.1. ...,I. a.” lit... :4 ‘3 {:5 3.1.3: . :1! .f X 3...! 13.... ~37 5). . s. 3 . it...) I. I; {610' .lvlsiqu. £15!) éul .. L? in... ‘ . o v p :9; .31}: .1 at. $§II slu.~ . ,.......c.:.un. a. . . :3 sniff! u 13.10. 5. not}: If). “34' .runl...u...! any! a (4942.19. , . . MWTHHSFE. :‘# .. . 9 #6190. .4 )5”. K 2.913;: )‘1:,:..;x\;3 3V. :1 .isiul . 33“: a... it. in. 5...»... z: t... ,‘ 51!— 0).}! {hint j 5 llio .\~ tn. «flux: 3.. . In. 3 .3}: t... . . .ile t u a... 1.3.! t. 1 ‘ .: .l. s K i .2“. I. :5 l3...” .9. 3 f: 5.9... 2‘ I 814‘ 1. .1 x ‘ 3:2?» 1. . a tit, 1r... A? x: I ‘HE&$ This is to certify that the dissertation entitled IN VITRO EFTECTS OF THE TRICHOTHEECENE \FOMITOXIN ON B AND T CELL FUNCTION presented by Roscoe L. Warner has been accepted towards fulfillment of the requirements for Ph. D. degree“, Food Science and Human Nutrition. Institute for Environmental Toxicology VW «444%.. Major professor - -“7 Date 7 I 6 3 MS U is an Affirmatiw Action/Equal Opportunity Institution 0-12771 IN VITRO EFFECTS OP THE TRICHOTHBCBNE VONITOXIN ON 3 AND T CELL FUNCTIONS BY Roscoe L. Warner 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 and Institute for Environmental Toxicology 1993 ABSTRACT IN VITRO EFFECTS OF THE TRICHOTHECENE VONITOXIN ON 8 AND T CELL FUNCTION BY ROSCOE L. WARNER The exposure of lymphocyte cultures to vomitoxin was used to determine the initial mechanism by which this toxin induces serum IgA elevation and IgA nephropathy in the mouse. Vomitoxin exposure did not stimulate, but inhibited, in a dose dependent manner DNA synthesis, protein synthesis and IgA, IgG and IgM production in lymphocyte cultures from the spleen and Peyer's patch. This inhibition of cell function occurred within the range of 10 to 1,000 ng/ml vomitoxin. Similarly, when purified splenic B cells were exposed to vomitoxin, inhibition, but not stimulation of IgA production was observed. In contrast, following 24 hrs of exposure to vomitoxin and the mitogen ConA, splenic CD4+/CD8+ cells were capable of inducing a 3 to 5 fold increase in IgA, but not IgG and IgM production, by splenic B cells as compared to control T cells exposed to mitogen only. Purified splenic CD4+ cells were also capable of causing a three to five fold increase in IgA production by B cells when precultured with ConA and toxin. CD4+/CDB+ and CD4+ cells that were exposed to toxin, underwent a delay in the proliferative response at day 3, but by day S and 7 there was no significant change in cell number or viability. CD4+ lymphocytes produced more 11-5 at vomitoxin concentrations which induced a delay in the proliferative response and increased IgA production. Inclusion of vomitoxin in ConA stimulated CD4+ 96-well plate cultures, resulted in significantly increased production of 11-4, 11-5 and 11-6. These results suggest that vomitoxin was capable of modulating an increase in IgA production by altering the regulatory capabilities of 004* cells. A similar effect at the PP level may be one of the underlying mechanisms for vomitoxin-induced IgA nephropathy. ACKNOWLEDGMENTS The author would like to express sincere gratitude to his academic advisor, Dr. James J. Pestka, for encouragement and support throughout the five-year odyssey of graduate study. Special thanks are expressed to Dr. R. Brooks for technical guidance in Immunology and tissue culture methodology and for helpful suggestions in preparation of this dissertation, and to Dr. S. Bursian for making Toxicology a fun topic of study. He also wishes to thank the rest of his guidance committee members, Dr. J. Linz and Dr. D. Dixon-Holland, for their valuable advice. In addition the auther would like to graciously thank Dr M. Witt-Wilson for preparation of the vomitoxin used in these experiments and Dr. J. Azcona for optimization of the 11-5 ELISA procedure. iv Dedicated to may father and in loving memory of my mother. For their love and encouragement throughout my life, and for instilling in me the desire to continually push ahead. TABLE OF CONTENTS Page LIST OFTABLES OOOOOOOOOOOOO ..... OOOOOOOOOOOOOOOOOOOOOOO ix LIST OFABBREVIATIONS O0..I.0.0.0.0....00.000.000.000... x11 CHAPTER I. LITERATURE REVIEW Rationale ......... ........ ........................ Immunotoxicology .................................. Autoimmunity .................................... .1 Autoimmune Diseases............................ .2 Chemical-Induced Autoimmunity ................. Hypersensitivity ................................ Type I Hypersensitivity ....................... Type II Hypersensitivity ...................... Type III Hypersensitivity ..................... Type IV Hypersensitivity ...................... Hypersensitivity Diseases ..................... Chemical-Induced Hypersensitivity ............. 10 Immunomodulation ................................ 12 Immunosuppression ............................. 12 .1 Chemical-Induced Immunosuppression .......... 13 .2 Drug-Induced Immunosuppression .............. 21 Immunostimulation ............................. 22 1 Synthetic Immunostimulants .................. 22 2 Natural Immunostimulants .................... 23 otoxins ........................................ 25 iosynthetic Pathway Classification ............. 27 ccurrence in Food and Feed Grains .............. 28 iological Effects .............................. 32 Aflatoxins .................................... 32 Ochratoxin .................................... 34 Zearalenone ................................... 36 Trichothecenes .................................. 37 Vomitoxin (Deoxynivalenol) .................... 41 .1 Occurrence in Food and Feed Grains .......... 41 .2 Biological Effects .......................... 43 egulation of IgA ................................. 47 B Cells ......................................... 47 Immunoglobulin Isotype ........................ 47 Haturation/Differentiation..................... 49 mmqqmmbhwuw UDUtthw(JUQNEOB)NBORJNrJth O O O O O O mcnhubuap O O O O O O utoaapraya 3 u< WCDUIO° ' héhfiUUNUNH OOO UMP ”- o o HO‘F‘HFJF‘HPHP‘HEJP‘HrdF‘Ht‘k‘Ht‘h‘HFJP*HEJP‘HPJF‘HFJF‘H HI‘F‘ hhthUUUUUUUUUUNNNNNNNNNNNNNNNNNNH HtHF* NH vi T Cells ........................ ..... ..... ..... .. 50 1 T Cell Development ............................ 51 l 1 Migration and Proliferation ................. 51 1 2 Differentiation and Selection ............... 51 2 Cell Type and Function ........................ 53 2 1 Cytolytic T Cells ........................... 53 2 2 Suppressor T Cells .......................... 53 2 3 T Helper Cells .............................. 54 2 3.1 Lineage ................................... 55 egulation of IgA Production .................... 58 Cytokines ..................................... 58 Il-l and Il-2 ............................... 58 we... .1 .1.1 .1.2 Il-4 ........................................ 60 .1.3 Il-S ........................................ 61 .1.4 Il-6 ........................................ 62 .1.5 .1.6 .1.7 .2 11-10 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 63 Transforming Growth Factor beta (TGFfl) ...... 64 Interferon gamma (IFN-y) .................... 65 mucosal Immune SYStem O O O O O O O O O O O O O O O O O O O O O O O O O 66 Hr-haHrarIHrah-Hiahawrah-Hrahap OOOOOOOOOOOOOOOOOOO itbdibnfidbbd>finhibédbhuhdbfiJib OOOOOOOOOOOOOOOOOOO UUUUUUUUUUNNNNNNNNN CHAPTER II. MATERIALS AND METHODS 2.1 General Experimental Design ....................... 70 2.1.1 Effect of in vitro vomitoxin exposure on Ig production, protein synthesis and cell proliferation of lymphocytes from spleen and PP.. 70 2.1.2 Effect of vomitoxin exposure on Ig production by B cells from spleen and PP ................... 71 2.1.3 Effect of vomitoxin exposure on the ability of total T (CD4+/CD8*) and T helper (CD4*) cell populations to induce Ig production by B cells.. 71 2.1.4 Effect of vomitoxin on cytokine production by CD4+ cells....................................... 73 General Procedures ................................ 74 1 Animals ........ ..... . ..... ...................... 74 2 Culture media ................................... 75 3 Lymphocyte preparation .......................... 75 4 Lymphocyte fractionation ........................ 75 4.1 B Cell preparation ............................ 76 4.2 T Cell preparation ... ...... ................... 77 4.3 CD4+ cell preparation ......................... 77 5 Esterase staining ............................... 78 6 Cell proliferation .............................. 78 7 Protein synthesis ............................... 79 8 19 quantitation ................................. 80 9 Il-2 quantitation ............................... 81 10 Il-4, Il-S, Il-6 Il-lo quantitation ............ 82 11 Purification and characterization of antibodies used in B cell preparation .......... 83 11.1 Clone growth and 19 purification ............. 83 11.2 Antibody characterization .................... 84 12 Statistics ..................................... 85 NNNNNNNNNNNNNNN 00000000000000. ”MN NNNNNNNNNNNNNNN NNN 0.. vii CHAPTER III. RESULTS [7H] TdR incorporation in lymphocyte cultures ..... ["C] Leucine incorporation in lymphocyte cultures . Ig production in lymphocyte cultures .............. Ig production by splenic B lymphocytes ............ T cell (CD4*/CD8+) stimulated 19 production ....... T cell (CD4*/CD8*) number, viability and [5H] TdR incorporation ............................ 7 T helper cell (CD4+) stimulated 19 production ..... 8 T helper cell (CD4+) number, viability and Il-5 production ................................... 3.9 T helper cell (CD4+) cytokine production .......... 3.10 T helper cell (CD4+) cytokine production ......... 3.11 T helper cell (CD4+) supernatant stimulated Ig production .................................... uuuuuu O‘UluhUNH CHAPTER IV. DISCUSSION DiscuSSion OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO CHAPTER V. SUMMARY AND FUTURE RESEARCH Summary and Future Research ............................ References OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO viii 87 87 90 96 96 101 101 105 105 108 113 121 135 140 LIST OF TABLES 1121: 1. Adverse immunologic side-effects of drugs in humans OO OOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 2. Xenobiotics known or presumed to produce allergic reactions in experimental animals or man . 3. National Toxicology Program tests for humoral- mediated and cell-mediated immunotoxicity ......... 4. Chemicals capable of inducing immunologic changes . 5. Examples of pesticides reported to modulate immunity in experimental animals ........... ....... 6. Polyketide-derived mycotoxins ..................... 7. Melvalonate pathway trichothecenes ................ 8. Macrocyclic trichothecenes ........................ 9. Reports of potentially toxic molds from foods ..... 10. Major classes of trichothecenes ........... ....... . 11. Growth and differentiation factors in the immune system OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 12. Effect of vomitoxin on [’H] TdR incorporation by Splenic lymphocytes OOOOOOOOOOOOOOOOOOOOOOOOOOOO 13. Effect of vomitoxin on [3H] TdR incorporation by PP lymphocytes OOOOO OOOOO OOOOOOOOOOOOOOOOOO OOOOO 14. Effect of vomitoxin on [“C] leucine incorporation by splenic lymphocytes OOOOOOOOOOOOOOOOOOOOOOOOOOOO 15. Effect of vomitoxin on [“C] leucine incorporation by PP lymphocytes OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 16. Effect of vomitoxin on IgA production by splenic lymphocytes .00... ...... ooooooooooooooooooooooooooo ix 11 14 15 18 19 20 20 29 39 59 88 89 91 92 93 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. Effect of vomitoxin on IgA production by PP lymphocytes 000......0.0......OOOOOOOOOOOOOOOOO.... Effect of vomitoxin on IgG production by splenic lymphocytes 0.0.0.0................OOOOOOOOOOOOOOO. Effect of vomitoxin on IgG production by PP lymphocytes .0...............OOOOOOOOOOOOOOOOOO.... Effect of vomitoxin on IgM production by splenic lymphocytes .0...0.0.0.0.0000.........OOOOOOOOOOOO. Effect of vomitoxin on IgM production by PP lymphocytes OOO.............OOOCOOOOOOOOOOOOOOO.... Effect of vomitoxin on Ig production by splenic B cells....OOOOOOOOOOOOO0.00.00.00.00......OOOOOOOO Effect of vomitoxin and ConA exposure on CD4+/CD8+ cell help for lg production in 96-well tissue culture plates ............................. Effect of vomitoxin on unstimulated CD4+/CDB+ s lenic lymphocyte, cell number, viability and [ ] TdR incorporation in 96-well tissue culture plates .........OOOOOOOOOOO......OOOOOOOOOOOOOOOOOO Effect of vomitoxin on ConA stimulated CD4*/CD8+ splenic lymphocyte, cell number, viability and [fl] TdR incorporation in 96-well tissue culture plates ......OOOOOOOOOOOOOOOOOO......OOOOOOOOOOOOOO Effect of vomitoxin and ConA exposure on CD4+ cell help for Ig production in 96-well tissue culture plates O00......OO......0..........IOOOOOOOOOOOOOOO Effect of vomitoxin and ConA stimulated CD4+ splenic lymphocyte, cell number, viability and Il-S production in 96-well tissue culture plates .. Effect of vomitoxin and ConA on interleukin production by CD4+ splenic lymphocytes cultured in 96-well tissue culture plate ................... Effect of vomitoxin on interleukin production by ConA stimulated CD4+ splenic lymphocytes in two day T flask cultures ....... ................... Effect of vomitoxin on interleukin production by ConA stimulated CD4+ splenic lymphocytes in seven day T flask cultures ........................ 94 95 97 98 99 100 102 103 104 106 107 109 110 111 31. Effect of vomitoxin on interleukin production by CD4+ splenic lymphocytes in seven day T flask cultures ............................ ..... ......... 112 32. Effect of 2—day supernatants from ConA and vomitoxin treated CD4+ splenic lymphocytes on Ig production by splenic B lymphocytes ................ 114 33. Effect of 2-day supernatants from ConA and vomitoxin treated CD4+ splenic lymphocytes on Ig production by PP B lymphocytes .................... 115 34. Effect of 7-day supernatants from ConA and vomitoxin treated CD4+ splenic lymphocytes on 19 production by splenic B lymphocytes ................ 116 35. Effect of 7-day supernatants from ConA and vomitoxin treated CD4+ splenic lymphocytes on 19 production by PP B lymphocytes ..................... 117 36. Effect of 7-day supernatants from vomitoxin treated CD4+ splenic lymphocytes on Ig production by splenic B lymphocytes .......................... 118 37. Effect of 7-day supernatants from vomitoxin treated CD4+ splenic lymphocytes on Ig production by PPBlYmphocytes O.............OOCOOOOOOOOOOOOOO 119 38. IC,o values for vomitoxin treated splenic and PP lymphocyte cultures 0....0..........OOOOOOOOOOOOOOO 122 xi List of Abbreviations Ag ...........Antigen APC ..........Antigen-presenting cell CD ...........C1uster of differentiation CD4+ .........Helper T cells CD4+/CDB+ ....Total T cells CD8+ .........Cytotoxic/Suppressor T cells ConA .........Concanavalin A CTL ..........Cytotoxic T lymphocyte DTH ..........Delayed-type hypersensitivity ELISA ........Enzyme linked immunosorbent assay ELISPOT ......ELISA-spot technique GM-CSF .......Granulocyte monocyte colony stimulating factor HLA ...... ....Human leukocyte antigen Ig ...........Immunoglobulin ip ...........Intraperitoneal iv ...........Intravenous LPS ..........Lipopolysaccharide MHC ..........Major histocompatibility complex PHA ..........Phytohemagglutinin po ...........Peroral PP ...........Peyer's patch PWH ..........Pokeweed mitogen SRBC .........Sheep red blood cell TCR ..........T-cell receptor TdR ..........Tritiated Thymidine xii CHAPTERI LITERATURE REVIEW 1.1 Rationale Vomitoxin (deoxynivalenol), a potent protein synthesis inhibitor, is a trichothecene mycotoxin produced by toxigenic fungi. The widespread occurrence of vomitoxin in food and feed grains, along with its toxicity, represents a potential threat to human and animal safety. Ingestion of this toxin by mice increases serum IgA and leads to pathologic effects similar to human IgA nephropathy. By characterizing the cellular mechanisms by which vomitoxin modulates IgA production, it should be possible to infer the potential risks that humans face upon exposure to this toxin in our food supply. Extended feeding of vomitoxin diet increases the polymeric to monomeric IgA ratio in serum and increases PP size and germinal center proliferation. At the same time, there are quantitative changes in the lymphocyte populations including increased IgA+ cells, CD4+ (T helper) cells and CD4+:CD8+ (T helper:T suppressor) cell ratios in the spleen and PP. Cytokines, produced by CD4+ cells, are known to closely regulate B cell maturation, proliferation, differentiation and immunoglobulin production. It is possible that vomitoxin-induced alterations in CD4+ cell number and production of cytokines in vivo are likely the cause of increased IgA production and this could be reproduced in vitro. To better understand the principles required to undertake the current research, it is helpful to review pertinent literature on immunotoxicology, mycotoxins 3 and the regulation of IgA production. 1.2 Immunotoxicology Immunotoxicology is a relatively new scientific focus formed by the "hybridization" of the older disciplines of immunology and toxicology. It was born after the recognition that the immune system can be a target for the toxic effects of natural and man-made chemicals. Such effects can be seen as changes in histology or cellular function or as an allergic or autoimmune response to unique antigens resulting from foreign chemical interactions. Immunosuppression and immunostimulation are key manifestations of the toxicologic effects of chemicals on immune cell function. 1.2.1 Autoimmunity The greatest challenge presented to the immune system in the battle to keep the body healthy is the recognition of self versus nonself. Through use of the Major Histocompatibility Complex, cells of the immune system are capable of recognizing "self". By both clonal deletion and clonal anergy, self-recognizing T and B lymphocytes are deleted from the lymphocyte pool prior to their maturation to functional competence. Autoimmunity is the result of the failure or the breakdown of the mechanisms which are normally responsible for maintaining self-tolerance. In addition, autoimmunity may sometimes be initiated by 4 chemicals, this response is not restricted to the compound itself but also involves self antigens (Fleuren et a1., 1985). 1.2.1.1 Autoimmune Diseases Autoimmune diseases are classified as either organ- specific or non-organ specific diseases (Golub and Green, 1991). Diseases which react to a single organ and produce antibodies directed toward specific antigenic components of the tissue are termed organ specific diseases. One example is autoimmune thyroiditis (Hashimoto's Disease) where antibodies are formed against the thyroglobulin and cytoplasmic antigens of the thyroid. Antibodies of non- organ specific diseases react to multiple antigenic sites from a variety of cells throughout the body. Systemic lupus erthyematosus (SLE) is a classic example of this disease type and is characterized by high titers of IgG autoantibodies to a wide variety of nuclear antigens, particularly dsDNA (Fialkow et a1., 1973). These autoantibodies result in high levels of immune complexes in the serum which eventually deposit in the basement membranes of different tissues, particularly in kidney glomeruli. 1.2.1.2 Chemical-Induced Autoimmunity While much is known of the pathology of autoimmune diseases little is understood about their etiology. In the few cases where an etiological agent of human autoimmune 5 disease can be identified, it is often a chemical, and most typically a drug (Table 1)(Miller et al.,1992). This does not mean however that chemicals are the main etiological agents of autoimmune disease. For several human autoimmune diseases there are predisposing genetic factors, particularly certain HLA genes. Myasthenia gravis disease is the formation of autoantibodies against the acetylcholine receptor at the neuromuscular synapsis after exposure to D- penicillamine, but it also has the predisposing factor of DRl and Bw35 of the human HLA (Emery et. a1. 1984). 1.2.2 Hypersensitivity Hypersensitivity is commonly referred to as an allergic reaction. This reaction only occurs when an individual has been previously exposed to an antigen and instead of mounting an immune response to the antigen, the body is sensitized to it. Symptoms range from hayfever and asthma for Type I hypersensitivity to leukopenia, glomerulonephritis and contact dermatitis for Types H, H], and IV respectively. Antibodies are the known mediators of Types.I, H and.DH hypersensitivity reactions while T cells regulate Type IV reactions. 1.2.2.1 Type I Hypersensitivity Type I, or immediate hypersensitivity involves fixation of IgE to mast cells followed by binding of the allergin to the antibodies. Antibody crosslinking results 6 Table 1 Adverse immunologic side-effects of drugs in humans‘ Disease Self-Antigen Inducing Drug Autoimmune Membrane and Halothane, chronic actice microsomes of Tienilic acid hepatitis, liver cells virus-negative Autoimmune Membrane of L-Dopa, haemolytic erythrocytes Captopril, anaemia Penicillins Bullous Basement D-Penicillamine pemphigold membrane of skin Goodpasture Glomerular and D-Penicillamine syndrome alveolar basement membranes Myasthenia Acetylcholine D-Penicillamine gravis receptor Systemic lupus erythematosus dsDNA, Antigens on leucocytes and erythrocytes Gold salts, Hydralazine, D- Penicillamine, Thiouracil fAdopted from Miller et al., 1992. 7 in degranulation of the cell, releasing vasoactive amines, arachidonic acid metabolites and cytokines. These mediators alter capillary permeability, smooth muscle contraction and attract and stimulate cells of the inflammatory response. 1.2.2.2 Type fli Hypersensitivity Cytotoxic hypersensitivity (Type D) involves the binding of antibodies, primarily 196 but sometimes IgM and IgA, to the surface antigens of cells. Binding results in the activation of the complement cascade or antibody- dependent cell-mediated cytotoxicity (ADCC) which ultimately leads to cell lysis. Transplant rejection or transfusion reactions are examples of Type H reactions in which antibodies against cell surface antigens bind to the cell 4-12 hr after exposure to the antigen. 1.2.2.3 Type DH Hypersensitivity Type III, or immune complex hypersensitivity, involves the formation of antigen-antibody complexes and their precipitation in tissues. Complexes lodged in tissues lead to activation of the antibody effector mechanism and subsequently to tissue damage. Antigens in this response often result from persistent infectious diseases or from DNA that has been released following a Type H response. 8 1.2.2.4 Type Iv Hypersensitivity Delayed-type hypersensitivity (DTH or Type Rn reactions are caused by antigen stimulation of T cells and their subsequent interaction with other cell types. Four classes of DTH have been characterized, each requiring approximately one day before the onset of symptoms. Contact hypersensitivity is mediated by CD4+ T cells and is characterized by the localization of the reaction in the epidermis and the involvement of macrophage as the primary effector cell. CDB+ T cells may also act as effectors if class I MHC has been altered in any way. CD4+ T cells are stimulated by the Langerhans cells of the skin (Golub and Green, 1991). In tuberculin-type hypersensitivity, macrophages are the primary effector cells recruited and stimulated by CD4+ T cells. Unlike contact hypersensitivity this response is not limited to a particular location in the body but is systemic in its effects. In addition both macrophage and dendritic cells are responsible for stimulating CD4+ T cells (Golub and Green, 1991). When the body encounters persistent antigens it mounts a granulomatous response. Both macrophage and fibroblasts are recruited and stimulated by CD4+ T cells to proliferate and produce collagen to help localize the antigen. Also found in the area of inflammation are multi- nucleated giant cells, formed by the fusion of several macrophage under the influence of GM-CSF (Golub and Green, 1991). Cutaneous basophilic hypersensitivity (CBH) is similar to contact hypersensitivity in that the reaction is localized in the epidermis. However, the primary effector cell in this response is the basophil (Charlesworth et.a1. 1989) not the macrophage. It is similar to contact hypersensitivity in that the onset of the reaction takes only 12-24 hours after exposure to the antigen and the duration of the response is approximately 24 hours (Golub and Green, 1991). 1.2.2.5 Hypersensitivity Diseases Allergic reactions to antigens differ from autoimmune responses in that they are restricted to the offending compound and not to self-antigens as well. The allergic reactions mounted against pollen and dust particles are typical Type I responses in which mast cell degranulation may lead to anaphylaxis, atopy or urticaria. The Type D disease, erythroblastosis fetalis is a reaction in which anti-Rh antibodies from a Rh‘ mother are produced in response to the Rh+ red blood cells of her fetus. Serum sickness is a classic example of a Type DH reaction. The antitoxin for diphtheria was once produced in horses and injected into humans to fight the disease. Some individuals became sensitized to components in the horse serum and the resulting antigen-antibody immune complexes led to glomerulonephritis. Delayed-type hypersensitivity is 10 typified by contact dermatitis produced by poison ivy. The response is localized in the epidermis and is mediated by CD4+ T cells and involves the macrophage as the primary effector cell. 1.2.2.6 Chemical-Induced Hypersensitivity Large numbers of compounds, both natural and man made are known to elicit allergic responses (Table 2). Exposure routes vary from contact to inhalation and ingestion. A response may occur after an accidental exposure at the work place, as in the case of vinyl chloride or after long-term inhalation of formaldehyde from housing insulation. In addition, large numbers of allergenic compounds can be found in the food supply. Each of the major food groups contain items with known allergenic components. For example, in the fruit and vegetable group, lectins are found in avocados, carrots, apples and bananas (Nachbar and Oppenheim, 1980). Asthma, angioderma and anaphylaxis are known to be produced by a paravalbumin found in fish (Elsayed and Bennich, 1975). Wheat used in breads contain the allergenic compound agglutinin and soya beans contain haemagglutinin and soya bean trypsin inhibitor (Shibasaki et al., 1980). While casein and B-lactoglobulin are the major allergens in cow's milk, over 20 additional proteins are known to be allergenic (Andersson and Soyn, 1984). It is only recently that human-made or industrial 11 Table 2 Xenobiotics known or presumed to produce allergic reactions in experimental animals or manfi Antibiotics: ampicillin, spiramycin, penicillin, sulfathiazole, neomycin sulfate Carrageenans from red seaweed Chemicals: antioxidants, chloramine, diisocyanates, dichlorphene, ethylenediamine, formaldehyde, insecticides, hexachlorophene, phenylglycine acid, pyrolysis products of PVC, phthalic anhydride, trimellitic anhydride Dusts: house, organic, cotton, wood Enzymes: hog trypsin, pancreatic papain, subtilin Food coloring: Ponceau 4R, Amaranth, Sunset yellow, Quinoline yellow, Tartrazine Lectins: apples, avacados, bananas, bran, carrots, green peas, jackbeans, kidney beans, lentils, maize, peanuts, pumpkin seeds, soybeans, wheat Metals: nickel, mercury, beryllium Proteins: casein, fi-lactoglobulin, ovalbumin, paravalbumin (fish), soya bean trypsin inhibitor, Other: castor beans, green coffee beans, natural resins, vegetable gums, monosodium glutamate, vanillin,geranium oil, peru balsam, cinnamon oil, orange oil, lemon oil, potatos, cabbages, eggs, ‘Compiled from Dean et.a1.(1986) and Miller et.a1. (1992). 12 chemicals have been shown to elicit allergic responses. Reactions may be generated against the chemical itself or against conjugates of the chemical and the body's proteins or cells. In certain individuals, aspirin is capable of producing a Type I reaction. Allergic asthma has been associated with the salts of the heavy metals chromium, cobalt, mercury and nickel as well as ethylenediamine, formaldehyde, isocyanates and insecticides (Chan-Yeung and Lam, 1986). T lymphocyte-mediated contact dermatitis can be induced by neomycin, diphenhydramine, chloramphenicol, benzocaine, sulphonamides, chromium, cobalt, gold, mercury and pesticides (Cronin, 1980). 1.2.3 Immunomodulation While modulation of the immune system is often thought of as detrimental, as in the case of accidental chemical exposure, intentional modulation is a useful therapeutic tool. Accidental modulation may arise from the use of inadequately or inappropriately tested therapeutic agents, food additives or industrial chemicals. In addition, certain environmental chemicals are also modulatory. Intentional modulation, through the use of therapeutic drugs, is a primary way to combat cancer, tumors and tissue rejection (Lebish and Moraski, 1987). Immunomodulation can be classified into the general categories of immunosuppression and immunostimulation. 13 1.2.3.1 Immunosuppression Suppression is commonly seen when a compound leads to the death of lymphoid cells or organs. Death may occur when a cell's metabolic pathway is inhibited or when it is unable to replicate DNA or produce necessary proteins (Miller et al., 1992). In animal experiments, cell death is related to lymphoid organ weights, histopathology and differential and total white blood cell counts. In addition, suppression of the immune system can be seen as alterations in cellular function, such as in the humoral- mediated and cell-mediated immune responses. As part of the National Toxicology Program's (NTP) testing format for detecting immunotoxic compounds, both humoral and cell- mediated analyses are performed (Table 3) (Luster et al., 1988). 1.2.3.1.1 Chemical-Induced Immunosuppression Several classes of chemicals are able to induce suppression of the immune system (Table 4). In the case of the halogenated aromatic hydrocarbons, PCB's show close correlation between human and animal models for humoral suppression by reduction of circulating antibodies and antigen-specific antibody responses (Thomas and Hinsdill, 1980; Tryphonas et al., 1989). Depression also occurs in the DTH response (Thomas and Hinsdill, 1980) and reduced natural killer cell (NRC) activity (Talcott et al., 1985). Polycyclic aromatic hydrocarbons (PAH) are the 14 Table 3 National Toxicology Program tests for humoral- mediated and cell-mediated immunotoxicity‘ W Antibody-forming cell response to T-dependent antigen SRBC Enumeration of Ig producing cells by ELISPOT Enumeration of Ig production by ELISA Proliferation response to mitogens Blastogenesis of spleen cells in response to LPS, ConA and PHA Enumeratidn of splenic B cells Missed Mixed lymphocyte response (MLR) Splenic T lymphocyte response to ConA Cytotoxic T lymphocyte assay (CTL) Delayed hypersensitivity response to KLH Enumeration of splenic T cells fAdopted from Luster et al. 1988 15 Table 4 Chemicals capable of inducing immunologic changes‘ __Eff29£§__ Class Chemical HMI CMI Aliphatic Ethyl carbamate + + hydrocarbons Dimethylnitrosamine + + Vinyl chloride ND + Dibenzofip-dioxins, TCDD + + dibenzofurans Heptachloro- + + dibenzo-p-dioxin Tetrachlorodibenzofuran + + Metals Arsenic + + Cadmium + + Lead acetate + + Mercuric chloride + + Methylmercury + i Polychlorinated Hexachlorobenzene + i phenols Pentachlorophenol + i Polycyclic Dimethylbenz[a]- + + aromatic anthracene hydrocarbons Methylcholanthrene + + Benzo[a]pyrene + i Polyhalogenated Aroclor 1248 + i biphenyls Aroclor 1254 + i (PCB, PBB) Firemaster FF-l + + Isocyanates Toluene di-isocyanate + + Gaseous air Ozone + + pollutants Nitrogen dioxide + + '+, Statistically significant; 1, equivocal effect; ND, not determined, Humoral-mediated immunity (HMI), Cell- mediated immunity (CMI), ‘(Miller et al., 1992) 16 byproducts of fossil fuel combustion. Included in this group are 3-methylcholanthrene (3-MC), benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA), all of which are carcinogenic and immunosuppressive. 3-MC is associated with depression of humoral T-cell-mediated serum antibodies in mice (Malmgren et al., 1952), suppression of mitogen-induced T-cell proliferation and cytotoxic T-cell function (Wojdani and Alfred, 1983). BP is capable of suppressing T- dependent antibody responses (Stjernsward, 1966 ) while DMBA reportedly suppresses both humoral and cellular responses (Ward et al., 1986). Dibenzo-p-dioxins occur naturally in the environment but can also be found as a contaminant in certain industrial processes. Of the dioxins 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) is the most immunotoxic and generally used as the model compound for this class. Studies have shown that TCDD is capable of suppressing the antibody response, delayed-type hypersensitivity, mitogen induced lymphoproliferation (Thomas and Faith, 1985) and the cytotoxic T-lymphocyte response (Clark et al., 1981; Clark et al., 1983). Immunosuppression by metals has been clearly demonstrated in animals but not conclusively in humans. Cadmium is capable of decreasing serum antibody concentrations and the number of antibody-forming cells in rabbits (Koller, 1973) and mice (Koller et al., 1975). Delayed-type hypersensitivity and mitogen-induced l7 lymphoproliferation were depressed when rats were chronically exposed to lead (Gaworski and Sharma, 1978; Faith et al., 1979). Little data exists demonstrating immunomosuppression in humans by pesticides (Hermanowicz and Kossman, 1984; Fiore et al., 1986; Kashysap, 1986; Goldman et al., 1990; Mirkin et al., 1990). Existing data are from accidental exposures, as in the case of ground water contamination by aldicarb (Fiore et al., 1986) and from occupational exposures, as in the study of 51 humans exposed to chlorinated pesticides (Wysocki et al., 1985). Animal research on the other hand has generated considerable information on the immunological effects of pesticides (Table 5). Mycotoxins are secondary metabolites produced by a wide variety of molds (Tables 6, 7, 8). These compounds are capable of depressing both cellular and humoral responses (CAST, 1989). This includes decreased migration of neutrophils (Buening et al., 1982) and phagocytosis by macrophage (Niyo et al., 1986), inhibition of platlet function (Gentry et al., 1987) and increased sensitivity to bacterial endotoxins (Tai and Pestka, 1988a). Outbreaks of infectious diseases which often coincide with high levels of mycotoxins in feed, include Salmonellosis in swine (Miller et al., 1978) and turkeys and air sacculitis (Escherichia 9911) in turkeys (Hamilton et al., 1982; Pestka and Bondy, 1989). The immunologic effects of mycotoxins are discussed 8 1 mama .Hm.u0 HOHHMS EOHM cmfiuflfloz. hwma Juice condo 096 an uncommon 0.: omnmouomo mmsoz numofioad msma maumcm 0cm umuuum uncommon mac Umosomm panama cmusuonnmu umumemnhmu hmma .Ho.uo comcc0h uncommon Om< no sowmmmunusm mmso: osmoouoanu uncommon Aumdv damn hmma .He.um umficumm mcfieuoulhoonaucm omnmwuomo mmsoz :fiuoaoflo mums msumnm can ummuum mmcommmu man awesomm uflnnmm Boo mmsHhoHcoosmth AQBOV uncommon mahoonmaaa a mama .He.um mummvom owxououao mo sowmmmummsm mmso: coanumam: Amen. suw>fiuflmcmmume>n mums Manama can ammuum mmsuacmaaamu cmoaeom panama cofisumumeasnuoz mouecmmocmocmmho mmocmueuom muomuum no unmassm mowoemm mmofiowumom .maeawce Heuseawummxe a“ unassaaw «Defiance ou couscous meoaofluuem mo meanauxm m ennea 19 Table 6 Polyketide-derived mycotoxins‘ Group Mycotoxins Genus Tetraketide Patulin Penicillium, Aspergillus Penicillic Penicillium acid Pentaketide Citrinin Penicillium Ochratoxin Aspergillus, Penicillium Hexaketides Maltoryzine Aspergillus Heptaketide Viomellein Aspergillus, Penicillium Rubrosulphurin Aspergillus, Penicillium Alternariol Alternaria Octaketides Rugulosin Penicillium Ergochromes Claviceps, Aspergillus, Phoma Nonaketides Citreoviridin Aspergillus, Penicillium Zearalenone FUsarium Asteltoxin Aspergillus Decaketides Aflatoxin Aspergillus Sterigmato- Aspergillus cystin 9Adopted from Smith and Moss, 1985 20 Table 7 Mevalonate pathway trichothecenes‘ Trichothecin Genus Trichodermol Myrothecium Trichodermin Trichoderma Trichothecin Trichothecium Crotocin Cephalosporin Diacetoxyscirpenol Fusarium T-2 Toxin Fasarium Nivalenol FUsarium Deoxynivalenol Fusarium Fusarenon Fu$arium iAdopted from Smith and Moss, 1985 Table 8 Macrocyclic trichothecenes‘ Trichothecene Genus Isororidin E Cylindrocarpon Roridins Myrothecium Satratoxins Stachybotrys Verrucarins Myrothecium Vertisporin Verticimonosporium iAdopted from Smith and Moss, 1985 ___" 21 in further detail in Section 1.2 of this manuscript. 1.2.3.1.2 Drug-Induced Immunosuppression Immunosuppressive drugs, while beneficial in controlling the immune system at therapeutic doses, can produce toxicity in other organs of the body when given at higher concentrations. Cyclosporin A, used to treat allograft rejections (heart and kidney transplants) at a concentration of 15 ug/kg/day, produces hepatotoxicity, nephrotoxicity and lymphomas at 25 mg/kg ( LDm, IV) in rats. This hydrophobic cyclic peptide functions by penetrating antigen-sensitive T cells and binding to the Il-2 gene enhancer region and inhibiting its production, thereby effectively stopping a T cell response (Abbas et al., 1991). Methotrexate acts as a folic acid antagonist, through inhibition of nuclotide biosynthesis. At concentrations of 17 mg/kg IP in rats, it produces bone marrow depression, hepatotoxicity, fetotoxicity and teratogenicity. Azathioprine is used to inhibit T cell mediated responses. This drug acts by competing with inosinic acid for the enzymes which synthesize adenylic acids, successfully inhibiting nucleic acid synthesis. Cyclophosphamide inhibits cell function by alkylating and cross-linking DNA. In addition, Ig synthesis in B lymphocytes of mice is depressed after exposure to 50 mg/kg (IV) of this compound (Karacic and Cowdery, 1987). 22 1.2.3.2 Immunostimulation Although stimulation has long been viewed as nonspecific, recent studies indicate that certain chemicals are able to directly stimulate specific cell types and their functions. Such functions may involve cell proliferation, differentation, cytokine and antibody production or the phagocytosis and killing of microorganisms. Although stimulation of the immune system may enhance the body's ablity to combate foreign antigens, it may also lead to overregulation, resulting in detrimental effects. 1.2.3.2.1 Synthetic Immunostimulants Immune stimulating synthetic chemicals are almost exclusively produced to aid in the fight against cancer. While they may not directly combat cancers and tumors, many compounds are able to assist in the body's recovery following chemotherapy. Imuthiol (sodium diethyldithiocarbamate), while having no effect on B cells, induces T cell differentiation (Renoux et al., 1984) and enhances Il-2 production (Chung et al., 1985). The compound LF 1695 (benzoylphenyl piperidine) is capable of inducing, in vitro, CD4+ and CD8+ markers, mitogen-induced proliferation and ConA-induced suppressor activity in both.T cell precursors and mature human T cells (Othmane et al., 1985). Azimexome (2-cyanaziridine derivative) is an effective antitumor compound that is not directly cytotoxic, but is capable of activating natural killer (NK) cells. In 23 addition, at high doses this compound leads to an increase of Lyt2+ suppressor cells (Bicker, 1981; Olsson and Bicker, 1982). Isoprinosine is an inosine-containing complex which enhances mitogen-induced proliferation of T cells (Vechi et al., 1975; Madden et al., 1976), the generation of T suppressor cells (Touraine et al., 1980), the maturation of pro-thymocytes (Renoux et al., 1979) and increases the production of Il-2 (Barasoain et al., 1986). Hydroerythranol induces T suppressor cells (Madden et al., 1982), T cell differentiation and proliferation (Wybran, 1980). Levamisole has been shown to promote T cell proliferation, cytolysis, cytokine production and antibody formation (Whitecomb et al., 1976) and it may function as a thymic hormone, inducing precursor cells to become functionally mature T cells (VanGhinckel and Hoebeke, 1976). Pimelautide (lauroyltetrapeptide) stimulates macrophage phagocytosis and superoxide anion release (Floc'h et al., 1984), is mitogenic to thymocytes, induces Il-2 production from CD4+ cells and inhibits proliferation of T suppressor cells (Dauvergne et al., 1985). 1.2.3.2.2 Natural Immunostimulants Many natural chemicals which have been found to stimulate the immune system are now synthesized and used in the fight against cancer. Bestatin is a dipeptide antibiotic isolated from Streptomyces olivoreticuli (Suda et al., 1976). It is capable of activating macrophage, 24 enhancing Il-1 production (Schoorlemmer et al., 1983; Talmadge et al., 1986), increasing the expression of Il-2 receptors (ll-2 R) on T cells (Noma et al., 1984; Kishter et al., 1985) and inhibiting the production of T suppressor cells (Umezawa, 1984). Forphenicinol is a forphenicine derivative which is capable of stimulating the phagocytic activity of of peritoneal macrophage (Ishizukaet al., 1982) and Il-1 but not Il-2 production, and inhibiting of suppressor T cell proliferation (Umezawa, 1984). Muramyl dipeptide (MDP), originally isolated from the cell walls of Mycobacterium tuberculosis (Merser et al., 1975) stimulates phagocytosis by macrophage (McCoy et al., 1985) and the release of superoxide anion (Pabst and Johnston, 1980). It is mitogenic to B cells (Damais et al., 1977) and aids T cell-mediated lymphoproliferation (Azuma et al., 1976) and cytotoxicity (Igarashi et al., 1977). Thymopentin is a pentapeptide corresponding to residues 32-36 of thymopentin which is capable of inducing T cell differentiation (Weksler et al., 1978). Fluorinated 4-quinolone antibiotics are capable of enhancing the production of Il-2 from phytohemagglutinin (PHA) stimulated human T cells (Reisbeck et al., 1989). Recently, a group of compounds, known as mycotoxins, have been found to stimulate the immune system. The trichothecene T-2 toxin is capable of enhancing Il-2 production in ConA-stimulated mouse and rat splenocytes (Holt et al., 1988b), stimulating blastogenesis of L-6 25 myoblasts (Bunner and Morris, 1988) and causing an increase in the number of antibody secreting cells in the spleens of mice (Cooray and Lindahl-Kiessling, 1987). The macrocyclic trichothecenes Roritoxin B, Roridin A and Baccharinoid B5 are able to enhance blastogenesis in mitogen-stimulated mouse lymphocytes (Hughes et al., 1989). Another compound in the group of trichothecenes is deoxynivalenol (DON, vomitoxin). This toxin is capable of enhancing PHA-induced blastogenesis of rat lymphocytes, Il-1 production by peritoneal macrophage (Miller and Atkinson, 1986), IgA production from the cell line CH12LX (Minervini et al., 1993), inducing an increase in the number of CD4+ cells and IgA+ cells in the spleen and PP of mice (Pestka et al., 1990b) and elevating serum IgA concentrations in mice (Pestka et al., 1989; Pestka et al., 1990a; Bondy and Pestka, 1991; Dong and Pestka, 1993). 1.3 Mycotoxins Mycotoxins are a structurally diverse group of toxic compounds found in food and agricultural commodities. They are produced by fungi from a wide variety of genera including Alternaria, Aspergillgs, Bysggghlamyg, W. Winn. 91W. Claims. 1212153113. mum. 2311121111919. 211911;. mm, W. mm, W. mm. mm. nishethssitm and W The toxins can be produced over a wider range of substrates, I I_____—_ # 26 moistures, pH's and temperatures. Fungal growth and toxin production can occur from the time of plant growth through transport, storage and processing of food and feed grains. Toxigenic fungi are ubiquitous in nature and found throughout the world. While some genera are plant pathogens (glgyiggpg, Egggrigm) which are capable of growth and toxin production in the seeds and leaves of plants others are saprophytic (Maggie. mm. 51232111293215) and produce toxin while growing on grains and plant material in storage (Smith and Moss, 1985). In addition the fungi can overwinter as mycelium or resting spores on plant debris or in the soil. In the spring, this material acts as an inoculum that can be distributed by wind, insects or the splashing of raindrops. While the ideal conditions required for growth and toxin production have been investigated in the laboratory, for a number of organisms such conditions may not hold true in the field. Normally plants are resistant to attack by pathologic organisms but under conditions of stress such "predators" can gain a foothold. Factors which predispose plants to infection and colonization are drought, flooding, temperature extremes and insect damage. In addition, field conditions such as poor fertility, high crop densities, crop growth stages and weed competition have been associated with fungal growth and toxin production (Tuite, 1979; Lacey, 1986). 27 1.3.1 Biosynthetic Pathway Classification While mycotoxins as a group are not readily classified by structure, they can be classified by their biosynthetic pathways (Smith and Moss, 1985). These secondary fungal metabolites are similar to alkaloids, antibiotics and terpenes in that they are not essential for the growth of the cell. On the other hand, primary metabolites such as amino acids, fatty acids, saccharides, nucleic acids and proteins are essential to the cell's growth. Polyketides are formed when acetyl coenzyme A and malonyl coenzyme A are coupled together in a head to tail fashion. If ketone groups at the C2 position are continuously reduced a paraffin chain is produced. If, however, these ketone groups are not reduced, then the resulting compound will undergo a series of condensation reactions leading to ringed compounds. These polyketide structures range from 4 to 10 ketides in size (Table 6). Mevalonate acid is an important intermediate compound used to synthesize sterols, steroids and carotenoids. After three acetyl coenzyme A molecules are joined to form melvonic acid, it undergoes pyrophosphorylation, decarboxylation and dehydration to form isopentyl pyrophosphate. From this compound, monoterpenes (Cm), sesquiterpene (Cu), diterpenes (Cm) and triterpenes (Cm) are formed. Included in the sesquiterpenoid group are the trichothecene mycotoxins (Table 7), derived from farnesyl pyrophosphate by several cyclizations and methyl group 28 migrations. Macrocyclic trichothecenes are produced when two hydroxyl groups on the parent structure are linked by long di- or tri-ester chains to form a large ring (Table 8). The combination of precursors from two or more biosynthetic pathways, mevalonic acid and amino acids are the origins of some of the tremorgenic mycotoxins. Aflatrem, produced by Agpgzgillgg filaygg, has a polycyclic skeletal structure originating from mevalonate-derived isoprene units and tryptophan. Roquefortine, produced by Benigillium roguefgrtii, is a diketopiperazine structure derived from mevalonate isoprene units, tryptophan and histidine which is substituted in the indole ring with an isoprene subunit. Several of the mycotoxins are originated exclusively from amino acids. The macrocyclic peptide structures of malformin C and cyclochlorotine are derived from the combination of several molecules each of the unusual amino acids D-phenylamine and B-phenylalanine respectively. Highly condensed polycyclic structures are derived from the combination of two amino acids, phenylalanine and serine in the case of gliotoxin and alanine and tryptophan for sporidesmin. 1.3.2 Occurrence in Food and Feed Grains Mycotoxins are found in many different food and feed commodities (Table 9) in countries throughout the world. Their occurrence and concentrations vary among commodities, 29 Table 9 Reports of potentially toxic molds from food' Commodity Genus Mycotoxin Wheat, Flour, Aspergillus Aflatoxins Bread, FUsarium Ochratoxin A Cornmeal, Penicillium Zearalenone Popcorn Deoxynivalenol Peanuts, Aspergillus Aflatoxins Pecans FUsarium Ochratoxin A Penicillium Trichothecenes Meat pies, Aspergillus Aflatoxins Cheese, Penicillium Ochratoxin A Cocoa powder Patulin Black pepper, Aspergillus Aflatoxins Red pepper, Penicillium Ochratoxin A Macaroni Dry beans, Aspergillus Aflatoxins Soybeans, Penicillium Ochratoxin A Corn, Barley, Patulin Sorghum Citrinin Refrigerated Aspergillus Aflatoxin and Frozen Penicillium Ochratoxin A Pastries Patulin Foods stored Penicillium Aflatoxins at home, Aspergillus Ochratoxin A Refrigerated, Patulin Nonrefrigerated Apples and Penicillium Patulin Apple products {Adopted from Bullerman, 1979, 1986 30 regions and years. Contaminations found in foods and feeds may arise indirectly, as when the toxin is produced in the field and survives processing, or directly when the finished product is infected with toxigenic fungi. Even with the identification of toxigenic fungi in a commodity, it cannot be deduced that the toxin(s) exists since: (1) The presence of fungi is no indication that toxin was produced; (2) Toxin may persist after the fungus can no longer be found: (3) Individual fungi are able to produce more than one toxin: (4) Individual toxins may be produced by more than one genera of fungi. Aflatoxins are a group of heterocyclic mycotoxins containing oxygen and a bisdifurano ring system. Of this group, aflatoxins B, (AFB,) , B,, G, and G, occur most frequently in corn, peanuts and cottonseed, while aflatoxin IL is excreted in the milk of animals after ingesting AFB, (Stoloff, 1980) and can carry over into cheese (Brackett and Marth, 1982). These compounds are produced primarily by Agpgrgillus flavus and A. parasitigus during periods of warm humid days and cool nights. In a study involving 159 samples of peanuts imported into the UK from 7 countries, 60% contained aflatoxin and of these 33% measured greater than 50 ppb (Anon, 1980). In Virginia, 536 samples of corn were analyzed over a five year period. Forty six percent were positive for aflatoxin, 70% of these exceeded the federal limits of 20 ppb. The mean value for all samples tested was 66 ppb (Shotwell and Hesseltine, 1983). 31 Ochratoxins are a group of isocoumarin derivatives linked to L-B-phenylalanine and produced by Agpgrgillus ggnrgggug (Pohland et al., 1990). They are naturally occurring in corn, wheat, sorghum, oats, rice, barley, rye, peanuts, hay, peas and green coffee beans (CAST, 1989). While these compounds are more commonly found in Scandinavian and Balkan countries, they are also detected in the United States (Pohland et al., 1990). Of 293 corn samples from the U.S., only one percent were positive for ochratoxin A (83-166 ppb) (Shotwell et al., 1971) while in 191 corn samples from Yugoslavia, 26% were positive and the concentration ranged from 45-5215 ppb. Zearalenone is a B-resorcyclic acid lactone mycotoxin produced by Fusarigm in corn, wheat and other cereal grains (Mirocha et al., 1977) . Cool (12-14°C) and wet weather are the ideal conditions for fungal growth and toxin production. Production will continue after harvest if the contaminated grain is stored under wet conditions. Zearalenone has been found in corn from Italy (Bottalico, 1979), France, Yugoslavia, England and the Americas (Shotwell, 1977). While this toxin is more common in cereal grains, it has also been found in hay and silage at 14 and 87 ppm respectively (Shotwell, 1977). In addition to animal feed, zearalenone has been found in breakfast foods, snack foods, popcorn and even cake and muffin mixes (Warner and Pestka, 1987). 32 1.3.3 Biological Effects It is estimated that one quarter of the world's food crop is affected by mycotoxins annually (Mannon and Johnson, 1985). These contaminated commodities are destined, although not intentionally, for human and animal consumption and the resulting health hazards have great economic impact. Animal risks include decreased feed efficiency (Bryden, 1982), fertility, reproduction, growth rate of young and milk and egg production (Edds, 1979). In addition to direct lethality by the toxins, there is an increase in carcinogenesis (Wogan and Newberne, 1967) and death by pathogenic microorganisms (Tai and Pestka, 1990). Humans are exposed to mycotoxins by their occupations as farmers, grain handlers and processors as well as by consumption of food products. There are instances where their consumption is suspected to cause human diseases (Peers et al., 1987; Yeh et al., 1989). 1.3.3.1 Aflatoxins Aflatoxins are potent hepatotoxins and hepatocarcinogens in experimental animals. They were originally discovered in the 1960's when 100,000 turkey poults died after ingesting contaminated peanut meal (Goldblatt, 1969) and when hatchery trout developed liver tumors (Goldblatt, 1969) after consuming contaminated cotton seed meal. Experimental hepatic neoplasia have been produced in trout (0.4 ppb for 9 months), ducks (30 ppb for 33 2-4 weeks; Carnagham, 1965) and mice (20 pg/week for 76 weeks; Dickens and Jones, 1965) . Aflatoxin B, tests positive in the Ames mutagenicity assay, producing point and frame shift mutations (Berry, 1988). Acute lethal doses (LD50) of AFB, have been calculated for rats at 6.0 mg/kg (ip) and 17.9 mg/kg (po), dogs at 1.0 mg/kg and 0.5 mg/kg (ip and po, respectively) and ducklings at 0.37 mg/kg po (Butler, 1964; Wogan, 1965). In comparison, AFM, and AFG2 are considerably less toxic (L050) to ducklings with LD,o of 16.6 mg/kg, po and 172.7 mg/kg, po (Lijinsky and Butler, 1966) respectively. Chronic exposure of cows to 120 ppb AFB, resulted in decreased breeding efficiency (2%) and milk production (28%). In addition, calves of lower birth weight and poor health were seen (Guthrie, 1979). Reduced weight gain was also seen in calves and steers after feeding AFB, (80 and 700 ppb, respectively) for 2 weeks and reduced milk production was noted after consuming a diet containing 2 ppm AFB,. Aflatoxin B, is also capable of decreasing cellulose digestion, proteolysis, fatty acid formation and general movement in rumens (Dvorak et al., 1977; Cook, et al., 1986). Aflatoxins are capable of causing immunosuppression in laboratory animals. These effects appear in both the celluler mediated and humoral responses. A reduction was demonstrated in the capability of macrophage to phagocytose particles and of complement to complete lysis (Richard et 34 al., 1978). The overall reduction in immune system capacity can be seen when an increase in microbial infections coincides with outbreaks of aflatoxicosis (Miller et al., 1978). 1.3.3.2 Ochratoxin Ochratoxins are strongly nephrotoxic to farm and experimental animals. Exposure to natural contamination leads to damage of the proximal tubules and impairment of urine and glucose concentrating ability (Krogh, 1977). Additionally, ochratoxin A causes inhibition of kidney gluconeogenesis (Meisner and Selanik, 1979) and of phosphoenolpyruvate carboxykinase activity. Ochratoxin A has also been shown to be hepatotoxic to rats, pigs and chickens (Purchase and Theron, 1968; Smith and Moss, 1985; Cook et al., 1986). Damage initially occurs as necrosis of the periportal cells (Terao and Ueno, 1978) followed by an increase in infiltration of lymphocytes (Smith and Moss, 1985). Damage to the cells ultimately results in an increase in cytoplasmic glycogen (Purchase and Theron, 1968; Warren and Hamilton, 1980; Warren and Hamilton, 1981). The oral LD,0 for day-old chicks was reported to be 3.6 mg/kg (Peckhan et al., 1971) and for day-old ducklings, 150 pg per animal. Single versus multiple (10 day period) dose LD,o tests were performed in 10 day old chicks and found to be 10.67 mg/kg and 13.97 mg/kg respectively. Male and female LD,o comparisons were performed in both rats and 35 guinea-pigs (Purchase and Theron, 1968) with little difference being seen (28 mg/kg and 20 mg/kg for male and female rats, respectively, and 9.1 mg/kg and 8.1 mg/kg for male and female guinea-pigs, respectively. Ochratoxin A toxicity (LDw) to six month old trout was 5.5 mg/kg (ip) (Doster et al., 1974). Ochratoxins reduce weight gain in steers, chickens and weanling pigs at concentrations of 1.0 mg/kg (14 days), 2-4 ppm, and 2.0 ppm respectively, in their diets (Pier, et. al., 1980a). Reduced egg production in chickens was seen at 2.0 ppm (Pier, et al., 1980a)and higher concentrations led to a decreased in eggshell quality and an increase in egg defects (Shirley and Tohala, 1983). Ochratoxin A is also a potent teratogen in rats (More and Galtier, 1974), hamsters (Hood et al., 1976), and mice (Hayes et al., 1974) producing cranial abnormalities. In addition, it is also teratogenic to chicken embryos when 0.7 ug/egg is injected at 48 hours of incubation. Defects included short and twisted limbs, short and twisted neck, exencephaly, exerted viscera and reduced body size (Gilani et al., 1978). Ochratoxin A is capable of producing single strand breaks in the DNA of rodent livers and kidneys (Kane et al., 1986). In addition, tumors in the liver and kidney of mice have been induced (Bendele et al., 1983; Kanisawa, 1983; Ueno, 1984). An increased incidence of urinary tract tumors in eastern European patients suffering from Balken 36 nephropathy has been reported (Stark, 1980; Austwick, 1978, 1981). It is plausible that the high incidence of this disease is due to the consumption of grains contaminated with ochratoxins. 1.3.3.3 Zearalenone Zearalenone is unique in its ability to produce estrogenic effects in farm and laboratory animals. This was first documented in 1962 when ovariectomized mice were fed corn inoculated with Qiberella 1g; (Stob et al., 1962). Dietary concentrations of as little as 1.0 ppm can produce hyperestrogenism in pigs (Kurtz and Mirocha, 1978), which is expressed as swelling of the vulva and enlargement of the mammary glands, hypertrophy of the nipples and, in severe cases, vaginal and rectal prolapse. These symptoms develop approximately 6 days after exposure to the toxin and subside 3-4 weeks after the animal is off the feed (Eriksen, 1968). Young male pigs undergo feminization after consuming zearalenone. In addition, testicular atrophy, swollen prepuce, mammary gland enlargement and decreased libido are seen. Diets containing 50-100 ppm zearalenone are capable of altering cycling, conception, ovulation and implantation of sows (CAST, 1989). Reduced litter size and viability been seen in pigs (Sundlof and Strickland, 1986). Zearalenone can cause a reduction in fertility in dairy cows, rats, mice, guinea pigs, turkeys and chickens (Smith and Moss, 37 1985). Studies conducted with Fisher 344 rats (National Toxicology Panel, 1982) or Wistar rats (Becci et al., 1982) did not demonstrate neoplasia producing capabilities of zearalenone at up to 2-3 mg/kg body weight. In the NTP study there were however hepatocellular adenomas seen in female B6C3F1 mice and pituitary adenomas in male and female mice of the same strain (NTP, 1982). Zearalenone is negative in the Ames assay for mutagenicity (Bartholomew and Ryan, 1980) and in the point-mutation assays using Sggghgzgmyggs ggrgyigigg (Kuczuk et al., 1978) and mouse lymphoma cells (Truhaut et al., 1985). Zearalenone at 3 pg/ml slightly increased sister chromatid exchange in human lymphocytes and was able to inhibit DNA synthesis at 30 ug/ml (Cooray, 1984). 1.3.4 Trichothecenes Trichothecene mycotoxins are a structurally related group of fungal metabolites sharing the same tetracyclic 12,13-epoxy-trichothec-9-ene skeleton. These toxins are produced by a number of genera including: ggpnglggpgrinm, Fusarinm. Errethesiun. Stashxbetrxs..Trishgthesium and Yentisimgnosngrium (Ueno, 1983). with Fusarium being the most commonly associated with their production. Since the discovery of trichothecin from Trighgthegigm zgfiggm in 1948 over sixty trichothecenes have been identified (Freeman and Morrison, 1984) and categorized into 38 four groups (Vesonder et al., 1977). Class A compounds are distinguished by either an hydroxy or acyloxy substitution at carbon eight and class B by keto derivitizations at carbon eight. Class C, the macrocyclics, are typified by an additional carbon chain connecting C-4 and C-15. This chain may contain as many as 15 additional carbons. Class D trichothecenes contain an epoxide group connecting carbons 7 and 8. Table 10 contains a partial list of compounds in each class. Trichothecenes are found in a wide variety of grains throughout the world (Africa, Canada, Japan, Russia, United States). In the midwestern region of the U.S. and Canada wheat, corn, barley and oats suffer infection from Egfigzigm during times of cool wet weather. Such was the case of the fall of 1972 which resulted in the corn crop being stored with excessive moisture (Vesonder, 1983). Moldy corn was fed to farm animals resulting in toxicosis. Physical symptoms included vomiting, feed refusal, diarrhea, reduced milk production in dairy cattle, and hemorrhaging in the liver, stomach, heart, lungs, bladder, kidney and intestines (Bamburg, 1983). The trichothecene T-2 toxin was isolated from corn responsible for the death of 20% of a Wisconsin dairy herd suffering from moldy-corn toxicosis (Hemorrhagic syndrome) (Hsu et al., 1972). Throughout the nineteenth century numerous outbreaks of toxicoses have, in retrospect, been associated with foodborne trichothecenes. One of the more prominent of 39 Table 10 Major classes of Trichothecenes' __Q1_e_s_§_A_ Trichothecene Trichodermol Trichodermin Dihydroxytrichothecene Verrucarol Scirpentriol Diacetoxyscirpenol Triacetoxyscripene T-2 Toxin _____§l§§§_9___ Verrucarin A Verrucarin B Roridin A Roridin D Baccharinols B1 Satratoxin K Satratoxin G Vertisporin Class B Trichothecolone Trichothecin Nivalenol Fusarenon-X 4,15-Diacetylnivalenol Deoxynivalenol 7-Acetyldesoxynivalenol 4,7-Didesoxynivalenol 7-Desoxynivalenol ___§lQ§§_D___ Crotocin Crotocol 7,8-Epoxyisororidin E 7,8-Epoxyroridin H Diepoxyroridin H Verrucarin K 'Adopted from Ueno, 1983 40 these toxicoses resulted in the human disease Alimentary toxic aleukia (ATA). Extensive outbreaks of this disease, which killed over 100,000 people, took place in the Soviet Union during World War H and involved the consumption of moldy grain (millet, wheat, barley) which had overwintered in the fields (Mayer, 1953a). The fungi responsible were identified as W W. E. me. E- trisinsm and mm mm. Physiological characteristics included skin lesions, leukopenia, agranulocytosis, necrotic angina, hemorrhagic diathesis, sepsis and exhaustion of bone marrow. T-2 toxin was eventually identified as one of the compounds responsible for these effects (Bamburg et al., 1969). Trichothecenes are potent protein synthesis inhibitors known to cause the breakdown of polyribosomes (Wei and Mclaughlin, 1974). They bind to the L3 component on 608 subunit of eukaryotic ribosomes (McLaughlin et al., 1977) and block petidyl-transferase activity (Wei et al., 1974; Carter and Cannon, 1978). These compounds can be divided. into three classes relating to their ability to inhibit chain initiation, chain elongation and/or chain termination. The chain initiation inhibitors can be further divided into those that inhibit the function of intact ribosomes and those that inhibit the formation of the 808 initiation complex. For some of the trichothecenes, concentration determines their ability to function as either elongation or initiation types (Carter and Cannon, 1978). In the case of 41 T-2 toxin, at 1 mM it acts as an elongation inhibitor and at 1 uM it functions as an initiation inhibitor (Liau et al., 1976). 1.3.4.1 Vomitoxin (Deoxynivalenol) Vomitoxin (3,7,15-trihydroxy-12,13-epoxytrichothec-9- en-8-one) was first identified as a contaminant of Japanese barley produced by E. rggggm (Morooka et al., 1972). Vesonder et a1. (1973) isolated the same compound from corn responsible for an outbreak of moldy-corn toxicosis in swine. The primary symptoms were feed refusal and vomiting, the later effect giving rise to the common name "Vomitoxin". 1.3.4.1.1 Occurrence in Food and Feed Grains Vomitoxin has been found in barley, oats, sorghum, corn, rice and wheat (Forsyth et al., 1977; El-Banna et al., 1983; COté et al., 1984; Hagler et al., 1984) in many countries of the world (Africa, Canada, Japan, Russia and U.S.). Under the right environmental conditions, infection with Fusarigm and toxin production can be extensive. Ninety-four percent of the samples from the 1983 fall wheat crops of Kansas and Nebraska contained 0.12 - 5.5 ppm vomitoxin (Hagler et al., 1984). Cold wet weather in the fall of 1983 provided excellent growing conditions for E. granimggrgm in the Illinois corn crop. Samples collected from 50 counties revealed 80 % contamination with vomitoxin at concentrations from 0.1 - 42 ppm (COté et al., 1984). 42 Feed grains for animal use are generally ground and mixed with supplements, thus diluting the toxin concentration. Grains intended for human consumption are ground and undergo additional processing. In the case of wheat the milled kernel is separated into three fractions: (1) straight flours, which are intended for use in cakes; (2) feed flour for use in bread and biscuit baking: and (3) bran, which consists of bran, shorts and red dogs and are used as bulking ingredients. Toxin concentration can vary in these different fractions (Hart and Braselton, 1983; Scott et al., 1983). Straight flour, constituting 62-76% of the grain, contained 4.3 ppm toxin, feed flour, averaging 2.8-4.6% of the grain contained 4-8 ppm vomitoxin and bran, averaging 21-30% of the grain, contained 5.2-5.5 ppm toxin. The final outcome of processing indicates that roughly 65% of the toxin ends up in the straight flour and 35% in the feed flour and bran fractions. Baking of foodstuffs (bread, cookies, cakes and Egyptian bread), with an average temperature of 350°F, does not destroy vomitoxin (El-Banna et al., 1983; Scott et al., 1983; Young et al., 1984) though the melting point is 152%: (306°F) (Betina, 1984) . It is therefore not surprising that vomitoxin has been found in 60% of the commercial breakfast cereals (Trucksess et al., 1986). Initial findings (El-Banna et al., 1983) demonstrated no change in toxin concentrations after contaminated flour was fermented during bread baking. However, a later study 43 found an 150% increase in vomitoxin after fermentation (Young et al., 1984). The authors speculated that precursors of vomitoxin may have been transformed by yeast enzymes. 1.3.4.1.2 Biological Effects Vomitoxin is a strong emetic factor in swine (Vesonder et al., 1973), requiring 50 mg/kg ip. and an oral dose of 100-200 mg/kg body weight to produce this effect (Forsyth et al., 1977). At concentrations of 2 ppm in feed grains (corn, wheat and sorghum), reduced feed intake (24%) and reduced weight gain (17%) have been reported (Forsyth et al., 1977; Friend et al., 1982; Pollmann et al., 1985). Chickens demonstrate no significant reduction in body weight gain or feed consumption on a diet containing 1.87 ppm vomitoxin in long-term studies (Hulan and Proudfoot, 1982) and at 116 ppm in a six-day study (Moran et al., 1982). Weight gain in mice is significantly reduced at 5 ppm toxin (Forsell et al., 1986). In addition to reduced weight gain, vomitoxin has been found to reduce fetal weight in pigs (Friend et al., 1983) and egg production in chickens (Trenholm et al., 1984). Day-old Peking ducklings are very sensitive to vomitoxin, requiring only 27 mg/kg body weight (subcutaneous) to achieve the LD,o (Yoshizaxa and Morooka, 1977). LD,o values for female B6C3F1 mice were calculated to be 40 mg/kg po and 78 mg/kg ip (Forsell et al., 1987) and in 44 male and female mice (ip), 70 mg/kg and 76.6 mg/kg respectively (Cole and Cox, 1981). Day-old broiler chicks are more resistant to the effects of vomitoxin and require 140 mg/kg to achieve the Ln” (Huff et al., 1981). Of the birds that died, there was extensive hemorrhaging throughout the intestinal tract, liver and musculature. Hemorrhaging was so extensive that the carcass were burgundy colored. Concentrations of greater than 116 ppm are capable of producing oral lesions in six-day-old chicks (Moran et al., 1982). These ranged from pinpoint foci to large, raised plaques which occurred on the inner margins of the beak, tongue and roof of the mouth. The size and age of an animal influences its susceptibility to vomitoxin. When pigs weighing 121 kg were fed a diet containing 0.1, 1.7 and 3.5 ppm vomitoxin for 52 days, no significant changes in weight or lesions of the organs were found (Pollmann et al., 1985). Pigs (21 kg) fed a diet containing 0.58-3.06 ppm vomitoxin for 28 days showed no significant effects in the stomach, liver, kidney, liladder, or uterus, however, at 2.38 ppm, heart size was Significantly decreased (Friend et al., 1982) . Smaller animals (7.7 kg) experienced weight change of the liver, heart and kidney after 21 days on diets ranging from 0.9-2.8 Ppm toxin (Pollmann et al., 1985) . Vomitoxin, while not teratogenic, does cause fetal 17€3absorption in rabbits (5%, 9%, 25% and 100% at ‘=400 m2; McGhee et al., 1990) , it has been estimated that IgA represents >60% of the total Igs produced per day in humans (McGhee et al., 1989). IgA in the gastrointestinal tract reacts with food antigens as well as infectious organisms and aids in their clearance from the system. 1.4.1.2 Maturation]Differentiation B cells originate from multipotential haemopoietic stem cells. Maturation and differentiation of these cells takes place in the bone marrow and then in the peripheral circulation. Throughout all stages of development they are strongly influenced by cytokines. As stem cells develop into pre-B cells, they express in their cytoplasm the u heavy chains of Ig molecules, and on their surface, a developmental marker known as B220. At this stage, the cells are still antigen unresponsive. The cell then selects a x or A light chain and assembles IgM molecules which are expressed on the surface of the cell. This membrane bound IgM is capable of binding antigen, but the cell is unable to proliferate or differentiate in response to it. The cell then coexpresses membrane IgM and 190 with the same antigenic specificity. At this stage of maturation the cell is known as an immature B cell. The cell then migrates out of the bone marrow and into the peripheral circulation and lymphoid tissues. Maturation 50 continues even in the absence of antigenic stimulation. When the cell becomes a fully mature B cell it is antigen responsive and with the aid of cytokines will become an activated B cell and can differentiate into an 19 producing cell. Besides antigen stimulation of membrane IgM (mIgM) or mIgD, B cells require Il-2, Il-4 and Il-5. These cytokines act as growth and differentiation factors and are important for the clonal expansion of cells. Interleukin 4 (Il-4) is required to initiate the entry of G0 resting B cells into cell cycle (G,) (Abbas et al., 1991) . It then stimulates the entry from G, into S phase and in conjunction with Il-5 increases the expression of Il-2 receptors (Il-2 R). In addition, it increases class H MHC molecules on the cell surface (Noelle et al., 1984). 1.4.2 T Cells T cells are also derived from a multipotential haemopoietic stem cell in the bone marrow. The cells migrate to the thymus, the tissue from which they derive their name, and undergo additional maturation and differentiation. Members of this diverse group of cells have the ability to recognize and kill nonself cells and to up or down regulate humoral or cell-mediated immune responses in the body. 51 1.4.2.1 T Cell Development 1.4.2.1.1 Migration and Proliferation Human stem cells in the bone marrow, under the influence of colony stimulating factors (CSF), differentiate within the micro-environment of sessile cells and enter the thymus as committed hematopoietic T-cell precursors. As the cells enter the cortical region of the thymus they are T cell receptor negative (TCR’) , CD3‘, CD4“, CD8' and cannot recognize or respond to antigens but they do stain positive for terminal deoxynucleotidyl transferase (Tdt). Early in their journey, these cortical thymocytes express the markers CD1, CD2, CD3, CD4, CBS and CD8. Double positive cells (CD4+/CD8*) constitute about 80% of these thymocytes, CD4+ are about 12% and double negative and CD8+ cells are each about 4% (Scolley et al., 1984) of the population. During this time, the cells are maturing and rapidly proliferating with a half-life of about 3 days (Bryant, 1972) in their passage by/through "nurse" cells and with the influence of the thymic hormones thymosin, thymopoietin, thymulin and thymic humoral factor. 1.4.2.1.2 Differentiation and Selection Within the cortex the cells start to rearrange the V, . D, J and C genes required to generate the TCR. If the gene rearrangements produce a TCR which is expressed on the cell surface, they undergo selection for their ability to 52 recognize foreign antigens in association with self MHC (class I and II, CDB+ and CD4+, respectively). This occurs in the cortex and medulla as the cells encounter "sentinel" macrophage, medullary epithelial cells, dendritic cells, Hassall's corpuscle and interdigitating cells. The TCR:CDB complex allows the cell to recognize foreign antigens which can range from food components in the gut to dust or bacteria. To ensure that the TCRs do not recognize self antigens and that the cell can recognize self MHC, a sequence of positive and negative selections occur. Positive selection ensures that cells whose TCRs bind self MHC molecules will live and replicate and those with no affinity for self MHC will die. This step eliminates all non-self MHC-restricted T cells and allows foreign antigen- specific, self-MHC-restricted T cells and the potentially harmful self-antigen-specific, self-MHC-restricted T cells to live. To eliminate the self-reactive clones, the body next goes through negative selection. This eliminates cells whose TCRs bind with high affinity to self antigens in association with self MHC by either clonal deletion or clonal anergy. More than 95% of the cortical thymocytes die before reaching the medulla (Abbas et al., 1991). As cells leave the thymus, they are fully functional T cells. They have the ability to recognize foreign antigens and to differentiate self from non-self. In addition, specific subsets of T cells are also produced. These include the T cytotoxic cells (CD8), T suppressor cells and 53 T helper cells (CD4). 1.4.2.2 Cell Type and Function 1.4.2.2.1 Cytolytic T Cells Cytolytic T cells (CTL) kill cells in the body which have altered or different MHC, generally by infection with viruses, bacteria or protozoa by tumor alteration or as a result of tissue grafts. Most of the CTLs express CD8 molecules on their surface (rare exceptions do express CD4), and specifically recognize foreign peptides occurring from intracellularly synthesized antigens associated with self class I MHC. In this process, the CTLs are triggered by recognition of a target antigen associated with class I MHC. Killing is accomplished by membrane-bound cytoplasmic granules containing the membrane pore-forming proteins perferin and cytolysin. Only the target cell is killed, bystander cells and CTLs themselves escape injury. 1.4.2.2.2 Suppressor T Cells The existence and function of these cells is still highly controversial. The inability to purify enough cells to perform biochemical analysis of receptors and secreted products or to produce clones has made much of what is known of these cells questionable. Suppressor T cells are thought to inhibit the activation phase of immune responses in the following ways. They may produce excessive cytokines such 54 as TGFB which at high concentrations inhibit T and B cell proliferation, or IFN-y which inhibits the proliferation of B cells in vitro. Suppressor cells may also absorb growth and differentiation factors by way of receptors and inhibit the stimulation of other cells in culture. They may possess cytolytic activity and lyse cells bearing the stimulating antigens in association with MHC molecules. This may be achieved by tumor necrosis factor (TNF) and lymphotoxin. 1.4.2.2.3 T Helper Cells. T helper cells (CD4+) are a vital population in the body. With the aid of their cytokines and the ability to recognize portions of protein antigens bound to self class H MHC, they help initiate specific immunity. They assist in lymphocyte maturation, differentiation, activation and proliferation, cell-mediated and humoral-mediated immune responses. In cell-mediated immune reactions, both the effector phase and cognitive phase are initiated through antigen recognition by T cells. In delayed type hypersensitivity (DTH) reactions, antigen activated T helper cells indirectly recruit monocytes and then activate them to help eliminate antigens. Activated CD4+ cells help CTLs to lyse virally infected or organ transplant cells which express specific antigen-MHC complexes. Under the influence of cytokines natural killer (NK) cells differentiate into lymphokine- activated killer (LAK) cells which lyse target cells in 55 graft-versus-host disease. In humoral-mediated immune responses, the effector phase is mediated by the binding of antibodies to antigen. CD4+ help occurs only after the T cells have been activated by the presentation of antigen and class B MHC by B cells. Il-4 initiates the entry of resting B cells into cell cycle. B cells are then stimulated into growth, proliferation and differentiation by Il-2, Il-4 and Il-5. Antibody synthesis and secretion are helped by Il-4, Il-5 and Il-6. Cytokines also function to switch isotypes in B cells. For example, Il-4, IFN-y, and TGF-fl induce switching to IgE, IgGfi and IgA, respectively. CD4+ cytokines also aid affinity maturation and development of memory B cells (Rizzo et al., 1992). 1.4.2.2.3.1 Lineage The concept of different subsets of CD4+ cells and their lineage has been dicussed for two decades. Cantor and Asofsky (1972) proposed T1 and T2 subsets of CD4+ cells which had the same lineage. Young ”naive" T1 cells could be driven to T2 "memory" cells by antigen. Swain (Swain et al., 1988b) demonstrated that T2 "memory" cells secreted mainly Il-2 and IFN-y and that T1 "naive" cells, upon stimulation with exogenous lymphokines, could produce Il-3, Il-4, Il-5 and IFN-y, but very little Il-2. This is consistent with Mosmann's (Mosmann et al., 1986) discription of Tm and T,,2 subsets and their classification by cytokine 56 production, Il-2, IFN-y and Il-4, Il-5, Il-6, Il-10, respectively. Within the thymus, single positive (CD4+8), heat stable antigen high (HSAW') cells, which are precursors to mature CD4+, do not proliferate when exposed to anti-CD3 and dendritic cells. However, upon maturing to CD4+8'HSA”' cells, they are able to respond to these stimuli (Bendelac and Schwartz, 1991). In addition, immature single positive thymocytes (CD4*8' HSAm and CD8+4' HSAW') are unable to produce Il-4, Il-5 and Il-10 but can produce small amounts of Il-2 and IFN-y. Upon maturation, CD4+8' HSA'“ cells acquire the capability to produce Il-4, Il-5, and Il-10 while CD8*4' HSA‘" thymocytes do not (Bendelec and Schwartz, 1991). Mature CD4+8' HSA'“ cells which leave the thymus and enter the peripheral circulation are termed "naive" CD4+ cells. They are unable to produce Il-4, Il-5, Il-10 and IFN-y when stimulated by anti-TCR, but can produce high levels of Il-2 (Swain et al., 1990). Four days after anti- TCR stimulation, the cells proliferate into an effector cell population. They express CD4 and Il-2 R but cannot produce cytokines or their RNA's until they re-encounter Ag/APC or mitogen. Upon restimulation, they synthesize mRNA and secrete lymphokines more rapidly and for a shorter time than resting CD4+ cells. Conditions under which the 4 day effector populations are produced determine the nature of the effectors and the 57 cytokines produced upon restimulation. Effectors grown in IL-2 alone or in Il-2 and TGFB produce cells which secrete Il-2 and IFN-y but not Il-4 and Il-5 (Swain et al., 1991). Effectors cultured with Il-2 and low levels of endogenous Il-4 generate a population which secretes very low levels of Il-2 and very high levels of Il-3, Il-4, Il-5, Il-6, IFN-y, GM-CSF and TGFB (Swain et al., 1990; Weinburg et al., 1990). The addition of Il-4 (>20 U/ml) to effector cultures suppresses development of cells secreting Il-2 and enhances the generation of a population secreting Il-4 and Il-5 in a dose-dependent manner (Swain et al., 1988a). The classification of cells resulting from long term cultures of T helper cells into T", and Tm, based on their cytokine secreting pattern (Mosmann et al., 1986), does not represent the full range of phenotypes that can be expressed by murine T cells (Kelso and Gough, 1988). The Tm, phenotype is characterized by the simultaneous secretion of Il-2, Il-4, Il-5, Il-10 and IFN-y (Firestein et al., 1989, Mosmann and Coffman 1989). This group is thought to represent either a transient multipotential stage of CD4+ T cells or a distinct phenotype which is fully mature. Although, antigen and Il-4 stimulation enhances the development of the TH2 phenotype and inhibits that of T,,, from Tm,clones (Abehsira-Amar et al., 1992). 58 1.4.3 Regulation of IgA Production 1.4.3.1 Cytokines Cytokines are a group of compounds, including monokines and lymphokines, produced by mononuclear phagocytes and activated T lymphocytes respectively, which are heterogenous proteinaceous mediators that exercise specific, receptor- mediated effects in target cells. They function in the effector phase of both the innate and acquired immunity to stimulate or enhance cell functions. Within the innate immune system, these mediators are elicited in response to invading microbes or to antigen stimulated T cells. In the acquired immune system, cytokines primarily regulate the activation, growth and differentiation of various lymphocyte populations. Cytokines possess the ability to affect cells from both nearby and afar ("paracrine" action) or to affect the mediator-producing cell themselves ("autocrine" action). 1.4.3.1.1 11-1 and 11-2 While Il-1 (Table 11) does not directly regulate the production of IgA, it does influence antibody production in general. Il-1 functions with polyclonal activators to enhance B cell activation, growth, differentiation and antibody production (Falkoff et al., 1984). It also enhances the proliferation of lectin-stimulated lymphocytes by stimulating production of Il-2 by CD4+ cells (Smith et (al., 1980). This is accomplished by inducing the synthesis 59 Hmmd .smwuu use asaou Sou“ pmumopé. COHGHHQHCH QGDEEH “canny auoufinfincfi sowuwuomm unwxouau uouomu cusoum muaooucmem .mnncououneucH .mnuouocu ocwucaoaaum Hauntm nouocu mswocccsmnmma .= uouocu cusouo HHmOIm .uouonu mcwocammu Haunts «luouocm nuaouo HHCOIB .Hluouocu vcwucasawum admonm .uouocu :u3oum damnim uouocu nusouu HHCOIB nouocu mcwuc>auoclaamolm .Hlnouocu owumwonoamm .uouocu oswuc>wuocumuaoonmahq mwamqluwmua mHHOO B ox ma mHHmo m .maamo a as mm mHHmo +460 map as oeuen mHHmo a .maamo Hmflaonuoecm He ~.m mummanonnsm .mmuaooco: ox GNumH mime .30 as 9. 3:3 He s.w mime Loo 99 9. 3an 230 .30 as ox 9...: amusoouuma .maamo 9 ex ea maamo m .mHHeo Oflkupsmo ox ha maaeo Hawamcufinm .muccmouoc: 3: xmlumumumum wuammaumwmmumqm Hanuemno razmH nhUB OHIHH mIHH «IHH NIHH nHuHH aHuHH .fieumhm asses“ on» Ca muouocu sawucwuceueuuwp use cusouu dd CHA‘B 60 of c-fos, which complexes with AP-1 (c-jun product) to form a transcriptional promoter (Abbas et al., 1991). Il-2 (Table 7), produced primarily by T,,,CD4+ cells promotes the progression of T lymphocytes from G, to S phase of cell cycle. This autocrine factor binds the Il-2R (p55/p70 complex) which is internalized and activates the cell by an as yet unknown mechanism (Meager, 1990). This in turn causes more Il-2 production, cell proliferation and differentiation. Coinciding with these events in the activated CD4+ is the production of other cytokines (ll-4, Il-S, Il-6, IFN-y and TGF-B). Antigen (and PWM) induced Il- 2R on B cells bind Il-2 and lead to cell proliferation and enhanced antibody synthesis. 1.4.3.1.2 11-4 Il-4 (Table 11) is produced by CD4+ cells and is an autocrine growth factor for Tm,cells (Mosmann and Coffman, 1989). Il-4 aids the entry of resting B cells into cell cycle (G0 to G,) (Rabin et al., 1985) . With the addition of anti-IgM this cytokine is capable of costimulating the growth of murine B lymphocytes (Howard at al., 1982), although it fails to stimulate significant production of IgM (Alderson et al., 1987a). With the addition of Il-5, Il-4 stimulates the expression of p70/75 and p55 chains of the Il-2R complex (Loughnan and Nossal, 1989). In mature B cell neoplasms, Il-4 alone suppresses the growth promoting effect of Il-2 (Karray et al., 1988), suggesting two different B 61 cell subsets. Il-4 has been shown to induce both 196, and IgE secretion by LPS-stimulated murine B cells in vitro (Vitetta et al., 1984; Coffman et al., 1986) . IgG, secretion is inducible in cells which lack expression of IgG (Snapper and Paul, 1987a), and secretion is preceded by the induction of germ-line transcripts of‘n and e RNA in the presence of LPS (Rothman et al., 1988; Berton et al., 1989). Thus Il-4 is able to induce class switching of IgG, and IgE rather than just stimulate their secretion. Production of IgG, peaks at 100 and 10,000 U/ml while that of IgE reaches a maximum at 10,000 U/ml of Il-4 (Snapper et al., 1988a). IFN-y is able to block this production in LPS stimulated B cells (Coffman and Carty, 1986; Snapper and Paul, 1987b). 1.4.3.1.3 11-5 Il-5 (Table 11), produced by activated Tm3CD4+ cells, is capable of stimulating the proliferation of murine B cells co-stimulated with dextran sulfate (0x8) and anti-Ig (Swain et al., 1983). IFN-y can inhibit Il-5 induced proliferation of BCL, cells and antigen specific B cells (Lohoff et al., 1989; Armitage et al., 1990). Spontaneous proliferation is promoted in large in vivo activated B cells but not in small resting B cells (Nakajima et al., 1985; O'Garra et al., 1986). Although, there is no co-stimulation Of cell proliferation in anti-Ig stimulated B cells in 62 assays designed to test the growth promoting ability of Il-4 (Swain et al., 1983; Harade et al., 1985). Such results indicate Il-5 acts only at the later stages of B cell activation. Although, recent reports (Wetzel, 1991a,b) indicate that a small portion of resting B cells, stimulated with nonspecific polyclonal activators are Il-5 responsive. Il-5 is capable of inducing proliferation and IgM secretion of antigen stimulated B cells (Alderson et al., 1987b), while IFN-y can inhibit antigen induced IgM secretion (Armatige et al., 1990). Il-5 stimulates polyclonal B cell differentiation when co-cultured with Il-4 in the absence of antigen (Armitage et al., 1990). Il-4 and Il-5 have a synergistic effect on the secretion of IgM and IgG3 in unstimulated murine B cells and on IgG,, IgE and IgA secretion by LPS-stimulated B cells (Coffman et al., 1988; Tonkonogy et al., 1989). Il-S enhances IgA secretion from LPS-stimulated B cells (Harriman et al., 1988). This ability is expressed in both splenic and Peyer's patch B cells, but only in surface IgA positive cells (Beagly et al., 1988; Harriman et al., 1988; Lehman and Coffman, 1988; McGhee et al., 1989; Tonkonogy et al., 1989). 1.4.3.1.4 11-6 Il-6 (Table 11) acts as a growth promoter in B cell hybridomas and plasmacytomas in mice (Poupart et al., 1987; Van Snick et al., 1987). While alone, Il-6 is a poor growth promoter of murine B cells, but it significantly stimulates 63 cell proliferation when in combination with anti-1g, 0x8 and Il-1 (Vink et al., 1988). Murine B cell differentiation is also stimulated by Il-6 alone or in combination with antigen, anti-IgM and DxS (Takatsuki et al., 1988). IgM synthesis is increased in Peyer's patch B cells upon addition of Il-l and Il-6 (Kunimoto et al., 1989). When the human Il-6 gene is placed in a transgenic mouse, it increases serum IgG, concentrations by 120 to 400 fold. Il- 5 and Il-6 by themselves increase IgA secretion from Peyer's patch B cells, but together they have a synergistic effect on its secretion (Kunimoto et al., 1989). Il-6, unlike Il- 2, is a late acting differentiation factor capable of increasing IgA secretion after it is added on day 4 of an 8 day culture (Muraguchi et al., 1988). IgA secretion is enhanced in IgA positive but not IgA negative Peyer's patch B cells (Beagley et al., 1989). Il-6 aids Il-4 in the production of IgE by B cells (Verchelli et al., 1989), and is secreted by B cells in response to Il-4 (Smeland et al., 1989). 1.4.3.1.5 11-10 Il-10 (Table 11), produced by Tm CD4+ cells, is capable of enhancing viability and the expression of class D MHC on resting B cells (Go et al., 1990). It promotes growth of murine thymocytes and murine peripheral T cells when used in conjunction with Il-2 and Il-4 (MacNeil et al., 64 1990). Il-10 possesses the ability to suppress cytokine production from murine T,,,CD4+ cells (Fiorentino et al., 1989) by inhibiting the Ag-presenting function of purified macrophage and monocytes (Fiorentino et al., 1991; Ding and Shevach 1992), while in human CD4+ cells, both Tm and Tm function is downregulated (Del Prete et al., 1993). Human Il-10 is able to downregulate the expression of class D MHC on human monocytes (de Waal Malefyt et al., 1991) and inhibits human T cell proliferation and Il-2 production in response to either anti-CD3 or mitogen in the presence of accessory cells (Taga and Tosato, 1992). Recently it was reported that Il-10 inhibits Il-5 but not Il-2-induced antibody secretion by B cells (Pecanha et al., 1992). 1.4.3.1.6 Transforming Growth Factor beta (TGFfl) TGFB (Table 11), produced by antigen activated T cells and B cells displays both growth-stimulatory and growth- inhibitory properties, depending on the target cell and culture conditions (Sporn and Roberts, 1988). It is capable of inhibiting the proliferation and differentiation of normal activated human B lymphocytes (Kehrl et al., 1986a, 1989) and T lymphocytes (Kehrl et al., 1987a). In contrast, both Epstein-Barr Virus (EBV) infected normal B cells and EBV+ Burkitt's lymphoma cell lines are unaffected by TGFB (Blomhoff et al., 1987) . TGFB blocks G,/S transition in anti-Ig-activated B cells but not early activation events (Smeland et al., 1987). MHC class H is upregulated in B 65 cells exposed to TGFB (Lee et al., 1987; Cross and Cambier, 1990). In LPS stimulated B cell cultures, TGFB induces the secretion of IgA while decreasing IgG and IgM secretion (Coffman et al., 1989a; Sonada et al., 1989). This increase in IgA is the regulation of isotype specific, B cell differentiation, expressed as TGFB-induced isotype switching (Coffman et al., 1989a; Sonada et al., 1989). IgA'B.cells, cultured with TGFB and LPS, induce the synthesis of mRNA encoding for a-chain. The cells then express IgA on their surface and subsequent secretion (Coffman et al., 1989a). Recent work by Ehrhardt et al. (1992) suggests that Il-2 and Il-5 can enhance the expression of surface IgA and IgA secretion. 1.4.3.1.7 Interferon gamma (IFN-y) IFN-y (table 11), produced by T,,,CD4+ and CD8+ cells, inhibits the proliferation of anti-Ig stimulated B cells either alone or in combination with Il-4 (Mond et al., 1985), and inhibits the activation effect of Il-4 on murine resting B cells (Rabin et al., 1986). In contrast, IFN-y has been shown to stimulate the proliferation of B cells when cultured simultaneously with anti-IgM, but it is not able to inhibit B cell blasts generated by the same polyclonal activator (Nakagawa et al., 1985; Defrance et al., 1986). High density B cells, stimulated in vivo, are 66 more responsive to IFN-y (Romagnani et al., 1986b). In addition IFN-7 increases class I and II MHC molecules on various cell types (Abbas et al., 1991). IFN-y enhances Ig secretion from activated B cells co- cultured with Il-2 (Bich-Thuy and Fauci, 1986; Delfraissy et al., 1988) and inhibits Il-4-induced production of IgE, but not IgM (Defrance et al., 1988a; Pene et al., 1988). While IgG, production is inhibited (Golub and Green, 1991) , IgG,,I is increased in LPS activated B cells by isotype switching (Snapper and Paul, 1987b; Snapper et al., 1988b). 1.4.3.2 Mucosal Immune System The mucosal immune system is composed of the gastrointestinal tract (GI), upper respiratory and genitourinary tracts, mammary, lachrymal and salivary glands. This is the first line of defense against microorganisms, allergens, foreign antigens and even its own bacterial flora. While IgG is the major isotype of the systemic immune system, IgA is the primary isotype in the mucosal system, comprising over 60% of the antibodies produced per day in humans ( Solomon, 1980; McGhee et al., 1989). The mucosal-associated lymphoreticular tissues (MALT) consists of the bronchus-associated lymphoreticular tissues (BALT) and the gut-associated lymphoreticular tissues (GALT). Within the GALT, the primary site of IgA induction is the Peyer's patch (PP). This microenvironment, adjacent 67 to the lumen of the small intestine, serves to sample intestinal contents, microorganisms and food antigens, and allows the body to mount an immune response against them. Within the PP are lymphocytes, macrophage, follicles (B cell zone) containing germinal centers and parafollicular areas (T cell zone). The B cell zone consists of small resting cells, blasts and IgA+ cells (60%, 40% and 5-8%, respectively), while the T cell zone consists of CD4*, CD8+ and CD48 cells (SO-60%, 25-30% and 5%, respectively) (McGhee et al., 1990). Mature B cells (mIgM+ and mIgD+) are switched to mIgA+ under the influence of CD4+ cells. They are then activated by contact with antigen, accessory cells and T cells (Il-4) and undergo active division (McGhee et al., 1989). Sixty to seventy percent of IgA+ B cells in the PP are thought to have committed to IgA synthesis at this site (Butcher et al., 1982). Terminal differentiation occurs elsewhere (Butcher et al., 1982; Cebra et al., 1984) making the PP a site of IgA induction and not an effector region for IgA synthesis. Following primary contact with antigen and accessory cells of the PP, these committed precursors of IgA plasma cells leave the PP by the efferent lymphatics and enter the systemic circulation through the thoracic duct. The circulating cells then home to their effector sites in the mucosal system by receptors for high endothelial venules (HEV) (Stoolman, 1989). The cells then proliferate and 68 differentiate into IgA secreting plasma cells under the influence of cytokines (Mestecky and McGhee, 1987). CHAPTERII MATERIALS AND METHODS 70 2.1 General Experimental Design The underlying hypothesis for this work is that direct interaction of vomitoxin with lymphocytes is the primary event in vomitoxin induced IgA glomerulonephritis. Initially, mixed lymphocyte preparations from spleen and Peyer's patch (PP) were exposed to vomitoxin and mitogens in vitro, and tested for alterations in immunoglobulin (19) production, cell proliferation and protein synthesis. Then, isolated splenic B cells, exposed to vomitoxin and lipopolysaccharide (LPS), were tested for changes in Ig production. Thirdly, the effect of vomitoxin on the capacity of total T cells (CD4+/CD8+) and T helper cells (CD4+) to enhance Ig production in B cells was evaluated. Finally, splenic T helper cell, exposed to vomitoxin and mitogen, were examined for alterations in cytokine production. 2.1.1 Effect of in vitro vomitoxin exposure on 19 production, protein synthesis and cell proliferation of lymphocytes from spleen and PP. The purpose of this experimental series was to test the effect of in vitro vomitoxin exposure on 19 production, protein synthesis and cell proliferation. Preliminary experiments were conducted to determine optimum concentration for each of the mitogens in the absence of vomitoxin. Cell preparations were made from pooled lymphoid tissues. Spleens (3 mice) and PP (8-10 mice) were divided 71 into two subfractions. Cell concentration in the first 'aliquot was adjusted to 5 x 105 cells/ml and used to test cell proliferation and protein synthesis in response to vomitoxin (100 pg/ml - 500 ng/ml and zero control) and the mitogens LPS (25 ug/ml) and ConA (5 pg/ml) and an unstimulated control. The second aliquot of cells was adjusted to 2.5 x 10‘ cells/ml and used to test 19 production in response to vomitoxin and mitogens at concentrations used to test cell proliferation and protein synthesis. 2.1.2 Effect of vomitoxin exposure on 19 production by B cells from spleen and PP. Experiments were conducted to determine the effects of vomitoxin on 19 production in B cells. B cells were purified from the lymphocytes of spleen or PP and 2.5 x 106 cells/ml were incubated in a tissue culture plate (Costar) along with LPS (25 ug/ml), vomitoxin (100 pg/ml - 500 ng/ml) or a media control for 7 days. The supernatants were harvested and 19 concentration determined by ELISA. 2.1.3 Effect of vomitoxin exposure on the ability of total T (CD4+ICD8+) and T helper (CD4*) cell populations to induce Ig production by B cells. Experiments were done to determine the capacity of CD4+/CD8+ cells or CD4+ cells exposed to vomitoxin to induce Ig production in added B cells as compared to control T 72 cells. CD4"/CD8+ or CD4+ T cells were isolated from the spleens of five mice and 200 ul/well (5 x 105 cell/m1) were incubated in the absence and presence of vomitoxin (12.5 ng/ml - 500 ng/ml) and the T cell mitogen ConA (5 ug/ml) in a 96 well tissue culture plate. After 24 or 48 hrs the cells were washed three times with 50 mM a-methyl mannoside to remove vomitoxin and ConA. Purified splenic B cells were then added (200 ul/well) at a concentration of 7.5 x 10’ cells/ml along with LPS (25 ug/ml) and an unstimulated control and the plate was incubated for an additional seven days. The supernatants were harvested and Ig concentration determined by ELISA. To determine whether the increase in IgA from B cells cultured with vomitoxin-treated CD4*/CDS+ or CD4+ cells was due to an increase in T cell number or viability, the following experiment was conducted. Total T and T helper cells were treated as described earlier and incubated in triplicate in a 96 well tissue culture plate. Cells were harvested at days 3,5,7 and 9 and cell number and viability determined by trypan blue dye exclusion on a hemacytometer (American Optical, Buffalo, NY). Supernatants from vomitoxin-treated T helper cells were tested for their ability to increase the Ig production of splenic B cells. CD4+ cultures were prepared as above with the following modifications. Ten (10) ml of cells (1x105 cell/ml) were cultured in a 25 cm2 Ifflask (Costar Corp., Cambridge,MA) with vomitoxin and either with or without 73 ConA. After 48 hrs the cells were washed free of toxin and ConA and recultured with fresh media for 5 days. In addition, 2 day cultures containing vomitoxin and ConA were also prepared, in the following step the wash solution contained a-methyl mannoside to remove ConA from these cultures. Supernatants were concentrated and washed three times using an Amicon concentrator with 10,000 MW cutoff, by centrifuging at 1,500 x g for 10 min., decanting supernatant and adding 7 ml of Hanks buffered salt solution. The concentrated supernatant was filter sterilized and incubated in a 200 ul/well culture at original concentrations with 1x105 splenic B cells/well and LPS (25ug/ml) . The plates were incubated 7 days and Ig determinations made by ELISA. 2.1.4 Effect of vomitoxin on cytokine production by CD4+ cells. Experiments were conducted to determine the effect of vomitoxin exposure on cytokine production by CD4+ cells. The first set of experiments was performed by culturing 200 pl of cells (5x105 cells /ml) in RPMI-1640 (10% FCS) containing ConA (5 ug/ml) and vomitoxin (200, 100, 50, 25, 12.5 and 0 ng/ml) in a 96-well tissue culture plate (Costar Corp.). After 48 hrs, vomitoxin and ConA were removed by three washes of a-methyl mannoside suspended in Hanks solution. The cultures were then reincubated for five more days using fresh media. At the end of 7 days incubation, the supernatants were harvested and stored at -80°C until 74 analysis. In the second set of experiments, 10 ml of T helper cells (1x105 cells/ml) were cultured in 25 cm2 T flasks of RPMI-1640 (10% FCS) containing vomitoxin (100, 50, 25, 12.5 and 0 ng/ml) and either with or without ConA (5 ug/ml). After 48 hrs, vomitoxin and ConA were washed from cells using a-methyl mannoside. The cultures were then reincubated for 5 days using fresh media. Cultures that were harvested at day 2 used a-methyl mannoside in the following step. Supernatants were washed and concentrated three times using an Amicon concentrator with a 10,000 MW cutoff. The supernatant was concentrated two fold, filter sterilized and 0.5 ml aliquots stored at -80° C until analysis. Il-4, Il-5, Il-6 and Il-10 were analyzed by ELISA and Il-2 by the bioassay using a CTLL-Z cell line. 2.2 General Procedures 2.2.1 Animals Female B6C3F1 [C57BL/6(H-2") x C3H/HeN(H-2‘)] mice, 8-10 weeks of age, were purchased from Charles River (Portage, MI). Animals were housed two per environmental cage (Nalgene,Rochester,NY) and fed powdered semipurified AIN-76A diet (ICN Nutritional Biochemical, Cleveland,OH) upon arrival. Animals were acclimated to cages, feed, a 12-hr light-dark cycle, and housing facility for 7 days prior to experiments. 75 2.2.2 Culture media Primary cells and cell lines were cultured in RPMI 1640 medium (Sigma) supplemented with 100 U/ml penicillin, 100 ug/ml streptomycin, 5 x 10" M 2-mercaptoethanol, 25 mM Hepes buffer, 1 mM sodium pyruvate, and 1 mM nonessential amino acids (Pestka et al., 1989). In addition, 10% (v/v) Fetal Bovine Serum (Gibco Laboratories, Chagrin Falls, IL) was added to the medium . All cultures were grown in a 37%: humidified incubator containing 7% C02. 2.2.3 Lymphocyte preparation Mice were sacrificed by carbon dioxide asphyxiation and the spleen and PP were aseptically removed (Pestka et al., 1989). Cells were released from connective tissue by teasing with sterile tissue forceps. PP preparations were then passed through a sterile 85-mesh stainless-steel screen to remove connective tissue. Cells were then washed in Hanks Buffered Salt Solution (Sigma Chemical Co., St. Louis, MO) and erythrocytes lysed with 0.83% ammonium chloride. Cell number and viability were determined by trypan blue (Sigma) dye exclusion on a hemacytometer (American Optical, Buffalo, NY). 2.2.4 Lymphocyte fractionation Macrophage was removed from preparations (where indicated) by gently mixing the cells for 1 hr with Myloclear (Biotex Labs. Inc., Edmonton, Alberta, Canada). 76 The mixture was gently layered onto Histopaque (Sigma) at a density of 1.119, centrifuged for 10 min. at 200 x g and the "Buffy Layer“ (Klaus, 1987) collected. Efficiency of macrophage removal was determined by esterase staining (section 2.2.5; Yam et al., 1971). 2.2.4.1 B cell preparation Enrichment of B cells was achieved by depletion of T cells. A mixture of 100 pl each of anti-Thy 1.2 (HO-13-4, Marshak-Rothstein et al., 1979), anti-Lyt 2 (SB-6.72, Ledbetter and Herzenberg, 1979) and anti-L3T4 (GK 1.5, Dialynas, et al., 1983) antibodies was suspended in 3 ml of RPMI containing 10% FCS and incubated with lymphocytes for 45 min on ice. The mixture was washed with 45 ml of cold Hanks solution and centrifuged for 10 min at 450 x g. The pellet was resuspended in 3 ml of baby rabbit complement (Pel-Freez, Brown Deer, WI) diluted 1:6 in RPMI 10% FCS and incubated for 45 min in a C02 incubator. The mixture was then washed with 45 ml of Hanks solution, centrifuged for 10 min at 450 x g and cells cultured in RPMI 10% FCS. Purity of cell preparation was determined by staining (Klaus, 1987) for B cells with affinity purified, fluorescein-labeled, goat anti-mouse Ig (IgA, IgG, IgM) (Organon Teknika, Durham, NC) diluted 1:100 and staining for T helper and T suppressor cells with phycoerythrin-labelled goat anti-mouse CD4*, diluted 1:50 and fluorescein-labelled goat anti-mouse CD8+, diluted 1:50 (Becton Dickinson, Sparks,MD), respectively 77 and viewed under a fluorescence microscope. 2.2.4.2 T cell preparation All steps were performed at room temperature. After removal of macrophage from the lymphocyte preparation, T cells are purified using Mouse T Cell Recovery Kit (Biotex, Edmonton, Alberta, Canada). The column was first washed with 15 ml of PBS and then activated with antibody specific for B cells (Goat anti-Mouse B220). After 2 hrs of incubation, the unbound antibody was washed off with 15 ml of PBS and the elution rate adjusted to six drops per min. A total of 1.5 to 2.0 x 10‘ lymphocytes was added in 1.5 ml of PBS and allowed to run into the column matrix. Unbound T cells were eluted from the column with 20 ml of PBS and cell purity was checked by staining for B cells and CD4+ and CD8+ cells. 2.2.4.3 CD4+ cell preparation Cell preparation using the Mouse CD4 Cell Kit (Biotex, Edmonton, Alberta, Canada) was the same as above except for the following modifications. Antibody used to activate the column contained both goat anti-mouse and sheep anti-rat 19. Cells were incubated on ice for 30 min with antibody specific for mouse CD8+ cells (rat anti-mouse) prior to passage through the column. Cell purity was checked by staining for B cells and CD4+ and CD8+ cells. 78 2.2.5 Esterase staining B and T cell preparations were checked for depletion of macrophage using esterase staining (Yam et al., 1970). Lymphocytes (1x10‘ cells in 0.75 ml media) were gently placed onto a sterile coverslip inside a small sterile petri dish and incubated for one hour in a 37° C, tissue culture incubator. The coverslips were washed quickly in cold distilled water and fixed in cold fixative solution for 30 seconds followed by a second washing under gently running distilled water for 5-10 seconds. Coverslips were then immersed in napthol and fast garnet GBC salt, staining solution for ten minutes, washed for 20-30 seconds in distilled water and then incubated in Mayer's hematoxylin counterstain for 8 min at 24° C. After the final wash in distilled water the coverslips were examined under the microscope for stained macrophage. Following macrophage depletion and cell purification, all cultures contained less than ten percent of their original macrophage numbers. 2.2.6 Cell proliferation Cell proliferation, as measured by mitogen-induced thymidine uptake, was performed in triplicate microtiter wells (Forsell, et a1. 1985). Lymphocytes (2 x 10‘ cells/well), in aliquots of 100 pl of RPMI-1640 (containing 10% FCS), were cultured in flat-bottomed 96-well plates (Costar) for 72 hr in a 37°C, 7% CO2 humidified incubator. Cultures were stimulated with the mitogens ConA (5 ug/ml), 79 LPS (25 pg/ml) and a media control. Vomitoxin (gift of Dr. M. Witt; Witt et al., 1985) was added at time of mitogen addition. During the final 6 hr of culture, 20 pl of [fin TdR (0.20 pCi) was added to each well. At the end of incubation, cells were harvested onto glass filters using a Titertek Cell Harvester (Flow Laboratories Inc., VA) and radioisotope incorporation was counted on a Minaxi Tri- Carb scintillation counter (United Technologies, Downers Grove, IL). 2.2.7 Protein synthesis Protein synthesis, as measured by mitogen-induced leucine uptake was performed in triplicate microtiter wells (Liao et a1, 1976). Lymphocytes (2 x 10‘ cells/well), in aliquots of 100 pl of RPMI-1640 (containing 10% FCS), were cultured in flat-bottomed 96-well plates (Costar,) for 72 hr in a 37%L 7% CO2 humidified incubator. Cultures were stimulated with the mitogens ConA (5 pg/ml), LPS (25 pg/ml) and a media control. Vomitoxin was added at time of mitogen addition. During the final 1 hr of culture, 20 pl of [“C] Leucine (0.10 pCi) was added to each well. At the end of incubation, 100 pl/well of cold 10% trichloroacetic acid was added and insoluble materials were harvested onto glass filters using a Titertek Cell Harvester (Flow Laboratories, Virginia) and radioisotope incorporation was counted on a Minaxi Tri-Carb scintillation counter (United Technologies, Downers Grove, IL). 80 2.2.8 19 quantitation Culture supernatants were assayed for IgA, IgG and IgM by enzyme-linked immunosorbent assay (ELISA). Immulon II Removawell microtiter strips (Dynatech Laboratories, Alexandria, VA) were coated by an overnight incubation at 4°C with 50 pl/well of goat anti-mouse IgA, IgG, or IgM (Cappel Worthington, Malvern, PA) diluted 1:2000 in 0.1 M bicarbonate buffer (pH 9.6). Plates were washed three times with 0.01 M phosphate buffered saline (pH 7.2, PBS) containing 0.2% Tween-20 (PBS-T, Sigma Chemical Co., St. Louis MO). Three hundred pl of 1% (w/v) filtered chicken egg albumin (Sigma Chemical Co., St. Lois, MO) in PBS (OVA- PBS) was added to each well to reduce nonspecific protein binding. Plates were incubated at 37°C for 30 min and then washed three times with PBS-T. Ig reference serum (ICN Immunobiologics, Costa Mesa, CA, or Bethyl Laboratories, Inc, Montgomery, TX) or samples were diluted in RPMI 1640 (containing 10% FCS) and 50 pl was added to appropriate wells. Plates were wrapped in aluminum foil and incubated at 37°C for 1 hr and then washed four more times with PBS-T. Next, 50 pl of goat anti-mouse Ig horseradish peroxidase (a,7 or p-chain specific, Cappel Worthington), diluted 1:500 in 1% OVA-T, was added to each well, incubated for 30 min at 37°C and then washed five times with PBS-T. Bound peroxidase was determined as described by Pestka et al. (1980). Absorbance was measured at 405 nm on an ELISA plate reader (Biotek Instruments, Inc., Burlington, VT or 81 Molecular Devices, Palo Alto, CA), and Ig quantified using either the ELISA (Birmingham, AL) or Vmax software (Molecular Devices) programs. 2.2.9 Il-2 quantification Interleukin 2 was measured by reduction of 3-(4,5- dimethylthiazol-z-yl)-2,5-diphenyltetrazolium bromide (MTT) to the blue derivative formazan by the cell line CTLL-Z (Mosmann, 1983). CTLL-2 cell cultures were fed every 3 days with RPMI 1640 (containing 10% FCS) and supplemented with 1 U/ml of Il-2 (ICN Biomedicals, Irvine CA). (Note: excess Il-2 produced a nonresponding population of cells unsuitable for assay use 1) Cell density at this stage was approximently 3x105 cells/ml, cultures were split to 3x10‘ cells/ml for feeding. Interleukin 2 (ICN Biomedicals, Irvine, CA) standards and samples were both assayed (50 pl/well) in triplicate wells of a 96-well tissue culture plate (Costar). Prior to the addition of standards and samples to the plate, 200 pl of each was added to a microcentrifuge tube containing 25 pl of lyophilized anti-Il-4 antibody from the cloned cell line 11B11 (ATCC, Rockville, MD). Rapidly growing, healthy CTLL- 2 cells were washed once in Hanks Buffer, resuspended in assay media at a concentration of 4 x 10’ cells/ml and aliquoted at 50 pl/well. After 20 hr at 37°C in a CO, incubator 10 pl of MTT (5 mg/ml) was added to each well and incubated for an additional 4 hr. at room temperature. Then , T 82 150 pl of 0.04 N HCl in isopropanol was added to each well and mixed several times aid dissolution of formazan crystals. Plates were read on an ELISA plate reader (Molecular Devices) at of 570 nm with a reference wavelength of 690 nm and Il-2 quantified using Vmax software. 2.2.10 11-4, 11-5, 11-6 and Il-10 quantitation 7“ Culture supernatants were measured for interleukins by ELISA. The procedure and monoclonal antibodies (capture with biotinylated detectors) were from Pharmingen (San Diego, CA). Standard Il-4 and Il-6 were from Cellular E- Products Inc. (Buffalo N.Y.), Il-5 was from Genzyme (Cambridge, MA) and Il-10 was from Pharmingen (San Diego, CA). Il-4 (Mosmann et al., 1990), Il-5 (Schumacher et al., 1988), Il-6 (Van Snick et al., 1988) and Il-10 (Thompson- Snipes et al., 1991) monoclonal antibodies were used for a basic ELISA procedure. Immunolon IV Removawell microtiter strips (Dynatech Laboratories) were coated by overnight incubation at 4°C with 50 pl/well of rat anti-mouse IL monoclonal antibody at a concentration of 1.0 pg/ml in 0.1 M sodium bicarbonate buffer (pH 8.2). Plates were washed three times with 0.01 M PBS (pH 7.2) containing 0.02% Tween 20 (PBS-T, Sigma Chemical Co.). To reduce nonspecific protein binding, 300 pl of 1% bovine serum albumin (BSA) in PBS-T was added to each well, incubated for 30 min at 37%: and then washed four times with PBS-T. Reference 11 or samples were diluted in RPMI 1640 (containing 10% FCS), and 83 50 pl were added to appropriate wells. Plates were wrapped in aluminum foil and incubated for 1 hr at 37°C and then washed four more times with PBS-T. Biotinylated rat anti- mouse 11 monoclonal antibody, suspended in 1% BSA PBS-T at a concentration of 1.0 pg/ml was added to each well and the plate incubated for 1 hr at room temperature. After five more washes with PBS-T, 50 pl of a 1.5 pg/ml solution of streptavidin horseradish peroxidase (Sigma) was added to each well and incubated for 60 min at room temperature. The plates were then washed five times with PBS-T and 100 pl of substrate, containing 3.3 mg ABTS (Sigma), 11 ml 0.1 M citric acid buffer (pH 4.35) and 10 pl 30% Hg», was added to each well until appropriate color developed. Absorbance was measured on an ELISA plate reader at 405 nm and Il quantified using Softmax. 2.2.11 Purification and characterization of antibodies used in B cell preparation. 2.2.11.1 Clone growth and Ig purification Cloned cells used in this procedure were purchased from ATCC (ATCC, Rockville, MD) and grown in RPMI 1640 containing 10% FBS. Antibodies against the T cell markers Thy 1.2, Lyt-2 and L3T4 were produced by the clones HO-13-4, 53-6.72 and GK 1.5 respectively. Cells were cultured for 10 days to produce the maximum amount of antibody per culture flask. The media was then centrifuged for 10 min at 450 x g to 84 remove the cells and the supernatant poured into a clean flask. Saturated ammonium sulfate was added to a final concentration of 50% (w/v) and then stirred slowly for 1 hr at 4° C. The mixture was then centrifuged for 20 min at 10,000 x g to pellet denatured proteins and the supernatant was discarded. The protein pellet was then rehydrated in PBS to 1/10 of the original volume and the precipitation procedure repeated twice more. After the final rehydration in PBS the solution was dialyzed overnight at 4° C against 4 liters of PBS using dialysis tubing with a 12,000 - 14,000 MW cutoff. The dialyzed antibody solution was centrifuged at 10,000 x g for 20 min to remove any permanently denatured proteins. The antibodies were then filter sterilized, aliquoted to sterile tubes and frozen at -20° C until needed. 2.2.11.2 Antibody characterization Each of the three antibodies was analyzed separately for capacity to kill T cells when combined with complement. Antibodies were diluted 1/50, 1/100 and 1/200 in RPMI-1640 (10% FBS) and 2 ml aliquots were combined with 2.5 x 107 spleen cells and incubated on ice for 45 min. Cold Hanks solution was then added to each aliquot to bring the volume to 50 ml and the mixture was centrifuged for 10 min at 450 x g. The supernatant was discarded and the cell pellet resuspended in 3 ml of a 1:6 dilution of baby rabbit complement (Pelfreez, Brown deer, WI) in RPMI-1640 (10% FBS) 85 and then incubated for 45 min at 37° C in a tissue culture incubator. After incubation the cells were washed with 45 ml of 37°C Hanks solution, centrifuged for 10 min at 450 x g and the supernatant discarded. The cell pellet was resuspended in 5 ml of RPMI-1640 (10% FCS) and the cell number determined by trypan blue dye exclusion on a hemacytometer (AO). Cell populations were then determined by fluorescent staining. T cells were stained with phycoerythrin labelled goat anti-mouse CD4+ and FITC labelled goat anti-mouse CD8+ (Becton Dickinson,St. Louis, MO). B cells were stained with FITC labelled goat anti- mouse 19 (IgA, IgG, IgM) (Organon Teknika). The dilution of each antibody (anti-Thy 1.2, anti-Lyt 2 and anti-L3T4) which produced the best T cell kill was then used in combination as an "antibody cocktail" for all further B cell preparations. 2.2.12 Statistics Differences between vomitoxin-treated and control groups, for data from two or more experiments were analyzed using Least Significant Difference (LSD) following two way analysis of variance (ANOVA) using the Microcomputer Statistical Program (C.S. Sciences, Michigan State University). Data comprising one experiment was analyzed by LSD following one way ANOVA. CHAPTERIII RESULTS 87 3.1 [3H] TdR incorporation in lymphocyte cultures To determine the effect of vomitoxin on [’H] TdR incorporation, as a measure of cell proliferation, cultures were prepared from spleen and PP lymphocytes and incubated for three days with vomitoxin and mitogens. Thymidine incorporation was inhibited in LPS, ConA and unstimulated spleen cultures from 10 to 1,000 ng/ml vomitoxin (Table 12). It was significantly decreased at 1,000 ng/ml vomitoxin in ConA and unstimulated cultures (P<0.01) and at 100 and 1,000 ng/ml in LPS stimulated cultures (P<0.01). Thymidine incorporation was inhibited in LPS, ConA and unstimulated PP cultures by 10 to 1,000 ng/ml vomitoxin and was significantly decreased at 1,000 ng/ml vomitoxin in ConA and unstimulated cultures (P<0.01) and at 100 and 1,000 ng/ml in LPS stimulated cultures (P<0.05 and P<0.01, respectively; Table 13). 3.2 [“C] Leucine incorporation in lymphocyte cultures To determine the effect of vomitoxin on [“C] Leucine incorporation, as a measure of protein synthesis, cultures were prepared from spleen and PP lymphocytes and cultured for three days with vomitoxin and mitogens. Leucine incorporation in spleen cultures was inhibited in a dose- dependent manner in LPS cultures, ConA cultures and unstimulated cultures from 10 to 1,000 ng/ml vomitoxin and in ConA was significantly decreased at 1,000 ng/ml vomitoxin cultures and unstimulated cultures (P<0.01 and P<0.05: 88 ..Ho.ovm:v usac> Houucoo Sony Aomnv maucmofiuficoflm Heuuap xmwueumc cuss cexuea mesac> .mucniamwuu 5” can musoawummxm mean» no Axum 3 some use even. denounce auafinuocofipcu can peanut—ca mums maauo use . «.303 Hun H01 .3 EL no 0359 Mao: w m >n pezoadou mun—on pm you mammoufia can saxovflnoxr €53 peusuaso 0.33 3.33 Hum maaoo noaxi mouxoocmfiwq. :om « Hma :wH a moa :«m a med ooo.a mam.a H ~¢e.n men.m~a a mmo.noa :mmm.~m a mmm.mm~ ooa ouc.~ H Hem.¢ Ham.HmH H «Hm.on~ «mm.n¢ a Nom.ono oH mo~.~ a mn~.v mme.omfl H mmm.na~ Nun.mv a Hem.woe a moa.n u «mn.m ban.mua a noH.Hm~ .mn¢.m4 H www.mms o poundsawumca «:00 meg .Ha\o:c .zmoc coaumummuoocH mes ~=e_ mammmwemu .meuhuonmaha oaseanm an cowucquuOOCw men. 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Leucine incorporation in PP cultures was inhibited in LPS cultures from 10 to 1,000 ng/ml vomitoxin and was significantly decreased at 1,000 ng/ml vomitoxin in LPS stimulated cultures (P<0.01); very little leucine incorporation was observed in ConA cultures and unstimulated cultures (Table 15). 3.3 Ig production in lymphocyte cultures To determine the effects of vomitoxin on Ig production, cultures were prepared from spleen and PP lymphocytes and cultured for seven days with vomitoxin and mitogens. IgA production in spleen cultures (Table 16) was inhibited in LPS, ConA and unstimulated cultures from 1 to 1,000 ng/ml vomitoxin and was significantly decreased at 1,000 ng/ml vomitoxin in LPS, ConA and unstimulated cultures (P<0.01). IgA production was inhibited in LPS, ConA and unstimulated PP cultures from 10 to 1,000 ng/ml vomitoxin and was significantly decreased at 100 and 1,000 ng/ml vomitoxin for LPS, ConA and unstimulated cultures (P<0.01, except P<0.05 for 100 ng/ml unstimulated cultures) (Table 17). IgG production was inhibited in a dose-dependent manner in LPS, ConA and unstimulated spleen cultures (Table 18). IgG was significantly decreased at 100 and 1,000 ng/ml vomitoxin in LPS cultures (P<0.01) and 1,000 ng/ml vomitoxin in ConA stimulated cultures (P<0.01) . IgG production was inhibited in a dose-dependent manner in LPS, ConA and 91 .xHo.ovm: “mo.ovsp msHe> Houusoo sosu Aomnv amuseoHuHsmHm smumwu xmwumume spa: uexses mmsae> .uueufiasfisu ca :5.» musmsflummxm muss» no Axum HV sees use even. .uussmeea >HH>HuoeoHueH use uoume>ses mes sHeuoss mHHeo use .Aaama see «u: m.ov eswosma Hora no amass ssos e e an umaoHHou 930: we sou msmooufis use saxouwsos. saws ups—$.30 use: 3H8: Hem maauo Hoaxi neuwoosmga. .eH H mm :4 H om :mm H mm ooo.H mH H «e nHH H mmH :nmm H «om ooH oH H mm mmm H own mom H moH.~ oH «H H on on H nan mew H nHo.~ H HH H mm men H mam .mms H oom.~ o umueassfiumso «sou man , , “He\mc. AssnHjeoHumHoaummqwldemmws Fog. qanmuaamu .ueuaoosmsaa OHseHmu us sOHueuomuoosH «swosuq Hora so saxouwso> no vacuum ed eunea 92 .xHo.cvm:V usHe> Hosusoo sosu Howdy hsuseuflussvsu suuusu sussuuue sass uuxses uusHe> .uueusasssu ss sss ussusssusxu uussu no Axum HV seus use euepa .uussueus ass>suoeoHues use uuuuu>ses we: swuuoss uHHuu use .AHHus sun so: m.ov ussusus ”org so uuHss ssos a e ha uusoHHou mssos we sou usumouss use stouHso> sass uusssaso usu: :Hus sum uHHuo node: uusaoossshq. on H H4 5 H mH :em H as coo.H mH H hm 8H H 44 HH H HnH ooH o H o4 NH H am He H mam oH HH H mm oH H «H sq H mnH H HH H on mH H mm .moH H mam o uuueasswuusb «sou man A AHa\mcc 12mm. cosumwuumuuusluqsmmuq Fox. assessed» .uuu>uosme>a mm as soHuesomsoosH ussosus Hoza so ssxossso> so Huuuum nu eases 93 Homes sHHceoHHHcmHm HmHHHe smHHuHme nuHa cusses musHe> uusss so Axum Hy seus use even. .AHo.ovm:v nusHe> Hosusou sosu .uueUHHsHsu sH sss unsusHsumxu .4mHnm soHsuseu as smH sou uunaaese usu3 usseuessumsm .uheu h sou usuvouHs use stosHso> ssHa uussuaso usu3 .HHua sum uHHuo Hosxmv uuuaoosnshq. we +l «ON 44 mod +l and H .mve H and HHH mhu wee Hom moo :mH H mm :HH H Hm mm H wed mm H hm NH H MNH om H and He H emH ms H an mN H and cm H era uuueHsEHuusD esou .Hssmeqlems ooo.H ooH OH .He\m:c qHHuHHauu .uuuhuossst oHsuHsu as soHsosuoss «OH so stouHso> so uouuum on eases 94 . HHo.ovs: “moévmp uusHe> Hosssou sosu Aomq. >Huse0HMHsmHu suuuHu qusuuue saws uuxses uusHe> .uueoHHsHsu sH sss mususHsumxu uussu so Axum Hy seus use even. .emHam sowsuseu ws svH sou uueaaese usu: usseuessussm .uaeu h sou usumouHs use stouHso> saws uusssHso usu3 :Hus sum uHHuo Honmv uuuhoosmsmq. :obH H Hem :mmH H mHm :omm H one .mou H can :osm H emo.H :omm.~H H Hs>.HH ~m~.¢ H Hom.n Hem.~ H «mo.m omo.oH H mem.mH mmo.n H omn.n HNH H mHe.m eme.m H mmm.mH nee H th.H nm~.~ H omH.¢ .eem.m H mmm.- uuseHssHuusD «sou mug stquqlems coo.H OCH CH .He\m:. qHsuuHsuN .uushuosse>H mm as sowsusuoss «as so stouHso> so suuusm as eases 95 .AHo.ovs:v uusHe> Hosusoo sosu Homqv wauseoHuHsmHu suumwu qusuHue ssHs uuxses uusHe> .uueoHHsHss sH sss upsusHsusxu uussu so Axum Hy seus use even. .mqum sonuseu an uma sou uunhaese usua usseuessussm .uaeu h sou usumouHs use stouHso> suHs uussuHso usu3 Asauz sue uHHuu HonmV uusaoosssan. mm H HH :mH H mm :~ H mH coo.H HH H Hm mm H Hp :mm H HeH OOH HH H HH He H HHH HHH H Ham oH m H mm HOH H HHH HmH H 844 H w H mm mH H omH .mm H HHH o uuueHssHuusD csou wen .Ha\o:c .Hasmuqlums qHsuHHeuN .uuuhoosasaa uHsuHmu ks soHuusuosm umH so stouHao> no souuum on eases 96 unstimulated PP cultures from 10 to 1,000 ng/ml vomitoxin (Table 19). IgG was significantly decreased at 100 and 1,000 ng/ml vomitoxin in LPS, ConA and unstimulated cultures (P<0.01, except P<0.05 for 100 ng/ml unstimulated cultures). IgM production was inhibited in a dose-dependent manner in LPS, ConA and unstimulated spleen cultures (Table 20). IgM was significantly decreased at 100 and 1,000 ng/ml vomitoxin in LPS cultures and ConA cultures stimulated cultures (P<0.05). IgM production was inhibited in a dose- dependent manner in LPS, ConA and unstimulated PP cultures (Table 21). IgM was significantly decreased at 100 and 1,000 ng/ml for LPS and ConA (P<0.01) and unstimulated cultures (P<0.05). 3.4 19 production by splenic B lymphocytes To determine the effect of vomitoxin on Ig production by B lymphocytes, cultures were prepared from spleen cells and cultured for seven days with vomitoxin and LPS. 19 production was inhibited in a dose-dependent manner (Table 22). There was a significant decreased in IgG and IgM at 100 ng/ml (P<0.05) and 1,000 ng/ml vomitoxin (P<0.01) and for IgA at 1,000 ng/ml vomitoxin (P<0.05). 3.5 T cell (CD4*/CD8+) stimulated Ig production To determine the effect of vomitoxin on the ability of splenic T cells to stimulate Ig production from splenic B cells, CD4+/CD8+ cells were exposed to vomitoxin and ConA 97 least sHuceoHHHcmHm HuHHHe smHHuHme nHHs eussea musHe> uussu mo Azmm Hy seus use eueo. . AHoévm: «mo.ovm.v musHe> Hosusoo sosu .useoHHaHss sH sss unsusHsusxu .«qum soHsuseu as omH sou uuuasese usua usseuessumsm .uheu b sou usumosHs use stouHso> ssHa uusssaso usu3 AHHuS sum uHHuo Honmv uus>oosms>q. \D 4*! ma 5 H mu men H Hon be and 'H HNH NmH +l uuseHsEHsusD HH mm 4‘! mm 55 'H own H wwu mum H mom mad H emu ¢GOU :m H :oHH H o>~.H H mom H .ems H was ha mnn h¢N.N nsm.H mnh.d HHsHmsqlmmH .uuumoosssaa mm us ooo.a OCH CH .Ha\ucc ussuuseuu soHuusuosQ me so stosHso> so souuum en eases 98 .AHo.ovm:v uusHe> Hosusoo sosu Amway hauseoHuHsoHu suuuHu qusuuue suHa uuxses uusHe> .uueOHHsHsu sH sss ussuswsusxu uussu no Axum Hy seus use even. .equm soHsuseu hm 20H sou uuuaaese usu3 ussesessumsm .uheu 5 son usuvouws use stosHso> suHs uussssso usu3 AHHua sum uHHuoHonmv uuu>oosss>a. mm H m4 :mm H ~HH :mn H mm coo.H H4 H oeH :msn H Hue :omm H ~4~.H ooH mmH H Ham 4mm H 4ms.H Hm~.~ H Hus.m oH cam H ems eem H me>.H «m4.~ H «no.4 H moH H o4e nun H oem.H .mp4.~ H m>~.4 o uuueassHuusD «sou mmq H .Ha\m:. HHeHmsHImu naeuuasuu .uuumoosssha uHsuHmu us soHsosuoss :OH so stouHso> so uuuuum cu eases . AHo.ovm: “mauve; musae> Hosusou sosu Howdy hasseonHsaHu suuuHu qusuHue ssHs uuxses uusHe> .useuHHsHsu sH sss unsusHsusxu uussu no Axum Hy seus use eueQ. .equm sowsuseu as 20H sou uunmaese usu3 usseuessussm .uheu 5 saw usuvouws use stosHao> ssHs uusssaso usus Aaaus sum uHHuunonmv uus>uossams. .4n H mm :4e H 40H :Hm H mm coo.H .oH H we :mm H nHH :HHH H m4~ ooH swm H ~44 mam H omm enH H 4HH.H oH p~4 H mom Hem H wmo o- H 4me.H H cm H mmm o4H H mes .mnn H mme.H o uuseHsEHuusD «sou was .Hs\ocv “Heumqqimus . qumwfledw .uusaoossE>H as as soHuusuoss 20H so stosHso> so suuuum HN eases 100 Table 22 Effect of vomitoxin on Ig production by splenic B cells‘ ngitgxin IgA Igg IgM_____ (he/ml) (he/m1) (he/ml) (he/m1) o 483 1 246b 3,056 1 1,088 7,985 i 276 1 320 i 40 4,093 r 1,311 8,268 r 1,576 10 264 1 143 3,319 i 537 9,238 i 754 100 120 t 76 872 i 153’ 2,255 i 846' 1,000 25 1 6‘ 2 i 1“ 48 1 9” 'Macrophage-free lymphocytes (5x105 cells per well) were cultured with vomitoxin and LPS (25 pg/ml) for 7 days and supernatants analyzed for Ig by sandwich ELISA. °Data are mean (1 SEM) of two experiments run in triplicate. Values marked with asterisk differ significantly (LSD) from control values (‘p .useoHHmHsH sH sss mssusHsusxu 03s «0 .2mm Hy seus use even. .«msqm squuseu as uunhHese ustsuHsuHsH use uusmu>ses usu3 ussesessumsu use .eHuus smusu suHs maeu m sou uussussous uHHuo us» use #50 uusmes usu3 «sou use stos suss use ussos we sou .stua my «sou use stouHso> sst uHeHs ussuaso usuuHu Hausa em e 5.. uussusso usu3 :Hus sue uHHuo HonHv uuuaoossfiha uusuuuuessosuez. H H mm mH H on s H mm :oHo.H H 4m~.H com 1 H mm :mmH H me4 :4 H HH4 m4 H can ooH Hm H nu mm H 4mm .ooH H mam 4m H mnH om HH H as :nm H 544 .mHH H ~4m mHm H HHm mm Hm H 54 4m H m4H mm H mum mm H H4H m.~H HH H m4 mm H~ HsH mm H omH .sm H «NH 6 xHa\=c xHa\ocv xHa\a. .Ha\mac xHa\m:. .QHHHH IIHHHHI IwudH IIHHHHI \ ededwssuu .uusess usssasu usuuHs Hauslom sH uussuasu uus>uosss>s uHsuHsu +4ou as soHsusuoss stsuHsussH so «sou use sHXOUHso> so suuumm em eases 110 .useoHHssu sH sss ussusHsusxu ssou.uo Axum Hy seus use even.- .«qum sowsuseu as ustsuHsuusH sou uuuasese usu: ussesessumsm .umeu a son Astma mu «sou use stosHso> ssHs uusssasu usus guess a. s3... saxssuo HonHV uuumoossshs uusunuuessosuez. 4.6 H 6.6 6.6 H H.6 6.6 H H.6 46 H 66 66.6 H 66.6 66H H.6 H 6.6H 6.6 H 6.6 6.6 H 6.6 6 H 6H 64.6 H 66.6 66 6.H H 4.6 H.6 H 6.6 6.H H 6.4 4 H 66 66.6 H 66.6 66 6.H H 6.6 6.4 H 4.6 6.6 H 4.6 4 H pH 66.6 H H6.6 6.6H H.6 H 6.6 6.6 H 4.6H 6.H H 6.4 6 H 6H .66.6 H 66.6 6 AH8\DV .Ha\meu xHe\=u .Hs\msc .Ha\ac .Hs\ac. IqHuHH _ IIHHHHI ImuHHI IHHHHnu IMHHHI queumssum u . u us .uussuasu guess a aeu 03» s« uuuuuosssas uwsuasu +4ou uuueHssHsu «sou as soHsusuoss stsuHsussH so stosHso> so Huuusm an eases 111 even. .AHo.ovs:v usHe> Hosssoo us» sosu “sway xssseuHustHu sumuHu qususue saws uuxses uusHe> .uueuHHmsu sH sss upsusHsumxu ssou no Axum Hy .«qum suHsuseu hm ustsuHsuusH sou uuuasese usu3 museuessussm CEO—H 0H0 .uaeu m sou eHuus suusm sH uussuHsous use «sou use stou no uusu uusues susu use us: we sou Aasxo: my «sou use stosHso> saws uussssso usus guess .H. s... Hs\uHHu0 6362; uusauosmsha uusunumessosoez. :46 H 64H 66 H 64 :66H H 466 6H6 H 666 :66.6 H 6H.6 66 H 66 6H H 66 H6 H 66H 66H H 66H 6H.6 H 6H.6 66 H 46 HH H 66 66 H H6H 66 H 6HH H6.6 H H6.6 66 H 66 4H H 66 66 H 66H 66 H 6HH 66.6 H 66.6 66 H 66 6H H 66 66 H 6HH 66H H 66H .6H.6 H 66.6 .Ha\a. 1H8\6:. st\:. xHa\6su .Hs\6. GHIHH DIHH WIHH fildH NIHH meHHmuHsuHsH OHsuHsu +vou uuueassHuu «sou .uussuasu xueau B aeu su>uu sH uuuhoossska OOH om xHa\6:. um soHuusuoss stsuHsuusH so stouHso> no uuuuum on eases 112 .uueuHHssu sH sss mususHsusxu ssou mo Ham .3 seus use even... .«mHnm sOHsuseu ans mstsuHsussH sou uuemaese usu3 ussesessussm .uzeu 6 son eHuus suusu sH uussussuus use stou no uusu uusues susu use uss m4 sou stosHso> suHs uussudso usu3 guess a sH Hs\u:uu 66:va uuuhuossfiaa uusuuumessosoez. 6.6 H 6.4 6 H 6H 66 H 66 6H H 6H 66H 6.6 H 6.4 6H H 66 66 H 66 6H H 66 66 6.6 H 6.4 6H H 6H 66 H 66 6 H 46 66 6.6 H 6.4 6H H H6 66 H 66 .6H H 66 6 xHa\o. xHa\6:6 xHe\s. 1H846sv .Ha\6:. ndHuHHI IIHHHHI lauds: IIHHHMI quumHeuN wswmmmdummmfl .uusssHsu seeds 9 >eu su>uu sH uuuauosmshs uHsuHsu +4su as soHuusuoss stsuHsuusH so stouHso> mo suumum Hm eases 113 3.11 T helper cell (CD4+) supernatant stimulated Ig production To determine the effect of vomitoxin on the ability of CD4+ lymphocyte supernatants to stimulate Ig production, cultures were prepared from spleen and PP lymphocytes and incubated for 7-days with CD4+ supernatants and LPS. While supernatants from 2-day, ConA-treated CD4+ lymphocytes had no effect on IgG and IgM production in splenic lymphocytes (Table 32), there was a significant decrease in IgA production (P<0.01) at 100 ng/ml vomitoxin. Supernatants from 2-day, ConA-treated CD4+ lymphocytes cultured with PP lymphocytes (Table 33) caused significant decreases in the production of IgM [(P<0.05), 100 ng/ml vomitoxin], IgG [(P<0.050, 50 ng/ml vomitoxin] and IgA [(P<0.01), 25, 50 and 100 ng/ml vomitoxin]. Supernatants from 7-day, ConA-treated CD4+ lymphocytes cultured with splenic (Table 34) or PP lymphocytes (Table 35) had no significant effect on IgA, IgG or IgM production. Supernatants from 7-day unstimulated CD4+ lymphocytes cultured with splenic (Table 36) or PP lymphocytes (Table 37) had no significant effect on IgA, IgG and IgM production. 114 .AHo.ovs:v usHe> Hosusou us» sosu Asmqv >Hsse0HuHsmHu suuuHu smHsuHue ssHs uusses musHe> .uuuxoossshs m suHs uueuHHssu sH uussuHsu mes useuessussu +4ou seem .Awusv uueuHHssu sH sss mususHsusxu +4ou ssou no Axum HV seus use eseP .«mHsm sOHsuseu as usHHssoamossssH sou uunasese usus usseuessussm .maeu 6 s0u “Hs\va mmv mmq use .«sou use stouHso> ssHs uheu a son uusssHsuv museuessumsu +4ou s35 uussuasu usu3 :Huz sum uHHuu 6oH x o.Hv uuuauossshH m uusulumessosuez. 66H.6 H 464.6 66H H 64H :666 H 666 66H 666.6 H 666.4 H6 H H6H 66 H HH6 66 666.66 H 666.66 66H H 66H 666.H H H46 66 66H.6 H 666.6 66H H 46H 6H4.H H 666.H 6.6H 666.6 H 646.6 66H H 66H .664 H 666 6 3565 2565 3565 3565 lineal: IImmHII IlemHIu . H s > zsssuosuozomss .uusauossshH m 0Hsussu >s soHsosuoss UH so uusaoosmsaa 0HsuHsu +4ou uuueusu ssxossso> use «sou Boss ussesessussu >euu~ so suuusm «6 eases 115 .xH6.6vm: 666.6 V6. Hosucoo 0:» sous 16666 aHuceoHHH66H6 HuHHHe smssmume 6HH: cusses 666H6> .muuaoonsaaH 6 69H: uueoHHsae sH uussuHsu mes sseuessussu +4cu sues .Amusv useOHHssu ss sss uususssusxu +4ou sacs uo Axum HV seus use ese .«mHsm suszuseu as usHHssonosssss sou uuuaHese usus uusesessussm .uaeu a sou .Hs\o mmv was use .«sou use ssxousso> sass maeu N uussussuv ussesessussu +4au s65: uussusso usu3 :Hus sun uHHuo HOH x o.Hv uuuauosssas m uusuluuessosuez. .666 H 666 66 H 6HH :66H H 664 66H 666 H 666 .66 H 64 :666 H 66H.H 66 666 H 6H6 66 H 66H :H66 H 666.6 _ 66 666 H 666.H 6HH H 46H 666 H 666.6 6.6H 646 H 666.H 66 H 66H .6H6.H H 666.6 6 .Ha\6:6 st\6:6 .He\6cc .Ha\6:6 .IHHJWI. IlmmHII IIHMHII susumwada mzssbmosuozD22H .uusauosssaH m mm as sasuusuoss OH so uuuauosssas essuHsu +4su uuueuss ssxouseo> use «sou sosu ussesessussu aeuum so sousum an eases 116 .uuuauosssaa m suss useusassu sH uusssaso mes usesessussu +4ou sues .Awusv useuHHssu sH sss upsusssusxu +4ou ssou so Axum Hy seus use eve .«mHsm sussuseu as usssssonossssH sou uuuasese usua musesessussm .uaeu 6 sou Aas\m 66v was use Assosuwa uaeu m use «sou use ssxouwso> ssHs uss we uussussuv usseuessussu +4au saws uussuasu usu3 :Huz sum uHHuu 60..” x o.Hv uuuauosssas m uusunumessosuez. 666.H H 646.6 664 H 666 666 H 666 66H 6H6.4 H 6HH.6 H66 H 664 666 H 666 66 666.6H H 666.6H 666.6 H 666.6 66H H 666 66 6HH.6H H 666.6H H66.6 H 666.6 666 H 666 6.6H 666.HH H 666.6H 666 H 666 .666 H 644.H 6 .Ha\6cc .He\66. .He\6:6 1H8\6:. IImmHII .IIHMHIw IlemHII . H > mzsssmosoozozms .uusaoosssas s owsuasu as sasuosuoss OH so uusauosssas assussu +4ou uuueusu ssxossso> use «sou soss usseuessussu aeuu6 so uouuum en essea 117 .uuuauosmsaH m suHs uueuHHssu sH uussssso mes usesessussu +4ou sues .Awnsv uueuHHssu sH sss upsusHsusxu +400 ssou so Axum Hy seus use even. .«mHsm s0H3useu as assassonossssH sou uuuaHese usuz usseuessussm .uaeu 6 sou AHs\vam~v was use AssosuHs uaeu m use «sou use stouHso> suHs uss o4 uussussuv usesessunsu +4au s35 uussussu usu3 :Hua sun uHHuu HOH x o.HV uusauosssas s uusuluoesmosuez. 666.H H 466.H 64H H 6H6 66H.6 H 664.6H 66H 666 H 66H.H 66 H 666 H66.6 H 644.6 66 666 H 666 HH6 H 666 666.6 H 666.6 66 664 H 666.H 666.H H 444.H 666.6 H 666.6 6.6H 664 H 66H.H 666 H H66 .666.6 H 666.6 6 xHa\6:6 st\6cc HHa\6:u .Hs\6:. Ilmmfllu llamalu IlemHII nsmumasda asspmosuozomms .uusauosseaH a mm as soHsusuoss UH so uuuauosssas essussu +4su uuseusu ssxossso> use «sou soss ussesessussu aeul6 so souuum an eases 118 ..6Hucc muuauonmaaH 6 69H; mueoHHsae sH uussussu mes usesessumsu +4ou suem .uueuHHssu sH sss upsusssusxu+4su ssom mo Axum Hy seus use use? .«mHsm sussuseu as usHHssonossssH sowH uueaaese usu3 museuessussm .uaeu 6 sou «Hs\v: may was use Assosusz uaeu m use stouHso> sst uss we uussuasov usseuessumsu +4ou sass uusssssu usu: :Huz sum uHHuo 63 x o..3 uuuauosssaa m uusuluvessosuez. 646.H6 H 644.66 666 H 664 666 H H66 66H 666.H6 H 664.6H 64H H 6H4 644 H 666 66 666.66 H 664.6H 66H H 666 666 H 666 66 666.6 H 644.6 66H H 466 .666 H 664 6 .Ha\6:6 .Hs\6:. .H84666 .Hs\6:c annual! Ilmmull unusual sfleqwdsda mzHHsmosuozszss .uusauosssaH s uHsuHsu as soHuusuoss uH so uuuauosssas OHsuHsu +4cu uuueuss stouHso> sass usseuessussu aeuu6 so uuuusm on eases 119 ue3 usesessussu +4su sues seus use eves. 6 sou Ass\ma mmv was use Assassss uaeu m use stossso> sass uss m4 uussHHsuv musesessumsu .uusauosmsaa m saws uueuHHssu sH uussuaso .Amusv useuHHssu sH sss ussusssusxu +4ou ssou mo Axum Hy .«mHsm sossuseu as usHHssoamossssH sou uunaaese usus ussesessussm .uaeu +4su sass uussuHsu usua :Hus sum uHHuo Hos x or: uuuaoosssaa s uusulumessosoes. 466 + H46.H 666 + 646 666 H 666.H 66H 666 H 646.H 666 H 66H 466 H 666 66 646 H HH6.H 66H H 66H 664.H H 666.6 66 6H6.H H 666.H 66H H 66H 66H6 H 6H4.H 6 1H846cu st\6:u .Ha\6:6 .He\6:6 IlsmHII IlmeII niemull. sflsumssqa zHHsuosoozszss so uusauosssas uHsuHsu +4su uuseusu stouHso> .uuuauosssaH m as as soHuosuoss 0H Boss ussesessussu aeul6 mo uuuuum 6n eusea CHAPTERIV DISCUSSION 121 The results presented here indicate vomitoxin did not stimulate cellular functions of non-fractionated lymphocytes, but rather inhibited them. Furthermore, while in vitro vomitoxin exposure did not directly stimulate an increase in IgA production by B cells, it could induce an IgA increase, by altering regulation in the CD4*population. Finally, vomitoxin also increased cytokine production by CD4+ cells without significantly increasing their overall cell numbers or their viability. Several aspects of this research require further discussion. Addition of vomitoxin to non-fractionated lymphocyte cultures from spleen and PP consistently inhibited TdR and leucine incorporation as well as IgA, IgG and IgM production in a dose-dependent manner (Tables 12-21). Relative inhibition could be roughly approximated as the dose required for 50% inhibition (ICm) of TdR and leucine incorporation and IgA, IgG and IgM production for spleen and PP cultures exposed to different mitogens (Table 38). Inhibition of cell function occurred in a dose dependent manner and had a critical threshold. While it varied among the mitogens and tissues tested, a dramatic inhibition of cell function occurred typically in the range of 10 and 1,000 ng/ml vomitoxin (Tables 12-21). This may be due to differential effects that toxin concentration exerts upon binding to the ribosome. For example in the binding of T-2 toxin to the ribosomes of rabbit reticulocytes (Liao et al., 1976), concentrations of 1 pM cause a breakdown of the .auH>Huoe no wow uHsHssH ou uusssvus soHsesssuusous .AoH use 4H .NH musseev ssHsuusses uHsu sH suHHseu uusHsuuuu usu3 uHuaHese ususvuussu use msHssuHsu uuauosseas. 122 66 44 46 666.H6 666 6660 mm 66 66 66H 66 66 66H 66 66 66H 66H 66H 66H «coo :uuHsm 44 6HH 666 66 66 was suuHsm new 66H 66H qusmuuuumumsfl sdflmuudusumsu newsman ummmuu 6505.3 8.; sea EL usaoosssaq .msugsnnsuH .uussuasu usauosssaa as use UHsuHmu uuueuss stosHso> sou uusae> SOH on edsea 123 polysomes into monosomes and at 1 mM the polysomes begin to freeze on the mRNA transcript. Since T-2 toxin is known to be an irreversible inhibitor of protein synthesis at high concentrations (10 pM; Liao et al., 1976), it is possible that at concentrations which cause a breakdown of polysomes, all of the cells may not be inhibited, allowing the resumption of detectable cell functions by using existing mRNA transcripts. At concentrations which freeze the ribosomes to mRNA no translation occurs and thus all cell function ceases. Vomitoxin and other trichothecenes might function in a similar fashion. Susceptibility of splenic lymphocytes to vomitoxin, as measured by TdR incorporation, appear to be similar across species lines, as seen by the IC” of 160 ng/ml for ConA (a T cell mitogen) and 140 ng/ml for PWM (a T and B cell mitogen) stimulated human lymphocytes (Forsell and Pestka, 1985) as compared to 170 ng/ml found in this study. In rat lymphocytes, 90 ng/ml is required for 50% inhibition (Atkinson and Miller, 1984; Miller and Atkinson, 1986). As determined by the IC,o values (Table 38), LPS stimulated cultures were generally more sensitive to the effects of vomitoxin than ConA stimulated cultures. There was generally a higher percentage of B cells in both the spleen and PP (50 and 55%, respectively; data not shown). These results were similar (50% for spleen, 49% for PP) to those found in control animals during a 12 week feeding study (Pestka et al., 1990a). It may be possible that the T 124 cell population, or a unique subpopulation, is less susceptible to inhibition by vomitoxin due to a difference in the ability to metabolize the toxin. The inability of vomitoxin to increase IgA production in non-fractionated lymphocyte cultures did not disprove the basic hypothesis. It was possible the presence of a heterogenous population masked any response that might have existed (Tables 16 and 17). Vomitoxin has been shown to marginally stimulate (140%) an IgA production from the cloned B cell line CH12LX (Minervini et al., 1993). It was therefore necessary to address the possibility that vomitoxin is able to produce an increase in IgA by directly regulating B cells. Vomitoxin was unable to stimulate an increase in IgA production in splenic B lymphocytes (Table 22), although there was a slight increase in IgG and IgM production at 1 to 10 ng/ml vomitoxin. As in the non- fractionated lymphocyte cultures, vomitoxin decreased Ig production at higher concentrations. The decrease in Ig production was probably due to the ability of vomitoxin to inhibit protein synthesis. Since T cells are responsible for the regulation of B cell maturation, differentiation and 19 production, this population might be responsive to vomitoxin and induce the increase in IgA production seen in animal studies (Dong et al., 1991; Bondy and Pestka, 1991). While splenic CD4+/CD8+ cells, exposed to vomitoxin for 24 hrs and then cultured for 7 days with splenic B lymphocytes, were able to induce a 3 125 to 5 fold increase in the production of IgA, relative to zero toxin control, no increase was seen in the production of IgG and IgM (Table 23). These results are similar to those of Bondy and Pestka (1991) who demonstrated a 5 fold increase in IgA production from cultures of lymphocytes isolated from mice fed a diet containing 25 ppm vomitoxin. When B cells from control mice were cultured with CD4+/CD8+ cells from treatment mice, there was a significant increase in IgA production relative to CD4+/CD8+ and B cell cultures from untreated mice. However, when B cells from treatment mice were cultured with CD4+/CD8+ cells from control mice there was only a slight increase in IgA production. Additionally, CD4+ lymphocytes were capable of increasing IgA, but not IgG and IgM production, when cultured with B lymphocytes following exposure to vomitoxin and ConA for 24 hours (Table 26). It is therefore reasonable to suggest that it is the CD4+ lymphocyte population which, following exposure to vomitoxin in vitro, and possibly in vivo, is capable of regulating an increase in IgA production from control B cells. Vomitoxin induced a delay in the proliferative response (dpr) of ConA stimulated CD4*/CDS+ cultures at concentrations responsible for increased IgA production (Table 24) and not in unstimulated cultures (Table 25). After three days of incubation of splenic CD4"/CDS+ lymphocytes with ConA and vomitoxin there was a significant decrease in total and viable cell numbers at 50 and 100 126 ng/ml vomitoxin, as well as a decrease in TdR incorporation at 100 ng/ml vomitoxin. Two days later, the values for both the total and viable cell counts were not significantly different from other toxin concentrations or the zero control, however there was a significant increase in the TdR incorporation for 100 ng/ml vomitoxin cultures. Finally,, at day seven, there was no significant difference in total and viable cell counts as well as TdR incorporation for control and vomitoxin treated cultures. The delay in the proliferative response represents a sudden increase in cell number and TdR incorporation at this one toxin concentration (100 ng/ml). It may suggest that CD4*/CD8+ cells or a subpopulation are initially inhibited by toxin binding to their ribosomes. The cells are apparently able to recover and proliferate to normal cell levels by some as yet unknown mechanism, such as, metabolism of the toxin or production of binding resistant ribosomes (via altered L3 component). It is possible that a suppressed cell population may produce increased cytokines or enhance IgA production via cognate B and T cell interaction. It is noteworthy that the vomitoxin concentration responsible for inducing elevated IgA production (Table 26) and the delay in the proliferative response of CD4+ lymphocyte populations, also produced a 4-fold increase in Il-5 production (Table 27). Since there was no significant increase, relative to zero control, in the total and viable cell counts at the day and vomitoxin concentration (50 127 ng/ml) responsible for increased Il-5 production at day 7 (1,101 U/ml), the output per cell must therefore have been increased. This increase in Il-5 may have been caused by quite different scenarios, such as selection of a unique CD4+ population or altered regulation of protein synthesis, more specifically, cytokine synthesis. The selection of a unique population of CD4+ cells may occur after the binding of vomitoxin to the L3 component of the ribosome. It is possible that a particular population, such as the THz subset, has the ability to produce the enzymes required to metabolize the toxin after prolonged binding. Upon metabolism of the toxin, from this population, the cells are able to replicate. One of the side effects of this unique population could be the ability to produce Il-5. Increased Il-5 production, by altered protein synthesis, may involve the dysregulation of a transcription nuclear factor, such as factor-kappa B (NF-KB). This protein participates in the regulation of multiple cellular genes and its regulation and effects are potentially modeled by the superinduction of Il-2 by cycloheximide (Shaw et al., 1988). NF-KB exists in the cytoplasm as an inactive precursor complexed with its inhibitory protein IxBa. Upon mitogen stimulation of T lymphocytes, IxBa can be rapidly degraded, resulting in the activation of NF-eB and expression of genes involved in the immediate early processes of immune, acute phase and inflammatory responses. 128 Cycloheximide (25 pg/ml) can inhibit the resynthesis of degraded IxBa in PMA activated Jurkat T cells, thereby allowing an increase in nuclear bound NF-xB (Sun et al., 1993). In addition, cycloheximide exposure (20 pg/ml for 4 hrs) can cause superinduction of Il-2 in PMA and ConA stimulated Jurkat cells by producing an increase in mRNA (Shaw et al., 1988). It may be possible that vomitoxin functions in the same manner. When ribosomal bound vomitoxin inhibits protein synthesis, mRNA might still continue to accumulate until the removal of vomitoxin. As the cell regains normal functions a burst of translation could result in large quantities of newly synthesized cytokines. Il-5 is not the only cytokine which affects specifically and non-specifically the production of IgA. B cell growth and differentiation capabilities are also attributable to Il-2, Il-4 and Il-6. It was therefore necessary to examine the ability of vomitoxin to increase the production of these cytokines. Vomitoxin induced a significant increase in Il-4, Il-5 and Il-6 production in CD4+ cells, cultured in 96-well plates (Table 28). The increase in Il-5 and Il-6 occurred at vomitoxin concentration(s) responsible for increased IgA production in T and B cell cultures (Tables 23 and 26), although Il-4 jproduction increased at a higher toxin concentration and I1- 10 was unaffected. The dose effect of vomitoxin, on «cytokine (ll-4, Il-5 and Il-6) production, was slightly 129 . decreased at 50 ng/ml, relative to 25 ng/ml and 100 ng/ml vomitoxin. This consistent decrease may be the result of 2 or 3 competing mechanisms. The increase in Il-5 and Il-6, acting synergistically, would explain the increase in IgA seen in T and B cultures (Tables 23 and 26) and probably that seen in in vivo feeding studies (Pestka et al., 1990a; Dong et al., 1993). Harriman et al. (1988) determined that Il-5 can increase the production of IgA in LPS-stimulated PP B cells (4x10‘ cell/well) , relative to LPS stimulated controls and is capable of increasing IgA production from mIgAl PP B cells and not mIgA'cells. While Il-6, in conjunction with Il-1, is a potent growth and differentiation factor for B cells (Vink et al., 1988), it is capable of increasing IgA production in IgA*, but not IgA’mmrine PP B cells (Beagley et al., 1989). Of paramount importance is the fact that Il-5 and Il-6 synergistically increase the production of IgA in PP B cells (Kunimoto et al., 1988). Although the production of Il-4 was only significant at 200 ng/ml vomitoxin, other concentrations demonstrated large increases (12.5, 25 and 100 ng/ml). These increases, coinciding with increases in Il-5 and Il-6, would also aid in the increase of Ig production. Il-4 induces isotype switching to 196, and IgE in LPS-activated murine B cells (Vitetta et al., 1984; Sideras et al., 1985; Coffman et al., 1986). Reports indicating that Il-4 aids the entry of B cells into cell cycle, growth and differentiation (Rabin et al., 1985; Howard et al., 1982) 130 and in conjunction with Il-S stimulates the expression of Il-2R on B cells (Loughnan and Nossal, 1989), suggests that Il-4 might assist in Ig production, and may help support an increase in IgA. Vomitoxin was able to induce a significant increase in the production of Il-5 and Il-10 from CD4+ cells following a 48 hr pulse with vomitoxin and ConA in 10 ml bulk cultures (Table 30). However, these cultures were unsuccessful in inducing a significant increase in the production of Il-2, Il-4 and Il-6. As stated earlier, Il-5 is known to increase the production of IgA. The significant increase in Il-10 seen after 7 days in culture (Table 30) would help to select for the TH2 subset of CD4+ cells. Since Il-10 is capable of down regulating the production of cytokines from Tm CD4+ cells (Fiorentino et al., 1989). These findings support the hypothesis in two ways. First, by inducing the decreased production of IFN- 7 (from Th, cells) there should be an increase in activation of B cells by Il-4 (Rabin et al., 1986), and second, by down regulating Tm cytokine production those cytokines produced by TMCD4+ cells should predominate. This scenario is supported by the fact that the production of Il-2 (a Tm_cytokine) was significantly decreased at vomitoxin concentrations responsible for significantly increasing Il-5 (a T,,2 cytokine) . The inability of the last experiments (Tables 32-37) to demonstrate an increase in IgA production by spleen and PP B cells, when cultured with supernatants from vomitoxin 131 treated.Th cells, may have several explanations. First, production of cytokines capable of inhibiting Ig production may have been induced following CD4+ cell exposure to vomitoxin. IFN-a at low concentrations enhances Ig production in PWM activated murine cells, but suppresses Ig production at high concentrations (Peters et al.,1986; Rodriguez et al., 1983). The production of TGFB may account for similar results. While TGFB is capable of acting as a switch factor in IgA'cells (Coffmann et al., 1989a; Sonoda et al., 1989) it has suppressive effects on lymphoid cells and has been shown to inhibit the production of several 19 isotypes in LPS-stimulated B cell cultures (IgM, IgG,, IgG,, and IgG,) (Coffmann et al., 1989b; Sonoda et al., 1989). In addition, the production of IFN-y may account for such effects, owing to its ability to suppress the secretion of IgG,, IgG,,” IgG3 and IgE (Snapper et al., 1988a, b). Second, the low concentration of CD4+ cells in the T flask may have been a critical factor in the response to vomitoxin and subsequent cytokine production. Lastly, there may be a requirement for T and B cell cognate interaction before the increase in IgA production can be seen. The potential effects of human exposure to vomitoxin in ‘the food supply can be modeled by the B6C3F1 mouse. ZFollowing 24 weeks of exposure to 25 ppm vomitoxin, there was a 17 fold increase in serum IgA production (Pestka et .al., 1989). Concomitant with this, there was an increase in ‘the germinal centers of the spleen, PP and MLN, as well as 132 an increase in the CD4+/CD8+ cell ratio in both the spleen and PP (Pestka et al., 1989). In addition, there was an overall increase in the number of B cells and the expression of IgA+ cells in the PP as well as an increase in the number of B cells producing Igs specific for bacterial antigens (Rasooly and Pestka, 1992). Such findings may be the result of dysregulation caused by an increase in the production of cytokines. It is possible that following the consumption of vomitoxin, an increase in Il-4, Il-5, Il-6 and Il-10 production is stimulated from T helper cells within the gut. These cytokines would then cause the expansion of both the B and T cell populations. Increased production of Il-10 could then select for the T,,2 subset by downregulating the T,,, subset, thus increasing their (Tmfi cell number and overall cytokine production. Through increased expression of Il-S and Il-6, IgA secretion by B cells would also by increased. Prolonged exposure of animals to mycotoxins compromises their entire immune system and makes them more susceptible to bacterial infections (Tai and Pestka, 1990). As a result of increased bacterial numbers the permeability of the small intestine (SI) may increase due to LPS production, allowing luminal contents to cross more readily. In addition, the leakage of LPS across the SI basement membrane barrier and into the periphery would lead to the polyclonal activation ‘of B cells throughout the body. At the same time, the production of antibodies specific for gut bacterial antigens ‘wuuld increase, as well as an increase in immune complexes 133 (Ic) for dietary components. As concentrations of IgA and IgA-Ic increase in the serum there is an increase in their deposition within the glomerulus of the kidney, which develops into IgA nephropathy. Such a senario may be happening in humans following chronic exposure to vomitoxin in the food supply and may be partilly responsible for human glomerulonephritis. CHAPTERV SUMNIARY AND FUTURE RESEARCH 135 Vomitoxin was not capable of increasing IgA production in unfractionated lymphocyte cultures of the spleen and PP. Cellular functions, such as TdR and leucine incorporation, IgA, IgG and IgM production were inhibited in a dose dependent manner, between the concentrations of 10 and 1,000 IgA production was not increased in splenic B ng/ml. lymphocytes, although IgG and IgM were slightly elevated at concentrations of 1 and 10 ng/ml. Splenic CD4+/CD8+ and CD4+ lymphocytes were able to induce an increase in the production of IgA by splenic B lymphocytes, after being cultured with vomitoxin and ConA. This 3 to 5 fold increase occurred only with IgA, no increase was seen for IgG or IgM. It is therefore probable ‘that vomitoxin induces an increase in IgA in mice by .altering the CD4+ mediated regulation of B cells. Vomitoxin produced a delay in the proliferative response of ConA stimulated CD4"/CD8+ and CD4+ at concentrations responsible for increased IgA production. In addition, CD4+ lymphocytes produced a 4-fold increase in 11- 5 at the vomitoxin concentration responsible for the delay «111 ‘the proliferative response and increased IgA production. ‘VTDDRitoxin was also responsible for increased production of 11‘4 and Il-6 from ConA-stimulated CD4+ lymphocytes. While the mechanism for these events are as yet unknown it may be silllilar to that of cycloheximide superinduction of Il-2. cycloheximide induces mRNA accumulation in the cell by Inhibiting protein synthesis, superproduction of I1-2 occurs 136 upon the resumption of protein synthesis. While vomitoxin was able to induce an increase in Il-4 and Il-6 in 96-well tissue plate cultures it was unable to do so in 10 ml cultures. Nevertheless, the increase in Il-S and Il-10, along with the decrease in Il-2, are similarly consistent selective effects on Tm cells. This selection may be induced initially by the binding of vomitoxin to T,,2 cells and then amplified by the inhibition of TH, cytokine production by Il-10. Additional experiments need to be performed to determine the ability of vomitoxin-induced cytokines, or some other soluble component, to induce IgA production. The first set of experiments should include the exposure of a range of CD4+ cells, up to 5x105 cells/ml, to vomitoxin (0 - 200 ng/ml) and ConA for 24 hrs. Following concentration, the supernatants should be cultured with a range of splenic B cells, up to 5x105 cells/ml for 7 days. Low cell numbers should be compensated for by the addition of irradiated filler cells. Follow-up experiments need to be performed to address the requirement of cytokines or soluble component, at: certain stages of B cell differentiation and not as an "all at once" mixture. This can be accomplished by using Transwells (Costar) . This product is essentially a well Wi‘thin a well. Here, CD4+ cells could be incubated for 24 hrs with ConA and vomitoxin, washed free of toxin and ConA and then added to the upper well. Then B cells could be p lficed in the lower well and incubated for 7 days. 137 Cytokines are able to pass from the top well to the bottom through a porous membrane. If the effect is extracellular then it would be expected that B cells will produce elevated levels of IgA. It is possible that vomitoxin stimulates expression of a unique surface bound component of the CD4+ cell and this in turn stimulates the increase in IgA. To address this potential, irradiated vomitoxin-exposed CD4+ cells will be cocultured with the B cells in the bottom wells of the Transwells. Cytokines will be provided by vomitoxin exposed CD4+ cells in the top well and the "unique signal" will be provided directly to the B cells in the bottom well. Detection of mRNA, from vomitoxin treated CD4+ cells could serve to detect earlier cytokine gene expression. Cells should be pulsed for different lengths of time using (iifferent concentrations of vomitoxin and ConA to determine the optimal time for stimulating the different cytokine genes. It should be noted that production of mRNA is not an .absolute indication that there will be cytokine secretion from the cell. The mechanism by which mycotoxins cause chronic toxicity has not been adequately investigated. In light of ‘talia current research, it is reasonable to assume that e""IDCJsure to vomitoxin, stimulates a unique population of lymphocytes, and that this population may cause dl’SI'egulation of the immune system which leads to chronic and acute effects. Since long term exposure to vomitoxin 138 leads to the eventual death of mice, cloned cell lines need to be produced in vitro. Splenic CD4+ cells (2x105 cell/ml) from B6C3F1 mice, in the presence of 1x106 irradiated (3,300 rad) syngeneic cells could be stimulated in weekly cycles with vomitoxin and ConA. 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