated at 51 :1 mole ratio Was coupled to immuno-affinity gel 15 (Bio-Rad Laboratories, Hercules, CA) according to manufacturer’s protocol. Briefly, TNP-BSA solution was concentrated using a Centriprep 10 concentrator (Amicon, Beverly, MA) and dissolved in 1 ml of MOPS coupling buffer (0.1 M, pH 7.5).ITNP-BSA ligand in coupling solution (1 ml) was added to 1 ml of immuno- affinity gel 15 and agitated sufficiently to make a uniform suspension. After incubation at 4° C overnight, TNP-BSA—Gel was centrifuged for 10 min at 450 x9 and used directly to absorb the Abs. For affinity purification, 1 ml of lgA-containing solution was incubated with 1 ml of TNP-BSA—Gel at 4° C overnight for affinity purification. Following incubation the mixture was centrifuged for 10 min at 450 x 9. After washing three times with PBS the bound lgA was eluted from the affinity gel with 0.1M glycine solution ( pH 2.3). The eluent was collected by centrifuging for 10 min at 450 x g, and then dialyzed against PBS. lgA concentration was measured by ELISA. Samples were concentrated with a Centriprep 10 concentrator (Amicon) and filter sterilized prior to injection. 4.3.5 Hematuria analysis Urine samples (approximately 2 ml/mouse/16 hr) were collected overnight in a metabolic cage at 2 wk intervals and centrifuged at 500 x g for 10 min. 139 Erythrocyte numbers in 10 random microscopic fields (x 45) were counted in the sediment and averaged as described by Dong et al., (1991). 4.3.6 ELISAs Blood samples were collected orbitally from ether-anesthetized mice every 2 wks. Serum lgA, IgG and lgM were quantitated by ELISA (Bondy and Pestka, 1991). Briefly, Immunolon 4 Removawell microtiter strip wells (Dynatech Laboratories Inc., Chantilly, VA) were coated overnight at 4° C with 50 pl/well of heavy-chain specific goat anti-mouse lgA, IgG or lgM (Cappel Worthington, Malvern, PA) at a concentration of 10 pglml in 0.1M bicarbonate buffer (pH 9.6). Coated plates were washed 3 times with 0.01 M PBS (pH 7.2) containing 0.2% Tween 20 (PBST) to remove excess capture Abs. Plates were incubated with 300 pl of 1% (wlv) BSA in PBS (BSA-PBS) at 37° C for 30 min to block nonspecific protein binding, and then washed 4 times with PBST. For lg determination, standard mouse reference serum (Bethyl Laboratories, Inc, Montgomery, TX) or serum samples were diluted in BSA-PBS and 50 pl was added to appropriate wells. Plates were incubated at 37° C for 60 min, washed 4 times with PBST, and then 50 pl of goat anti-mouse lgA, lgG or lgM horseradish peroxidase conjugates (Cappel Worthington, Malvern, PA) detection antibodies, diluted 1:1000 (for lgA) and 1: 500 (for IgG and IgM) in 1% (wlv) BSA-PBS, was added to each well. Plates were incubated at 37° C for 30 min and washed 6 times with PBST. Bound peroxidase was determined with 2,2-azino-bis (3-ethylbenzolin-6-sulfonate) (ABTS) substrate [0.4mM ABTS, 50mM citrate buffer (pH 4.0), and 1.2mM hydrogen peroxide) as I40 described previously by Pestka et al., (1980). Absorbance was measured at 405 nm and lgA, lgG and IgM were quantitated by using the Vmax Software (Molecular Devices). For detection of circulating IgA-IC, sera were precipitated using 3.5% (w/v) polyethylene glycol (PEG 6000) as described by Dong et al., (1993) and then quantitated by lgA ELISA as described above. For detection of casein Ig-IC, Immunolon 4 Removawell microtiter strip wells were coated overnight at 4° C with 50 pl/well of capture sheep anti-casein Ab (Cortecs Diagnostics, Deeside, UK.) at a concentration of 10 pglml in 0.1M bicarbonate buffer (pH 9.6). Following BSA-PBS blocking step, standard mouse reference serum or serum sample was diluted in BSA-PBS and 50 pl was added to appropriate wells. Bound lg was quantitated by ELISA as described above. For casein inhibition studies, lmmunol 4 Removawell microtiter strip wells were coated overnight at 4° C with 50 pl/well of capture sheep anti-casein Ab (Cortecs) at a concentration of 10 pglml in 0.1M bicarbonate buffer (pH 9.6). Coated plates were washed 3 times with PBST to remove excess capture Abs, and 100 pl/well of free casein (100 pglml) in PBS was added to each well. Plates were incubated at 37° C for 30 min, and washed 4 times with PBST to remove unbound casein. Following BSA-PBS blocking step, standard mouse reference serum or serum sample was diluted in BSA-PBS and 50 pl was added to appropriate wells, and then quantitated by ELISA as described above. 4.3.7 Quantitation of mesangial lgA, lgG, lgM and C3 141 BALBIC and B6C3F1 mice were humanely killed at 3, 4 and 6 wks following injection with lgA Abs or lgA-secreting hybridoma cells. Kidneys were removed and immediately frozen in liquid nitrogen then stored at -80° C for section. These kidneys were sectioned into 7 pm slices on a cryostat (Riechert-Jung, Cambridge Instruments, Buffalo, NY) and stained with fluorescein-labeled goat anti-mouse lgA, lgG, lgM and C3 Abs (Sigma) according to the procedure of Valenzuela and Deodhar, (1981). Sections from each mouse were analyzed under a Nikon epifluorescence microscope as described by Greene et al., (1994a; 1994b). Immunofluorescence staining intensities of the kidney mesangium (10 glomeruli/section/mouse) were measured using an ITM Densitometric Video Camera (Waltham, MA) and analyzed by JAVA image analysis system software (Jandel Scientific, San Rafael, CA). 4.3.8 Electron microscopy Electron microscopy examination was done at department of pathology in MSU. BGC3F1 mice were humanely killed 6 wks after injection with lgA Abs. Kidneys were removed and fixed in 2.5% glutaraldehyde in 200 mOsm. 0.1M sodium phosphate buffer at a pH of 7.4 and rinsed with a 0.1 M phosphate buffer (4 x 15 min per rinse). Samples were post-fixed in 1% Osmium tetroxide, rinsed with 0.1M phosphate buffer (2 x10 min per rinse). Dehydration of samples through a series of graded ethanols was followed by propylene oxide with infiltration and embedding in Polybed-Araldite resin. Samples were polymerized for two days at approximately 74° C. One micron sections were prepared using an LKB Ultrotome 142 and stained with 1% Toluidine Blue for examination by light microscopy. Areas selected for ultramicrotomy included a minimum of three representative glomeruli per animal. Thin sections (70-90 nm) were cut with a diamond knives and placed on 300 mesh copper grids. Sections were contrasted with 2% aqueous Uranyl - acetate and lead nitrate. All sections were examined at 60 KV on a Philips 301 electron microscope. 4.3.9 Statistics The data were analyzed by Danaid’s test or Student-Newman-Keuls (SNK) test following one way ANOVA using SigmaStat Statistical Analysis System (Jandel Scientific, San Rafael, CA). A p value of less than 0.05 was considered statistically significant. 143 4.4 RESULTS Effects of monoclonal lgA Abs on BGC3F1 mice. Monoclonal lgA Abs had been previously produced from PP hybridomas of VT-fed mice and demonstrated to be polyreactive or autoreactive due to their ability for binding to various self and non-self Ags (Rasooly et al., 1994). To assess the potential of VT-induced lgA to induce experimental nephropathy in mice, several different polyreactive lgA Abs were used in this study. TNP was chosen for efficient lgA purification since all injected polyreactive lgAs have been previously demonstrated to react with TNP (Rasooly et al., 1994). Furthermore, MOPC 315 was used in this study as a murine lgA positive standard. Rasooly et al., (1994) also observed that MOPC 315 can strongly react with an Ag panel including DNA, PC, casein, inulin, TNP, thyroglobulin, collagen and cardiolipin. This suggested that MOPC 315 might also be polyspecific. The effects of injection of purified monoclonal lgA Abs on serum IgA, IgA-IC, lgG and lgM were assessed in B6C3F1 mice at 2 wks intervals. lgA levels were significantly increased 2- to 4-fold in treatment groups 12-1-B5 at 2 wks, in 3-1-G5, 8-2-F6, 12-1-85, 25-1-D5 and MOPC 315 at 4 wks and in all treatment groups at 6 wks as compared to 'control groups (Table 4.1). IgA-IC levels were significantly increased by 5-fold in treatment groups 11-1-B5 and 12-1-B5 at 2 wks and by 2- to 5-fold in all treatment groups at 4 and 6 wks as compared to control groups (Table 4.2). Injection of IgA appeared to have the greatest effect on serum lgG levels which where enhanced significantly 2- to 4-fold at 2 wks, and 2- to 17- fold at 4 and 6 wks in treatment as compared to control groups (Table 4.3). lgM Table 4.1. The effects of monoclonal lgA Abs administration on serum IgA accumulation in B6C3F1 mice“ 144 lgA lug/ml) Material injected wk 2 wk 4 wk 6 PBS (control) 323 i 30 320 :i: 23 334 :t 22 3-1-G5 515 :l: 61 1311 1: 232° 1330 1 325° 7-1-E4 399 1 55 689 :t 140 966 :I: 222° 8-2-F6 555 :t 61 900 1 140° 822 :1: 96° 11-1-B5 625 t 141 579 :t 76 784 :t 75° 12-1-B5 832 :I: 67° 759 1 15° 850 t 83° 25-1-D5 464 t 122 1029 1 221° 1369 1 313° 42-2-B5 427 .4; 48 723 i 146 882 i 224° MOPC 315 425 i 74 923 1 218° 997 :1: 69° ' Mice were injected i.P. twice per wk with monoclonal lgA Abs or with an equal amount of PBS. b Data reported as mean i SEM (n=3). ° Indicates significantly different (p < 0.05) from control group (injected PBS). Table 4.2. The effects of monoclonal lgA Abs administration on serum lgA-IC formation in B6C3F1 mice“ 145 lgA-IC (pglml) Material injected wk 2 wk 4 wk 6 PBS (control) 1.5 :I: 0.2 1.7 t 0.4 2.5 t 0.8 3-1-65 3.8 :l: 0.6 6.4 319° 7.3 t1.4° 7-1-E4 2.4 i 0.9 5.8 i- 1.2° 6.3 :i: 03° 8-2-F6 2.5 :I: 0.4 6.9 :t 0.1° 8.0 i 2.1° 11-1-B5 8.3 i1.5° 4.8 t 04° 11 :1: 09° 12-1-85 8.2 1 03‘ 9.8 i 2.5° 9.3 i1.7° 25-1-D5 3.5 $1.7 4.2 i1.1° 5.2 :1; 05° 42-2-B5 4.6 :I: 2.3 8.9 i 0.7° 9.1 i O.6° MOPC 315 4.0 i 0.5 6.8 2 04° 13 i 21° ° Mice were injected i.p. twice per wk with monoclonal lgA Abs or with an equal amount of PBS. b Data reported as mean 1 SEM (n=3). ° Indicates significantly different (p < 0.05) from control group (injected PBS). 146 Table 4.3. The effects of monoclonal lgA Abs administration on serum lgG accumulation in B6C3F1 mice“ lgG (mg/ml) Material injected wk 2 wk 4 wk 6 PBS (control) 3.7 i 0.3 3.3 1 0.4 3.6 :I: 0.2 3-1-G5 8.3 :1: 16° 16.5 :- 08° 20.9 :I:1.7c 7-1-E4 8.2 i 03° 14.1 :I: 25° 15.0 1 40° 8-2-F6 8.1 :l: 1.4° 8.4 1 16° 10.3 1 20° 11-1-B5 14.7 :I: 32° 56.0 1: 255° ' 63.9 :8 308° 12-1-B5 13.3 1 20° 36.7 1: 105° 42.0 :11.4° 251.05 15.0 a; 18° 29.7 a: 16° 34.9 1. 30° 42-2-B5 9.1 9 20° 12.6 1 27° 13.4 :1: 37° MOPC 315 7.9 :1: 12° 18.8 :I: 86° 23.9 3 111° ° Mice were injected i.p. twice per wk with monoclonal lgA Abs or with an equal amount of PBS. . b Data reported as mean i SEM (n=3). ° Indicates significantly different (p < 0.05) from control group (injected PBS). 147 levels were significantly increased (2- to 5-fold) in most treatment groups at 2 to 6 wks as compared to control groups (Table 4.4). These observations demonstrated that VT-induced lgA Abs can induce elevation of serum lgA, lgG, lgM levels and lgA-IC formation. Since casein is the primary dietary protein found in the AlN-76A diet, the effects of injection of IgA Abs on casein lg-IC formation were also assessed in serum of mice. lgA-casein complexes (Table 4.5) and lgG-casein complexes (Table 4.6) levels were significantly elevated in groups treated with 3-1-G5, 11-1-B5, 12-1- B5, 25-1-D5 and MOPC 315 after 6 wks as compared to control groups. These complexes were inhibitable (90-95%) by free casein in the ELISA. The findings demonstrated that injection of lgAs can induce casein lg-IC formation, and suggested that casein may play a role in formation of these IC. Glomerular injury was determined at 2, 4 and 6 wks by enumeration of erythrocytes in the urine. All treatment groups showed significant elevation in the numbers of erythrocyte as compared to control groups with maximal effects being observed 6 wks after injection (Table 4.7). A significant increase in mesangial lgA, lgG, lgM and C3 deposition was observed in all treatment groups as compared to control groups (Table 4.8). Electron microscopy further revealed that there was marked electron-dense mesangial deposition in treatment animal after 6 wks (Fig. 4.1a), whereas electron-dense deposits were not observed in control animal (Fig. 4.1b). These results suggest that VT-induced lgA Abs could contribution to the pathological effects in this murine model. 148 Table 4.4. The effects of monoclonal lgA Abs administration on serum lgM accumulation in B6C3F1 mice“ 19M (Pg/ml) Material injected wk 2 wk 4 wk 6 PBS (control) 128 i 6.6 188 :I: 21 139 t 26 3-1-65 512 i 65° 325 i 26 510 2 256° 7-1-E4 334 :I: 45° 424 :1: 39° 417 1: 62° 8-2-F6 284 :1: 26° 323 i 57 393 :I: 39° 11-1—85 694 1 33° 729 1 96° 1136 1 296° 12-1-85 516 2 19° 967 :1: 242° 728 i 98° 25-1-05 353 :1: 26° 347 :i: 92 525 1 77° 42-2-B5 237 i 28° 388 t 61 503 1- 226° MOPC 315 300 1 36° 612 2 73° 537 2 75° ° Mice were injected i.p. twice per wk with monoclonal lgA Abs or with an equal amount of PBS. b Data reported as mean i SEM (n=3). ° Indicates significantly different (p < 0.05) from control group (injected PBS). 149 Table 4.5. The effects of monoclonal lgA Abs administration on serum lgA-casein complexes formation in B6C3F1 mice” lgA-casein complexes (nglml) Material injected w/o casein blocking w/ casein blocking° PBS (control) 0.6 :I: 0.3 < 01‘ 3-1-G5 44 t 4.3“ 2.8 i 04° 11-1-85 51 19.1d 5811.1e 12-1-B5 73 i 1.7d 6.0 :1: 06° 25-1-05 87 1- 9.2d 9.1 i1.4° MOPC 315 '54 s 14.6“ 2.9 2 07° ' Mice were injected i.p. twice per wk for 6 wks with monoclonal lgA Abs or with an equal amount of PBS. b Data reported as mean i SEM (n=3). ° Data were obtained by ELISA. Coated capture sheep anti-casein Ab was first blocked with free casein and then serum samples were added. ° Indicates significantly different (p < 0.05) from control group (injected PBS). ° Indicated significantly different (p < 0.05) from without casein blocking group. 150 Table 4.6. The effects of monoclonal lgA Abs administration on serum lgG-casein complexes formation in B6C3F1 mice"-b lgG-casein complexes (nglml) Material injected w/o casein blocking w/ casein blocking° PBS (control) 0.5 :I: 0.3 0.4 :i: 0.2 3-1-G5 167: 17.1“ 9.7 $1.29 11-1-85 512 282.7“ 19.3 245° 12-1-B5 2192573“ 14.4:2.1° 25-1-05 347 i 19.8“ 14.8 3. 09° MOPC 315 177 1: 34.7“ 7.7 313° “ Mice were injected i.p. twice per wk for 6 wks with monoclonal lgA Abs or with an equal amount of PBS. b Data reported as mean i SEM (n=3). ° Data were obtained by ELISA. Coated capture sheep anti-casein Ab was first blocked with free casein and then serum samples were added. “ Indicates significantly different (p < 0.05) from control group (injected PBS). ° Indicated significantly different (p < 0.05) from without casein blocking group. 151 Table 4.7. Erythrocyte counts infurine of B6C3F1 mice injected with monoclonal lgA Abs“ Erythrocyte/field Material injected wk 2 wk 4 wk 6 PBS (control) 2.3 :1: 0.4 4.1 :i: 0.1 6.7 :i: 0.2 3-1-G5 8.1 :I: 27‘ 13.9 i 15° 20.9 i 04° 7-1-E4 13.0 110° 18.0 :t1.0° 28.0 218° 8-2-F6 9.0 217° 17.3 $1.5c 25.7 i 07° 11-1-B5 16.1 $1.2c 21.7 1 07° 26.6 1 04° 12-1-B5 25.8 :I: 11° 33.1 :1: 04° 48.7 11.7" 25-1-D5 17.6 i 03° 22.3 :I: 1.4° 29.6 :1: 10° 42-2-B5 11.3 1 07° 17.2 i 09° 24.3 1 03° MOPC 315 19.6 i 24° 25.1 :I: 11° 28.4 11.9“ “ Mice were injected i.p. twice per wk with monoclonal lgA Abs or with an equal amount of PBS. ' b Data reported as mean 1 SEM (n=3). ° Indicates significantly different (p < 0.05) from control group (injected PBS). Table 4.8. The effects of monoclonal lgA Abs administration on lgA, lgG, lgM and 152 C3 deposition in the kidney of B6C3F1 mice8 Relative immunofluorescenceb Material injected lgA lgG lgM C3 PBS (control) 30 :l: 0.9 35 1: 1.4 20 :I: 0.4 51 1 0.5 3-1-G5 80 i1.9° 84 i 05° 44 115° 107 2 25° 7-1-E4 56 :I: 23° 58 1.» 23° 35 1' 25° 109 1 16° 8-2-F6 56 :1: 22° 68 .+. 28° 34 :1: 40° 100 :1: 17° 11-1-B5 62 i 03° 87 1 12° 53 1 07° 130 :1: 06° 12-1-85 61 :I: 30° 83 1 39° 52 9 33° 122 :1: 09° 25-1-05 64 :l: 06° 83 104° 39 3: 03° 119 i 06° 42-2-B5 52 :I: 05° 64 1 09° 33 :l: 04° 105 :1: 05° MOPC 315 56 1.- 3.2° 74 1 13° 36 303° 118 :I: 09° ° Mice were injected i.p. twice per wk for 6 wks with monoclonal lgA Abs or with an equal amount of PBS. b Data reported as mean i SEM of relative immunofluorescence on scale of 0-255 per pixel. Ten glomeruli measured per individual mouse/per group (3 mice/group). ° Indicates significantly different (p < 0.05) from control group (injected PBS). Figure 4.1. (a) Electron micrograph of a glomerulus from a B6C3F1 mouse injected with 1 mg VT-induced monoclonal lgA (12-1-B5) twice per wk for 6 wks. Electron- dense deposits (D) are seen under the glomerular basement membrane (GBM) and within the mesangial region (M). MC; mesangial cell; BC, blood capillary; E, endothelial cell; PFP, podocyte foot processes. (b) Electron micrograph of a glomerulus from a control mouse injected with PBS for 6 wks. Electron-dense deposits were not observed in mesangial region. (magnification x 11,000). 154 Effects of monoclonal lgA Abs on BALBIC mice. To assess whether the above immunopathological effects could be induced in the original strain from which lgA-secreting hybridomas were produced, BALBIC mice were also injected with purified monoclonal lgA Abs and serum lgA, lgA-IC, lgG and lgM were monitored at 2 wks intervals. lgA levels were increased 2-fold in treatment groups 12—1-B5 and MOPC 315 at 2 and 4 wks as compared to control groups (Table 4.9). Relatively, significant increases in lgA-IC (7- to 12-fold), lgG (10- to 44-fold) and IgM (2- to 4- fold) serum concentrations was also observed in all treatment after 2 and 4 wks as compared to control groups (Table 4.9). Consistent with B6C3F1 mice, all treatment groups had higher numbers of erythrocyte than control groups after 4 Wks (Table 4.10). Mesangial lgA, lgG and C3 deposition were significantly increased in all treatment groups as compared to control groups (Table 4.11) but no differences were observed in lgM deposits. All these observations suggest that BALBIC mice exhibit similar immunopathological effects of nephropathy with B6C3F1 mice after injection of VT-induced lgA Abs and these lgAs also can induce the experimental nephropathy in this strain of mice. Thus the effect of lgG observed in B6C3F1 mice was not simply due to an anti-allotype response. BALBIC mice response to lgA-secreting hybridoma cells. To further verify the effects of lgA Abs on inducing experimental nephropathy, BALBIC mice were also injected with lgA-secreting hybridoma cells. Serum lgA, lgA-IC, lgG and lgM were monitored at one wk intervals upto 3 wks. lgA levels were significantly increased in groups treated with 11-1-85 and 12-1-85 (37- to 190-fold) as well as 155 Table 4.9. The effects of monoclonal lgA Abs administration on serum lgs accumulation and lgA-IC formation in BALBIC mice“ Material injected wk lgA (pg/ml) lgA-IC lgG (mg/ml) lgM (pg/ml) (119/ml) PBS (control) 0 743 1 114 1.2 1 0.3 1.2 1 0.2 388 1 32 .2 728 1128 2.1103 1.1 102 320 112 4 631 132 2.3105 1.1 10.2 249 114 121-35 0 671 152 0910.1 1.4 10.2 334 113 2 1670 1 308° 15 1 12° 25.2 1 56° 1104 1164° 4 1296 1 209° 15 119° 44.7 1 205° 994 1 48° 25-1-05 0 765 1 126 2.0 1 0.4 2.0 1 0.7 324 1 40 2 1043 1 97 14 113° 22.2 112.4° 890 1 79° 4 877 196 15 122° 27.9111.7° 11981177° MOPC 315 0 998 1 47 2.4 1 0.4 2.2 1 0.6 524 1 40 2 1467 1 246° 15 1 12° 10.2 1 40° 805 1 85° 4 1067171° 24 167° 26.017.8° 11851297° ° Mice were injected i.p. twice per wk with monoclonal lgA Abs or with an equal amount of PBS. b Data reported as mean 1 SEM (n=3). ° Indicates significantly different (p < 0.05) from control group (injected PBS). 156 Table 4.10. Erythrocyte counts in urine of BALBIC mice injected with monoclonal lgA Abs” Material injected Erythrocyte/field PBS (control) 7.5 1 0.9 12—1-B5 40.7 1 07° 25-1-D5 44.5 1 25° MOPC 315 26.7 1 33° a Mice were injected i.p. twice per wk for 4 wks with monoclonal lgA Abs or with an equal amount of PBS. b Data reported as mean 1 SEM (n=3). ° Indicates significantly different (p < 0.05) from control group (injected PBS). Table 4.11. The effects of monoclonal lgA Abs administration on lgA, lgG, lgM and C3 deposition in the kidney of BALBIC miceal Relative immunofluorescenceb ' Material injected lgA lgG lgM C3 PBS (control) 35 1 0.2 41 1 0.6 28 1 0.5 60 1 0.7 12-1-85 71 11.2° 100 118° 29 1 0.6 166 1 36° 25-1-D5 77 1 04° 101 1 17° 33 1 0.6 154 1 26° MOPC 315 67 1 09° 91 1 09° 32 1 0.4 142 1 30° ° Mice were injected i.p. twice per wk for 4 wks with monoclonal lgA Abs or with an equal amount of PBS. b Data reported as mean 1 SEM of relative immunofluorescence on scale of 0-255 per pixel. Ten glomeruli measured per individual mouse/per group (3 mice/group). ° Indicates significantly different (p < 0.05) from control group (injected PBS). 157 11-1-85 and MOPC 315 (27- to 44-fold) after 2 to 3 wks respectively (Table 4.12). lgA-IC levels were also significantly increased in groups treated with 12-1-B5 and 25-1-D5 (17- to 60-fold) and 11-1-B5 (20-fold) after 2 to 3 wks respectively (Table 4.13). Significant increases in lgG levels were observed in groups treated with 25- 1-D5 or MOPC 315 but not 12-1-85 or 11-1 ~85 (Table 4.14). IgM levels were not affected in any of the treatment groups (data not shown). All treatments resulted in increased numbers of erythrocytes in the urine (Table 4.15) as well as mesangial lgA, IgG and C3 deposition (Table 4.16). These findings suggest that some of the immunopathological effects of nephropathy were also induced in BALB/C mice by injection of lgA-secreting hybridoma cells. 158 Table 4.12. The effects of lgA-secreting hybridoma cell injection on serum lgA accumulation in BALBIC mice"b ~ lgA (119/ml) Cell line injected wk 0 wk 1 wk 2 wk 3 Medium (control) 388 1 27 498 1 77 431 1 40 543 1 35 11-1-85 343 1 24 648 1 125 159911 7782d 240301 3372d 12-1-85 367 1 114 15111 405° 822571 196446 Nd° 25-1-05 336 1 144 560 1 139 3459 1 1540 2621 1 637 NS-1 266 1 94 194 1 60 157 1 20 297 1 32 Mopc 315 408 1 36 623 1 113 521 1 98 15180 112766 a Mice were injected i.p. once with lgA-secreting hybridoma cells (1 x 107) or with an equal amount of NS-1 cells. b Data reported as mean 1 SEM (n=3). ° Not determined (mice died before 3 wks). d Indicates significantly different (p < 0.05) from control group (injected medium). 159 Table 4.13. The effects of lgA-secreting hybridoma cell injection on serum lgA-IC formation in BALBIC mice“ lgA-IC (pg/ml) Cell line injected wk 2 wk 3 Medium (control) 0.8 1 0.3 1.2 1 0.5 11-1-85 3.0 1 0.6 25 1 5.3d 12-1-B5 48 1 13° Nd° 25-1-DS 14 1 6.7“l 4.6 1 0.9 NS-1 0.8 1 0.4 0.8 1 0.2 MOPC 315 1.7 1 0.7 2.5 1 0.9 ' Mice were injected i.p. once with lgA-secreting hybridoma cells (1 x 107) or with an equal amount of NS-1 cells. b Data reported as mean 1 SEM (n=3). ° Not determined (mice died before 3 wks). f d Indicates significantly different (p < 0.05) from control group (injected medium). 160 Table 4.14. The effects of lgA-secreting hybridoma cell injection on serum IgG accumulation in BALBIC mice“ lgG (119/ml) Cell line injected wk 0 wk 1 wk 2 wk 3 Medium (control) 502 1 66 493 1122 451 1 141 446 1 202 11-1-85 391150 366 127 300 184 77 113 12-1-85 486 1 113 1542 1 772 407 1 80 Nd° 25-1-D5 A 781 1 24 770 1 230 711 1 219 1083 1133“ NS-1 331 118 666 1274 571 1 256 365 1103 MOPC 315 437 1 78 952 1 262 1233 1 204° 180 1 88 ‘ Mice were injected i.p. once with lgA-secreting hybridoma cells (1 x 107) or with an equal amount of NS-1 cells. b Data reported as mean 1 SEM (n=3). ° Not determined (mice died before 3 wks). ° Indicates significantly different (p < 0.05) from control group (injected medium). Table 4.15. Erythrocyte counts in urine of BALBIC mice injected with lgA-secreting hybridoma cell“ Cell line injected Erythrocyte/field Medium (control) 4.5 1 0.2 11-1-B5 14.2 1 06° 12-1-B5 17.7 113° 25-1-D5 20.5 1 06° NS-1. 10.3 1 13° MOPC 315 24.1 1 16° ' Mice were injected i.p. once for 2 wks with lgA-secreting hybridoma cells (1 x 107) or with an equal amount of NS-1 cells. b Data reported as mean 1 SEM (n=3). ° Indicates significantly different (p < 0.05) from control group (injected medium). l6] Table 4.16. The effect of lgA-secreting hybridoma cell injection on lgA, lgG, IgM and C3 deposition in the kidney of BALB/micea Relative immunofluorescence” Cell line injected lgA lgG IgM C3 Medium (control) 34 1 0.7 40 1 0.5 31 1 0.4 53 1 0.2 11185 127 1 03° 73 1 05° 34 1 0.6 80 1 02° 12-1-B5 148 1 24° 71 1 07° 29 1 0.5 86 1 08° 25-1-D5 98 1 04° 77 1 05° 33 11.2 92 1 11° NS-1 33 1 0.4 66 1 09° 55 11.2 95 1 08° MOPC 315 90 1 08° 80 113° 31 1 0.5 93 1 01° a Mice were injected i.p. once for 3 wks with lgA-secreting hybridoma cells (1 x 107) or with an equal amount of NS-1 cells. D Data reported as mean 1 SEM of relative immunofluorescence on scale of 0-255 per pixel. Ten glomeruli measured per individual mouse/per group (3 mice/group). ° Indicates significantly different (p < 0.05) from control group (injected medium). 162 4.5 DISCUSSION The results presented in this study demonstrated that excessive lgA exposure can increase serum lgA and lgA-IC levels, glomerular lgA deposition and hematuria in both B603F1 and BALBIC mice. These observations are very similar to previous reports that exposure to dietary VT induces hyperelevated serum lgA and circulating lgA-IC concentrations, and increased glomerular lgA deposition and hematuria in 86C3F1 mice (Forsell et al., 1986; Pestka et al., 1989; Dong et al., 1991; Dong and Pestka, 1993; Greene et al., 1994a; 1994b) and in BALBIC mice (Rasooly and Pestka, 1994; Greene et al., 1994a). Dong and Pestka, (1993) suggested that persistent stimulation of IgA secretion caused by VT results in renal injury and consequently lgAN. The results presented herein suggest that elevation of serum lgA production, lgA-IC formation and mesangial lgA deposition may play critical role in nephropathy severity. Since kidney damage is revealed as hematuria, it was notable that both lgA- injected BBC3F1 and BALB/C mice showed increases in the parameter as early as 2 wks. This result is consistent with previous studies (Rasooly et al., 1994) where hematuria was found in BALBIC mice at 4 wks after injection of VT-induced monoclonal lgA Abs. The presented results also indicated that 12-1-85 lgA-injected B6C3F1 mice exhibited the highest numbers of erythrocyte in their urine as compared to other lgA-injected mice.,This finding suggests that this monoclonal lgA Ab was particularly effective in inducing severe hematuria and may have predominant contribution to pathogenic effects in this experimental model. 163 Elevated lgA-containing IC have been well demonstrated in the circulation of patients with lgAN (Lesavre et al., 1982) and in several experiment animal studies of lgAN (Rifai et al., 1979; Emancipator et al., 1987; Montinaro et al., 1991; Dong et al., 1991; Dong and Pestka, 1993). Woodroffe et al., (1980) have reported that dysregulation of lgA production against dietary Ag or pathogens leads to form circulating lgA-IC and subsequent deposition in the kidney result in glomerular damage. Consistent with these findings, our laboratory has previously reported that serum lgA-IC levels were significantly increased in VT-fed B603F1 mice (Dong et al., 1991; Dong and Pestka, 1993). Since VT-induced lgA Abs have been previously demonstrated to be polyreactive and autoreactive Abs due to their ability to bind to various self or non-self Ags (Rasooly et al., 1994), it seems possible that the lgA Abs produced as a consequence of VT exposure might react with some foreign or self Ags to form IC and subsequently deposit in mesangial regions, and that lgA-IC may play a potential role in the pathogenesis of this experimental model. Casein is a dietary protein source that is present at a high levels (30%) in AIN-76A diet. Previous studies have demonstrated increased production of anti- casein lgA Abs as a consequence of exposure to VT in mice (Pestka et al., 1990), suggesting that casein is a candidate Ag for induction of lgA secretion and potential IC formation. Rasooly and Pestka, (1994) demonstrated that casein can inhibit VT- induced serum IgA from binding other Ags such as sphingomyelin and cardiolipin, and suggested that this lgA may be polyreactive. This contention was further demonstrated by studies that lgA eluted from kidney sections of VT-fed mice shows 164 a marked reactivity with several other Ags such as TNP, PC, inulin, DNA and casein (Rasooly and Pestka, 1994). Rasooly et al., (1994) also reported that approximately 30% of the over 120 VT-induced monoclonal lgA Abs can react with casein. Collectively, these observations strongly suggest that casein was capable of binding lgA. In this study, casein complexes containing IgG or lgA were significantly increased in B6C3F1 mice after injection of lgA Abs and specificity was verified by 90-95% inhibition when free casein was present prior to addition of the serum samples. These results suggest that injected polyreactive lgAs have an ability to directly bind the dietary Ag casein. These could form large pathogenic IC with longer persistence in the serum, which might lead to glomerular injury and hematuria. The IgA and lgG-casein complexes f0rmation is shown in Figure 4.2. As dietary Ags gain access through mucosal membranes, it is possible that the presence of IgA at intestinal surfaces further promotes absorption of casein and thus deposition in the glomerular mesangium. In support of this contention, Russell et al., (1986) have observed that dietary protein Ags such as casein could be deposited in association with lgA in the glomerular mesangium of patients with lgAN. Woodroffe et al., (1980) also demonstrated similar lC formation to dietary Ag ‘ which resulted in glomerular damage. Notably, these IC also contain lgG isotype. Therefore, our observations suggest that casein as a dietary Ag may play a potential role in contribution to pathogenic effects by forming IC with lgA or lgG in this experiment model. Although lgA is the predominant isotype deposited in the kidney mesangium 165 36358 533.09 .cozmcte 5388 £33.09 new <9 NV 2:9... £030 . on. M“ <2 Y. 338.an0 533.3. «96358 £980.09 new (a. 691.2 166 of IgA patients, codeposition of lgG and C3 is often detectable in patients with lgAN(D’Amico, 1983). Furthermore, mesangial codeposition of lgG or C3 is also reported in several experimental animal studies of lgAN (lsaacs et al., 1981; lsaacs and Miller, 1982; Rifai and Millard, 1985; Emancipator et al., 1987; Emancipator and Lamm, 1989; Montinaro et al., 1992). This suggests that the codeposition event may be responsible for the alterations in glomerular function in lgAN. Accumulation of C3 in glomeruli is important, since IgA-IC can activate the alternative complement pathway (Gotze and Muller-Eberhard, 1971). Stad et al., (1993) demonstrated that injection of polymeric lgA Abs into rats led to the mesangium deposits of C3 in association with lgA-IC. These findings suggest that alternative pathway activation may play a pathogenic role in glomerular injury and lgAN. My observation of mesangial 03 accumulation further supports previous reports that mesangial C3 deposition occurred in lgA-injected mice (Rasooly et al., 1994) and VT-fed mice (Greene et al., 1994a; 1994b), and suggests that this mediator might play a pathogenic role in VT-induced lgAN. In this study, injection with lgA Abs into mice caused an extensive increase in serum lgG and lgM as well as mesangial lgG deposition. This result conflicts directly the previous observations (Pestka et al., 1989; Dong et al., 1991; Dong and Pestka, 1993; Greene et al., 1994a; 1994b) in which serum IgG and IgM concentrations as well ,as mesangial lgG deposition were decreased in VT-fed BGC3F1 mice but is in agreement with a previous report (Rasooly et al., 1994 ) that increased of mesangial lgG deposition in BALBIC mice was observed after injection 167 of VT-induced lgAs. One potential explanation is that persistent elevation of serum lgA-casein complex formation and inadequate clearance might be immunogenic to lgG production. The observation of increased serum lgG-caseintcomplex formation supports this possibility. Alternatively, it is possible that lgA might function as the Ag in the immune response. Berger, (1979) has suggested that lgA may aggregate and form deposits in the glomerular mesangial regions, serving as an Ag. Thus another potential possibility is that injected lgA may be an Ag to induce an lgG anti- lgA idiotype immune response. Stall, (1996) reported that anti-idiotype Abs are often induced when monoclonal Abs are used as an immunogen. For example, induction of anti-idiotype Ab synthesis occurs following injection of anti-CEA (carcinoembryonic antigen) monoclonal Ab into mice (de-Moraes et al., 1992) or anti-LPS monoclonal Ab into hamster (Field et al., 1993). Another possibility is that the extensive lgG response is caused by different lg allotypic specificities (Stall, 1996). However, since elevation of serum IgG was observed in both 86C3F1 and BALBIC mice, it seems likely that this is not an anti—allotype response. Further clarification of the existence of lgG anti-lgA idiotype immune response by injection of lgA Abs into mice is warranted. Rasooly et al., (1994) have demonstrated that approximately 80% of the monoclonal lgAs from PP hybridomas of VT-fed mice were reactive with more than one of self and non—self Ags that included casein, TNP, sphingomyelin, thyroglobulin, PC, DNA, inulin, collagen, and cardiolipin. Furthermore, one Ag can inhibit binding of some monoclonal lgAs to another Ag. These observations suggest |08 that these monoclonal lgA Abs were polyspecific and autoreactive. The cross- reactivity of injected monoclonal lgA Abs with an Ag panel is shown in Table 4.17. In this investigation, lgA Abs 3-1-65, 11-1—B5 and 12-1-85 showed more effects than other lgA Abs on immunopathologic parameters of lgAN including the elevation of serum lgA, lgA-IC, lgG, IgM levels and mesangial lgA, lgG, C3 deposition with hematuria. These three lgA Abs had been previously demonstrated to react with most Ags (Table 4. 17). Such lgAs might easily form IC with self or non-self Ags which are nephritogenic. Collectively, these findings suggest that polyreactive lgAs 3-1-G5, 11-185 and 12-185, especially 12-1-85, are particularly effective in inducing the experimental nephropathy, and that a particular lgA Ab specificity may contribute the pathogenic effects in this experimental animal study of nephropathy. In this study, injection of VT-induced monoclonal lgAs into BALBIC mice resulted in increasing mesangial lgG and C3 deposition and hematuria. This observation is consistent with a previous report (Rasooly et al., 1994) that increase of mesangial IgG and C3 deposition and hematuria was found in BALBIC mice after injection of VT-induced lgAs. In addition to these same observations, the results presented herein also showed that injection of VT-induced lgAs can elevate serum lgA, lgA-IC, lgG and IgM levels as well as glomerular lgA deposition in BALBIC mice. Thus, presented results further demonstrate that VT-induced polyspecific lgAs might be pathogenic in this experimental murine model. It is notable that increases in serum lgA, lgA-IC and lgG levels as well as 169 mesangial lgA, lgG and C3 deposition with hematuria also occurred in BALBIC mice after injection of lgA-secreting hybridoma cells. Higher levels of serum lgA in treatment mice are probably due to lgA-secreting hybridoma cells can grow and continually produce lgA in_1i10, which would lead to augmenting serum IgA and lgA-IC levels as well as accumulation of lgA in mesangium. These results further support the evidence that lgA-mediated nephropathy in mice was induced by injection of lgA Abs. In conclusion, the results presented herein demonstrated that injection of VT- induced monoclonal lgA Abs into both B603F1 and BALBIC mice can induce the elevation of serum IgA, lgA-IC, lgG, lgM levels and cause mesangial lgA, lgG, C3 deposition and hematuria. One potential dietary Ag, casein, appeared to form lC with lgA or lgG and these lC may participate in the pathogenesis of nephropathy. These studies showed that, with the exception of IgG effects, lgA-induced murine nephropathy model to be mechanistically similar to VT-induced lgAN. 170 Table 4. 17. ELISA reactivity of representative monoclonal lgA supernatants (10 ug/ml) with Ag panel (from Rasooly et al., 1994) Clone DNA PC Casein lnulin TNP Thyro Collag Cardio 3-1—GS + + + + +++ ++ nd nd 7-1 {4 ++ + ++ + +++ +++ + ++ 8-2-F6 + + + + ++ ++ nd + 1 1-1-B5 nd +++ nd nd ++ ++ nd + 12-1-85 ++ nd ++ ++ ++ ++ + ++ 25-1-D5 nd nd nd nd +++ nd nd nd 42-2-B5 nd nd nd ++ +++ + nd nd MOPC 315 +++ ++ I ++ +++ +++ ++ +++ + PC=phosphorylcholine Thyro=thyroglobuli Collag=collagen Cardio=cardiolipin Values are reactivity of each clone to each Ag as expressed by absorbance: + = low binding (OD<0.1); ++ = medium binding (OD = 0.1-0.5); +++ = high binding (OD> 0.5); nd = no detectable reactivity. CHAPTER 5 SUMMARY AND FUTURE STUDIES 171 172 In this study, I evaluated possible early and late mechanisms for VT-induced lgAN. This investigation suggests that the superinduction of cytokines lL-5 and lL-6 expression may, in part, be responsible for upregulation of lgA production in mice exposed orally to VT. I further demonstrated that both MD and to a lesser extent CD4‘ T cells may be responsible for upregulation of lgA production in mice exposed orally to VT. For Md), this is likely to involve both secretion of soluble mediators such as IL-6 and cognate interactions. Furthermore I also demonstrated that injection of VT-induced monoclonal polyreactive lgA Abs into mice can induce the elevation of serum lgA, IgA-IC, lgG and lgM concentrations as well as cause mesangial lgA, lgG, C3 deposition and hematuria. One potential dietary Ag, casein, appeared to form IC with lgA or lgG and these IC may participate in the pathogenesis of nephropathy. This study showed that, with the exception of lgG effects, lgA-induced murine nephropathy model to be mechanistically similar to VT- induced lgAN. It has been known that the costimulatory signals such as interactions between CD28 on the T cells and B7 on the M111 are required for induction of T-cell activation and for regulating immune responses. Based on the observation that VT could induce secretion of lL-6 by M¢ for regulating lgA production, it is possible that induction of expression of cell surface molecule B7 also occurred on VT- treated Md) and that the provided a costimulatory signal for regulating lgA response. Furthermore, cognate interactions between Md) and T cells appeared necessary for VT-induced enhancement of IgA production, Therefore, further investigation of the 173 potential for VT on inducing expression of B7 molecule on Md) is warranted. Flow cytometry in conjunction with phenotypic immunostaining can be used to detect the expression of cell surface molecule B7 on Md). 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