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'1 {Kg}; j amount; mam MATERIALS: ' ~:""~T.-f-,.~,,,W ‘ Place in book return to raove cw; «Ju- - .- charge fro. circulation records ‘6 ‘ K423“ RAJI CELL ROSETTE INHIBITION: A SCREENING ASSAY FOR DETECTION OF IMMUNE COMPLEXES IN SERUM By Susan Kathryn Codere A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1979 ABSTRACT RAJI CELL ROSETTE INHIBITION: A SCREENING ASSAY FOR DETECTION OF IMMUNE COMPLEXES IN SERUM Susan Kathryn Codere The diagnosis and management of immune complex (IC) diseases has been advanced by the measurement of circulating immune complexes (CIC) in serum. At present, most clinical laboratories do not offer tests for CIC, mainly due to the complexity of available methods and the inability of one test to detect all types of IC. The Raji cell rosette inhibition (ROS-I) assay was developed and evaluated for detection of CIC in serum. IC prepared in vitro were shown to inhibit Raji rosette formation with antibody coated (EA) and antibody and complement coated (EAC) target cells. A survey of 223 serum specimens obtained from patients with collagen vascular disorders, and healthy blood donors revealed some sera also inhibited Raji rosette formation. Sixty-three percent of sera from patients with systemic lupus erythematosus and mixed connective tissue disease, 60% with rheu- matoid arthritis, and 41% with other rheumatoid disorders were positive. A comparative study of the Raji ROS-I with two other CIC assays, elec- trophoresis and the Raji radioimmunoassay, showed 60% and 69% correla- tion, respectively. Raji ROS-I is a simple and sensitive assay for the detection of CIC. It is anticipated, however, that this assay will be used as one of a panel of tests for CIC, since a single CIC assay cannot detect all varieties of IC. DEDICATION To Tom--thanks for standing by me and for always being there. And to all my friends in the lab-- thanks for your support and for your help. ‘ ii ACKNOWLEDGEMENTS I wish to express my sincere appreciation to all those who helped with the completion of this study. I would especially like to thank my Major Professors: John R. Kateley, Ph.D., Director of Immunology, Edward w. Sparrow Hospital, for his continuing guidance, patience, for the many opportuni- ties he has made available to me, and for his endless support. Maria J. Patterson, Ph.D., Director, Clinical Microbiology Program, Department of Microbiology and Public Health, Michigan State University, for her advise, academic counseling, encouragement and flexibility in allowing me to incorporate unique clinical experiences into my program. I would also like to thank: Wilford E. Maldonado, M.D., Director of Laboratories, Edward W. Sparrow Hospital, for the use of reagents, equipment, and supplies, and for his help in securing patient specimens. Norman B. McCullough, M.D., C. Wayne Smith, M.D., John R. Kateley, Ph.D., and Maria J. Patterson, Ph.D., my advisory committee, for their review of my thesis and for their guidance. I also greatly appreciate the assistance and encouragement given me by my colleagues: Carol Block, Sally Chirio, Jane Crane, Ruth Heckman, Chris Heiler, Robin Insalaco, Chris Jones, Bill LaRue, and Peggy Weber. TABLE OF CONTENTS LIST OF TABLES .................................................. LIST OF FIGURES ................................................. INTRODUCTION .................................................... LITERATURE REVIEW ............................................... MATERIALS AND METHODS ........................................... Serum ..................................................... Raji Cells ................................................ Preparation of Aggregated Human Gammaglobulin ............. Preparation of Hypertet-Tetanus Toxoid Immune Complexes... Indicator Cells for the Raji Cell Rosette Assays .......... Raji Cell Rosette Inhibition (ROS-I) Assay for Detecting Immune Complexes in Human Sera ......................... Cytochalasin B ............................................ Colchicine ................................................ RESULTS ......................................................... Raji Cell Culture Requirements ............................ Influence of Colchicine and Cytochalasin B on Raji Cells.. Collection and Storage of Serum for Raji Rosette Inhibi- tion Assay ............................................. Determination of Normal Range for Raji Cell Rosette Inhibition ............................................. Utilization of Normal Sera to Calculate Percent Inhibition and Establish a Normal Range in the Assay .............. Raji Rosette Inhibition Using Serum from Healthy Blood Donors ................................................. Raji Rosette Inhibition Using Serum from Patients Submitted for Diagnostic Tissue Biopsies ............... Raji Rosette Inhibition Using Serum from Patients with Collagen Vascular Disorders ............................ iv Page vi viii Page Raji Rosette Inhibition Using Serum from Patients with Rheumatoid Arthritis ................................... 42 Raji Rosette Inhibition Using Serum from Patients with Rheumatoid Disorders ................................... 42 Raji Rosette Inhibition Using Serum from Patients with Digestive Tract Cancers ................................ 45 Raji Rosette Inhibition Using Miscellaneous Patient Sera.. 45 Comparison of Three Techniques for Evaluation of Circu- lating Immune Complexes ................................ 48 DISCUSSION ...................................................... 51 BIBLIOGRAPHY .................................................... 57 APPENDIX ........................................................ 67 TABLE A #wm 10. ll. 12. l3. 14. IS. LIST OF TABLES . Some Diseases Associated with Circulating Immune Complexes.. Immune Complex Diseases Associated with Endogenous Antigens. Immune Complex Diseases Associated with Exogenous Antigens.. Methods for Detection of Immune Complexes ................... . Methods for Detecting IC Bound to Tissues ................... . Antigen-specific Methods for Detecting IC in Biological Fluids ...................................................... . Antigen Non-specific Methods for Detecting IC in Biological Fluids ...................................................... Influence of Fetal Calf Serum Concentration on Raji Cell Number and Viability ........................................ Influence of Fetal Calf Serum Concentration on Raji Cell EA Rosette Formation ........................................ Influence of Time, Temperature and Serum Concentration on Raji Cell Viability ......................................... Influence of Colchicine and Cytochalasin B on Raji Cell Viability and Rosette Formation ............................. Influence of Frozen and Thawed Normal Human Serum on Raji EA Rosette Forming Cells ............................... Calculation Of-EA Rosette Inhibition ........................ Raji Rosette Inhibition Using Serum from l2 Negative Con- trols to Establish the Normal Range for the Assay ........... Healthy Blood Donors Evaluated for Circulating Immune Complexes by Raji Rosette Inhibition ........................ vi Page 9 IO l2 13 l4 IS 25 26 28 31 32 37 38 4O TABLE Page l6. Kidney, Skin and Muscle Biopsy Patients Evaluated for Immune Complexes by Tissue Immunofluorescence and Raji Rosette Inhibition .................................... 4l l7. Collagen Vascular Disease Patients Evaluated for Immune Complexes by Raji Rosette Inhibition ....................... 43 18. Rheumatoid Arthritis Patients Evaluated for Immune Com- plexes by Raji Rosette Inhibition .......................... 44 l9. Patients with Rheumatoid Disorders Evaluated for Immune Complexes by Raji Rosette Inhibition ....................... 46 20. Miscellaneous Patient Sera Evaluated for Immune Complexes by Raji Rosette Inhibition ................................. 47 2l. Comparison of Three Techniques for Evaluation of Circu- lating Immune Complexes .................................... 49 22. Comparison of Three Assays for Evaluation of Circulating Immune Complexes ........................................... 50 vii LIST OF FIGURES FIGURE l. Raji cells with EA rosettes ................................ 2. Baseline determination for Raji rosette inhibition ......... 3. Daily variation in baseline HEAC and EA values used to determine Raji Rosette inhibition .......................... viii Page 27 34 35 INTRODUCTION Immune complexes (IC) are produced when antibodies (Ab) combine with their corresponding antigen (Ag). Immune complexes play an essen- tial role in the normal immune response by providing a mechanism for clearance and destruction of many antigens such as foreign serum pro- teins, drugs, microbial antigens from viruses, bacteria, and parasites, and autologous antigens. The fate of immune complexes is dependent, in part, on the site of formation, the nature and concentration of the antigens and antibody involved, and the size of the complexes. Most IC in the circulation are readily cleared by the reticuloendothelial system (RES), particular- ly by liver Kupffer cells. 'Large complexes, usually formed in antibody excess, and IC that fix complement, are rapidly cleared from the circu- lation. These complexes are rarely associated with disease in contrast to smaller, soluble immune complexes usually formed in antigen excess or non-complement-fixing complexes which tend to persist in the circulation. These smaller complexes are cleared to some extent by the spleen, but often become fixed to the renal glomeruli during the filtration of blood, or in blood vessel walls or choroid plexus. Complexes formed in the extravascular spaces are not cleared as rapidly as those formed in the circulation and, thus, are more likely to be deposited in the tissues. Under certain conditions, immune complexes may trigger a sequence of pathologic events in tissues and organs throughout the body. Pathogenic immune complex-mediated tissue injury via plasma mediators, either by activation of the complement system or by attachment to mono- nuclear cells with immunoglobulin (Fc) receptors or complement receptors (C3b or C3d) has been clearly demonstrated in animal models for serum sickness (Dixon, l963) (24). Similar glomerular, vascular, and articu- lar lesions in human diseases are also thought to be mediated by IC. However, the pathological expression of the formation of immune complexes seems to be relatively rare in comparison with the frequent occurrence of such IC in the circulation or in extravascular spaces. Consequently, the finding of complexes in any disease does not necessarily imply that they have a pathogenic role. Chronic immune complex-associated diseases may be classified according to the antigens involved. For example, IC associated with rheumatoid arthritis (RA; Immunoglobulin Ags.), systemic lupus erythe- matosus (SLE; nuclear Ags.), malignant diseases and other autoimmune disorders (cellular Ags.) involve endogenous antigens. In contrast, immune complex-associated diseases involving exogenous antigens include serum sickness (accidentally induced), diseases resulting from the inhalation or digestion of environmental antigens, and infectious dis- eases and their sequelae such as serum hepatitis (viral), post-strepto- coccal glomerulonephritis (bacterial), malaria (protozoal), and Schisto- somiasis (helminthic). Irrespective of the antigen derivation of IC, there is a similarity in the pathologic tissue damage mediated by immune complex deposits. Several approaches have been used to demonstrate the occurrence of immune complexes in human diseases; however, the two most-used procedures include, I) the detection of IC bound to tissues by histo- logic and electron microscopic techniques, and 2) serological analySis of samples from various biological fluids. Immunofluorescence and immunoperoxidase techniques are routinely used to detect immunoglobulins and/or complement deposits in tissue sections in the absence of other plasma proteins (albumin and fibrino- gen). The presence of such deposits is circumstantial evidence of immune complex involvement. Conclusive proof that immunoglobulin-con- taining deposits are IC requires the identification of the antigenic component in the immune complex. Elution studies have been used in some instances to identify specific antibodies (Woodroofe and Wilson, l977) (l04). Additionally, antigen identification has been accomplished by immunofluorescence studies. However, these are not standard procedures and consequently, are not routinely performed. Due to the profound role of IC in certain diseases and the imprac- tical nature of repeating tissue biopsies for diagnosis and monitoring patients with IC, many investigators have designed procedures to detect circulating IC (CIC). Several recent studies have compared the specific- ity, sensitivity, and reliability of techniques for the direct demon- stration of IC in serum. Assays for CIC may be grouped into two major categories: antigen- specific methods which permit the selective detection of IC for a single antigen; and antigen non-specific methods which are used to detect CIC independent of the nature of the antigen involved. Antigen non-specific methods can be further subdivided into procedures which identify IC either on the physical properties of the IC (size and solubility changes) or their biological properties (interaction with complement, anti- globulins, or with cells). All of the antigen non-specific methods, however, will detect non-specifically aggregated immunoglobulins, as well as IC. The specificity of each antigen non-specific method for IC varies according to the nature of the immune complex and to the influ- ence of interfering factors. Additionally, the difficulty in standard- izing some of the required reagents (aggregated IgG, Clq, Raji cell cultures) and the complexity of some of the proposed methods (radiolabel- ing Clq or rheumatoid factor (RF), isolating and characterizing mono— clonal RF) render some of the methods inapplicable for use in routine clinical laboratories (Lambert and Casali, l978) (51). In an effort to gather information on the role of 1C in disease and to evaluate methods being used for their detection, the World Health Organization (WHO) organized a "Scientific Group on the Role of Immune Complexes in Disease” which met in September, 1976. Their report reviews current knowledge concerning IC and pathogenesis and makes recommendations for laboratory diagnostic tests, clinical studies and basic research in this area (WHO TRS, 1977). Later, following these recommendations, the WHO established a collaborative study to evaluate and compare the specificity and sensitivity of 18 different methods for detecting IC in serum. Results of this study indicated that the most sensitive methods for the identification of sera containing IC were the solid-phase conglutinin-binding test (KgB-SP) and the Raji cell assay (Raji-RIA), followed by the solid-phase mRF inhibition assay (mRF-I), the solid-phase (Clq-binding test (ClQ-SP), the Clq-binding assay (ClQ-BA), the Clq deviation test (ClQ-DV) and the platelet aggregation test (PAT). Of these methods, five depend on a reactivity with comple- ment and two depend on the recognition of immunoglobulin (Ig) aggregates by Fc receptors on platelets or by rheumatoid factor. Six of the seven recommended methods require radiolabeling of Igs, RF, or Clq by the investigator. The comparative data compiled in this study suggest that there are different types of IC depending on the disease and each method displays a particular pattern of reactivity (52). The purpose of this research study was to review the available literature concerning the detection of immune complexes in sera and to develop an assay to detect CIC which would be simple, yet sensitive, to be offered as a routine screening assay in a clinical laboratory. LITERATURE REVIEW The role of immune complexes (IC) in the pathogenesis of tissue lesions was suggested as early as l9ll by Von Pirquet (97). Since that time, experimental models have been developed which clearly demonstrate the pathogenic role of immune complexes in serum sickness and their involvement in similar glomerular and vascular lesions in human diseases (Dixon, 1963) (24). In the past several years, research in the area of immune complex- associated diseases has become very popular. Many investigators have reported new methods for the detection of immune complexes in tissues and in biological fluids and have implicated immune complexes in the pathogenesis of many diseases. In l976, a World Health Organization Scientific Group met to discuss the role of immune complexes in disease. Their report reviews much of the current knowledge concerning immune complexes and lists methods currently used to detect IC. In addition, this report makes recommendations for future research for investigators interested in immune complexes (WHO, 1977) (105). Immune complexes appear transiently in many infectious diseases and allergies where they play an important role in the normal immune response to such foreign antigens. They are commonly formed when anti- bodies are produced against antigens still persisting in the circulation or extravascular spaces or released from host cells or invading organ- isms. These transient IC may be responsible for some complications, such as glomerulonephritis, in acute diseases. However, the pathogenic role of IC is probably more important in chronic diseases where the antigens involved are continually produced and released. Immune complex-associated diseases may be classified according to the antigens involved. Table 1 lists some diseases associated with immune complexes. In Tables 2 and 3 the same diseases are listed along with the antigens and antibodies involved, the associated pathology, and the methods used to detect IC in the respective diseases. Several approaches have been used to demonstrate immune complexes in human diseases. In general, these methods can be divided into two groups, 1) methods for the detection of IC bound to tissues, and 2) methods for the detection of IC in biological fluids (Table 4). In Tables 5, 6, and 7 the groups are further subdivided into specific assays. Eighteen of the antigen-nonspecific methods for detecting CIC were recently evaluated by the World Health Organization (Lambert et al., l978) (52). Results of this study indicate that the most sensitive methods for the discrimination of sera containing immuno- globulin aggregates from normal sera were the KgB-SP, Raji-RIA, mRF-l, ClQ-SP, ClQ-BA, ClQ-DV, and the PAT. Three of these seven assays, the Raji cell radioimmunoassay, the solid-phase Clq-binding test, and the Clq—binding assay, are widely used and potentially applicable for specialized clinical investigation. The Raji cell radioimmune assay (Theofilopoulos and Dixon, l976) (92) employs cells from the Raji lymphoblastoid cell line which exhibit receptors for IgG Fc of low avidity and large numbers of receptors for C3b, C3d, and Clq. For the assay, Raji cells are incubated with serum TABLE 1. SOME DISEASES ASSOCIATED WITH CIRCULATING IMMUNE COMPLEXES INVOLVING ENDOGENOUS ANTIGENS Immunoglobulin Antigens Rheumatoid Arthritis Mixed Cryoglobulin Diseases Hypergammaglobulinaemic Purpura Nuclear Antigens Systemic Lupus Erythematosus Specific Cellular Antigens Tumors Autoimmune Disorders INVOLVING EXOGENOUS ANTIGENS Iatrogenic Antigens Serum Sickness Drug Allergy Environmental Antigens Inhaled--Extrinsic Allergic Alveolitis Ingested--Dermatitis Herpetiformis Antigens from Infectious Organisms Viral--Hepatitis --Dengue Hemorrhagic Fever Bacterial--Post-Streptococcal Glomerulonephritis --Leprosy Protozoan--Malaria --Trypanosomiasis Helminthic--Schistosomiasis --Onchocerciasis INVOLVING UNKNOWN ANTIGENS Chronic Immune Complex Glomerulonephritis Vasculitis Louowu vmoumsaozc .mco_oocoe : mas .c_ouoLaoo_o:cOD_L u mzx .xuusm u Em ._oo>_m oco_>;uo>_oa u cum _ mm .pw m_.ou mcocuo m— .m Loanu umcmmum mEm_cm;ooE Lan0c_uLmu mo_ucocm__mz mo. .szxz o>_uomoc >_Looa u.u so. comumu_a_oocao>cu oEOm mm onm c. >Lm> u. wc__:no_m_uc< mm co_um_:oc_o c. 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Cell-bound 196 is then measured using radio- labeled anti-human 196. Results are evaluated using a standard curve prepared using normal human serum (NHS) and aggregated human gamma- globulin (AHG) in varying amounts. The Raji cell assay is sensitive and quantitative, but detects only IgG-containing IC and involves radiolabeled anti-human IgG which is not commercially available at present. Other IC which fix complement, but do not contain IgG, would also bind to Raji cells; however, these IC would not be detected since they would not bind the labeled anti-human IgG. In the solid-phase Clq binding test (Hay, Nineham and Roitt, 1976) (36,37), EDTA-treated serum samples are incubated in plastic tubes coated with Clq. IC bind to the solid phase Clq by virtue of the affin- ity of immunoglobulin Fc regions for Clq. The amount of IC bound to Clq is determined using a radiolabeled anti-human IgG. This method, like all methods involving Clq, has the disadvantage that only complement- fixing classes will bind to the Clq. Additionally, only bound IC con- taining 196 will react with the labeled anti-human IgG. In addition to requiring the iodination of anti-human 196, this assay also requires the purification of Clq. The Clq binding assay (Nydegger, l974; Zubler et al., 1976; and Zubler and Lambert, l976) (71,108,l09) measures the binding of radio— labeled Clq to IC. EDTA-treated serum is incubated with ‘25 I-Clq and polyethylene glycol (PEG). Complement-fixing IC bind to Clq and are precipitated by the PEG while free Clq remains soluble. The Clq binding activity'is the percentage of protein-bound radioactivity which is 18 precipitated after centrifugation. This assay also detects only IC which fix complement, but differs from the Raji cell assay and the solid- phase Clq binding test in that it does not employ anti-human 196. Therefore, this assay detects any IC which react with the Clq. However, complexes already saturated with Clq in vivo may not be detectable by any Clq assays unless EDTA or heat treatment removed previously bound Clq. In addition to the Clq purification necessary for the ClQ-SP test, 1251 or other iso- the ClQ-BA also requires labeling of the Clq with topes, making this test less adaptable to the routine clinical labora- tory setting. An additional technique evaluated in the WHO study which has particular relevance to the present study was the inhibition of complement-dependent lymphocyte rosette formation. Ezer and Hayward isolated B lymphocytes from human adenoid tissue and evaluated complement-dependent rosette formation by these cells following their incubation in sera from healthy controls or patient populations (26). More recently, Kammer and Schur (46) used human peripheral blood lymphocytes and HEAC (erythrocytes coated with antibody and complement) and EA rosette inhibition to test for CIC. In both procedures, HEAC rosette inhibition occurred when C3b-bound complexes attached to lymphocytes bearing C3b receptors (HEAC rosette inhibition). The Schur modification includes inhibition of rosette formation with HEAC by C'-bound IC, as well as inhibition of rosette formation with EA cells by complexes which do not bind C'. This modified method detects IC of the IgG and IgM classes with or without bound C'; however, 19 with CIC involving 19A or IgE, only complexes with C' fixed by the alternative pathway would be detected. The assay developed for this study combines concepts employed by the Raji cell assay, as well as those of the rosette inhibition assay, since Raji cells are used as the resetting cells and IC binding both Fc and C3b receptors are detected. MATERIALS AND METHODS Sew A total of 223 sera included in this study were obtained from healthy laboratory personnel; individuals donating a unit of blood for transfusion; patients with rheumatoid disorders including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA); patients on whom kidney, muscle, or skin biopsies were performed; and other patients on whom specimens were submitted to the Immunology Laboratory, E. W. Sparrow Hospital, for routine analysis. Venous whole blood was collected in vacutainer tubes and allowed to clot at room temperature (22° C). After 30 minutes the clotted blood was placed on ice for an additional 30 minutes. Serum was separated from the clot by centrifugation at 3500 x g at 0° C in an RC-3 refriger- ated centrifuge (Dupont-Sorvall, Wilmington, DE). Serum was stored at -70° C in 200 pl and 1 ml aliquots until tested. Raji Cells Raji cells, a lymphoblastoid cell line derived from Burkitt's lymphoma, were purchased from the American Type Culture Collection (Rockville, MD). Raji cell cultures were initiated from an aliquot of cells frozen in DMSO-supplemented tissue culture medium. Frozen cells were thawed at 37° C, washed twice by centrifugation with RPMI l629 (GIBCO, Grand Island, NY) at 37°C in 5% C02. A continuous supply of cells was maintained by passing an aliquot of cells every third day. 20 21 For the assay, Raji cells were harvested 72 to 96 hours after passage, washed two times in RPMI, and counted in a hemocytometer. Cell viability was determined by trypan blue exclusion. An average viability was 94%. Preparation of Aggregated Human Gammaglobulin Cohn fraction II (Sigma Chemical Co., St. Louis, MO) was used to obtain aggregated IgG following a technique reported by Theofilopoulos and Dixon (90). One gram of Cohn fraction II was dissolved in 10 ml PBS, pH 7.2, and centrifuged at 146,000 x g for 90 min. in a Sorvall Ultracentrifuge. The upper third of the supernatant fluid was separated and used as the source of aggregated IgG. The protein concentration, determined by the Biuret method, was 8.2 g/dl. The immunoglobulin solu- tion was heated in a 63° C water bath for 30 min. and stored in 25 pl aliquots at 4° C. Preparation of Hypertet-Tetanus Toxoid Immune Complexes One ml (400 Units) of Tetanus Immune Globulin (Cutter Laboratories, Berkeley, CA) was mixed with 1 m1 (200 Units) of tetanus toxoid (Michigan Department of Public Health, Lansing, M1) at 4° C. After a one-week incubation period, the tetanus-anti-tetanus complexes were centrifuged at 1800 x g for 20 min, in a Sorvall RC-3 refrigerated centrifuge. The supernatant was removed and stored at 4° C. Immune complexes with mouse complement were prepared by incubating 0.25 ml aggregated IgG or 0.25 ml Hypertet-toxoid complexes with equal amounts of mouse serum for one hour at 37° C. Mouse serum was obtained from adult ICR female mice. The serum was prepared in a manner which would maintain its complement activity. 22 Indicator Cells for the Raji Cell Rosette Assays Human 0 erythrocytes were washed 4X with RPMI and resuspended to 2.5%, in RPMI. One hundred pl of rabbit serum with anti-human erythro- cyte antibody were incubated with 2 m1 (1:20 v/v) of the erythrocyte suspension for 40 min. at 37° C. Following incubation, the erythrocytes were pelleted by centrifugation, the supernatant removed, and 200 pl of mouse serum added. The cell pellet was resuspended in the mouse serum and the mixture incubated for 30 min. at 37° C. The complement- sensitized erythrocyte-antibody complexes (HEAC) were washed 7X with cold RPMI and resuspended to 5%. Antibody-coated erythrocytes (EA) also were used in this study. Sheep erythrocytes sensitized with hyperimmune (IgG) rabbit anti-SRBC were purchased from Cordis Laboratories (Miami, FL) for the assay. EA cells were washed 5X with RPMI and resuspended to 5% (v/v) concentration. Target cells were used within one week (HEAC) or two weeks (EA) from date of preparation (11,28). Raji Cell Rosette Inhibition (ROS-I) Assay for Detectipg Immune Complexes in Human Sera Fifty p1 of washed Raji cells at a concentration of 15 x 10° cells per ml were incubated with 100 p1 of human sera from healthy volunteers or hospitalized patients for one hour at 4° C. The Raji cell viability exceeded 90%. After the incubation of Raji cells and serum, the cells were washed twice with RPMI at 130 x g in an IEC Clinical Centrifuge (Damon, Needham Hgts., MA) and resuspended to 2 x 10° cells per m1. HEAC and/or EA target cells (0.1 m1 of 0.5% v/v in RPMI 1629 medium) and 0.1 m1 Raji cell suspension (2 x 10° cells/ml in RPMI) were mixed in 23 10 x 75 mm glass tubes. The Raji cell-target cell suspensions were centrifuged for 8 min. at 120 x g in a Sorvall GLC-2 centrifuge. Following centrifugation, the tubes were placed in the refrigerator at 4-6° C. Raji cells and HEAC target cell mixtures were incubated for 30 min. while Raji cells with EA target cell mixtures were incubated for 12 hr. At the time of assay, the Raji cell-target cell pellets were resuspended in 0.05% (wt/vol) trypan blue. The viability of Raji cells was determined microscopically by trypan blue exclusion (> 85% excluded the dye). Calculation of Raji cell rosette formation and rosette inhibition was based on 200 viable Raji cells. Generally, 75 to 150 Raji cells formed rosettes with HEAC cells and 90 to 130 formed rosettes with EA cells. Cytochalasin B A 100 ug/ml stock solution of Cytochalasin 8 (Sigma Chemical Co., St. Louis, MO) was prepared in RPMI. One ml aliquots of stock solution were frozen at -70° C. The stock was diluted 1:20 with RPMI to yield a 5 pg/ml working solution. Colchicine A 50 pg/ml working solution of colchicine (Sigma Chemical Co., St. Louis, MO) was made by adding 1 mg colchicine to 20 m1 RPMI 1929 medium. RESULTS Raji Cell Culture Requirements The recommended fetal calf serum (FCS) concentration for Raji cells was 10% (v/v). Experiments were undertaken to determine whether a 1% or 2% FCS concentration would be adequate for immune complex evalu- ation. The number and viability of Raji cells cultured in 1%, 2%, or 10% FCS were evaluated on days 3 and 4 of culture. Additionally, Raji cell rosette formation with EA target cells, with and without pretreat- ment with normal serum or aggregated IgG, was performed on the third day of culture. The Raji cells cultured in 10% FCS for 3 days had three to four times the number of cells as cultures supplemented with 1% or 2% FCS. For Raji cells cultured four days, an even greater difference was noted (Table 8). Satisfactory cell viability (> 90% V) was maintained in three-day cultures with either 2% or 10% FCS concentrations; however, for cells cultured for four days, only cells incubated in 10% FCS yielded viabilities greater than 90%. Portions of Raji cell cultures supplemented with 2% or 10% FCS were adjusted to 15 x 10° cells/ml for use in the Raji cell ROS-I assay. Raji cells (50 p1) were incubated with RPMI, aggregated IgG, or 100 pl of normal serum at 4° C for 60 minutes. EA rosette formation was greatest in Raji cells cultured in 10% FCS (Table 9). Figure 1 shows representative Raji cells with EA rosettes. 24 25 TABLE 8. INFLUENCE OF FETAL CALF SERUM CONCENTRATION 0N RAJI CELL NUMBER AND VIABILITY Cell counts x lO'° /m1 FCS Concentration Day 3 Day 4 1% 3.2 (87)° 5.0 (84)61 2% 6.5 (94) 5.0 (85) 10% 16.7 (95) 41.0 (96) aNumber in parentheses indicates the percentage of viable cells in 200 cells counted using trypan blue dye exclusion. TABLE 9. INFLUENCE OF FETAL CALF SERUM CONCENTRATION ON RAJI CELL EA ROSETTE FORMATION Raji Cell Percent (%) EA Rosette Forming Cells Pretreatment 1% FCS 2% FCS 10% FCS None 18 (83)a 23 (90) 34 (90) RPMI --- 3O (83) 41 (93) A99 196 --- 1 (83) 2 (83) NS-O1 --- 16 (81) 42 (91) NS-O7 --- 21 (86) 4O (68) aCell viability evaluated by trypan blue dye exclusion. 27 Figure 1. Raji cells with EA rosettes. 28 The influence of time and temperature on Raji cell viability was evaluated by a vital dye staining procedure. For the assay, 7.5 x 10° Raji cells in 50 p1 RPMI 1629 were incubated at room temperature (22° C) or in the refrigerator (4° C) with either RPMI (100 p1) or NHS (100 p1 or 200 p1). Four incubation periods, ranging from 10 minutes to 120 minutes, were evaluated (Table 10). Preliminary experiments revealed that serum interfered with the trypan blue diffusion into non-viable Raji cells, and thus, Raji cells were washed once in RPMI to remove serum prior to viability determinations. The viability of Raji cells was not influenced by the incubation temperature; however, cells incu- bated in NHS were consistently less viable, albeit only slightly, com- pared with cells incubated in RPMI. Influence of Colchicine and Cytochalasin B on Raji Cells Microtubules and microfilaments within the cell play an important role in the movement of surface receptors and surface immunoglobulins in lymphocytes (i.e., capping and patching). A similar phenomenon may occur in Raji cells. Cytochalasin B and colchicine are known inhibitors of microfilament and microtubule function, respectively (96). Treatment of Raji cells with cytochalasin B or colchicine may therefore result in higher percentages of rosette forming Raji cells. Studies were undertaken to determine the influence of cytochalasin B and colchicine on Raji cell number, viability, and rosette formation. Three-day cultures of Raji cells were washed once with RPMI 1629, resus- pended in RPMI, divided into three equal volumes and incubated with 20 ml of RPMI, or 50 pg/ml colchicine in RPMI, or 5 pg/ml cytochalasin B 29 TABLE 10. INFLUENCE OF TIME, TEMPERATURE AND SERUM CONCENTRATION ON RAJI CELL VIABILITY Time of Incubation Incubation Percent (%) Viability (min.) Temperature RPMI NHS (100 pT) NHS (200 pl) 10 22°C 92 -- 100 4°C 95 98 100 30a 22°C 96 92 4°C 96 91 60a 22°C 96 88 4°C 96 88 120a 22°C 92 89 86 4°C 90 89 88 aWashed 1X after incubation. 30 in RPMI for 10 minutes at 20° C. Following incubation, the cells were washed once with RPMI and resuspended in equal volumes of RPMI. Cell counts, viability, and rosette formation with EA and HEAC target cells were then determined (Table 11). The cell counts and viabilities were similar for RPMI and colchicine-treated Raji cells, while the cyto- chalasin B had a slight cytotoxic effect on the cells. The percentage of HEAC and EA target cells were comparable for all three groups and thus neither cytochalasin B nor colchicine increased rosette forming cells (RFC). Collection and Storage of Serum for Raji Rosette Inhibition Assay Since current methods of immune complex detection cannot differ- entiate between non-specifically aggregated immunoglobulins and true IC, it was important to determine the influence of repeated freezing and thawing of normal serum on rosette formation and Raji cell viability. Venous blood from a normal serum donor was incubated at room temperature for 30 minutes and then centrifuged at 3500 x g for 15 min- utes at 4° C in an RC-3 refrigerated centrifuge. The serum was divided into six aliquots. The serum aliquots were frozen and thawed from one to six times, in a dry ice-ethanol mixture. Serum samples were tested for ROS-I. The number of RFC when incubated with RPMI, aggregated IgG, Hypertet-toxoid complexes, and NHS frozen and thawed one through six times are shown in Table 12. Three cycles of rapid freezing and thawing of serum appeared to have little effect on rosette formation. Thereafter, however, ROS-I increased although Raji cell viability was not affected by freezing and 31 TABLE 11. INFLUENCE OF COLCHICINE AND CYTOCHALASIN 8 ON RAJI CELL VIABILITY AND ROSETTE FORMATION Percent (%) Rosette-Forming Cells Raji Cell Treatment HEAC EA RPMI 152 (5)a 117 Colchicine (50 pg/ml) 156 (93) 104 Cytochalasin B (5 pg/ml) 163 (93) 97 aPercent viable cells determined by trypan blue dye exclusion. 32 TABLE 12. INFLUENCE OF FROZEN AND THAWED NORMAL HUMAN SERUM ON RAJI EA ROSETTE FORMING CELLS EA RFC/200 Percent Freeze-Thaw Cycle Raji Cells Inhibition RPMI Control 78 -- l 79 -- 2 76 (4)a 3 76 (4) 4 67 (15) 5 63 (20) 6 55 (30) aNumbers in parentheses represent percent-inhibition of rosette forma- tion compared to sera frozen and thawed once. 33 thawing. To assure optimal results from patient sera, specimens were aliquoted and thawed only once immediately before testing. Determination of Normal Range for Raji Cell Rosette Inhibition The normal range for Raji Cell ROS-I was determined by computing the average of the RFC obtained for Raji cells incubated with the negative control sera (NHS) used. Three or four negative control sera were run concurrently with positive controls and patient sera. The number of RFC per 200 viable Raji cells counted was recorded and the average of the three or four values for the negative controls was designated zero percent inhibition. All patient values were compared to the amount of rosette inhibition exhibited by normal sera. Four examples of baseline determination for HEAC and EA rosettes are given in Figure 2. Representative assays performed between March 30, 1979 and May 24, 1979 are presented. The mean values are also presented and vary for HEAC rosettes from 94 to 150 RFC, and 102 to 133 RFC for EA rosettes. The daily variation in baseline values for 20 HEAC assays and 28 EA assays is depicted in Figure 3. The baseline HEAC rosette values ranged from 76 to 154 RFC, and baseline EA rosette values ranged from 62 to 156 RFC. Utilization of Normal Sera to Calculate Percent Inhibition and Establish a Normal Range in the Assay Sera from twelve healthy individuals were selected as negative control specimens. The serum was aliquoted and stored at -70° C. 34 HE AC Rosettes 03 16011 '— O2 ....... --—-~«---146 1‘0T 1‘00 03 U, 5 ... ....... c2--- - --- L) .- 01 ' if 60» [_l . . c ,, ll 2 .D 2 > 1600 02 04 c, —— g -4FL--—Q1 -------- {pub-‘50 “ 140 u ’ 01 u. C! 120 04 1601 ”W 07 09 ------ -- ---0-r145 14011 01 1200 Normal Control Sera EA Rosettes 150), 03 ,1 1304 """" 02"""‘""‘°’ 01 11011 1800 03 .1 1400 ------------- ~1----119 O2 1 ‘°°’ T1 11 03 12041 m 04 ‘00“ ----- 1-4-4 -------- «~41-‘02 02 01 120‘1 07 04 ------ <-¢-------- ~ 1 4 1000 0‘ 09 o 80‘? Normal Control Sera Figure 2. Baseline determination for Raji rosette inhibition: Four examples using 3 to 4 NHS per assay. Mean value indicated by broken line. 35 «.5: .o 39:52 on Os Op p ON 0. — PtPPPDPPFDPPPPPFPPPPFDDPDPPPP DDPPPFPP DDDDDDD bebb 1144111111541111414 11‘1J11ddi dd‘uui1fidddddqdddldd szOmOC ‘W “3:301 U zp_mo 1311031198 .IQBIA DOE/3:181 .DI'A “111.308 .m mczmwu 36 Baseline, zero percent inhibition, was calculated for each assay by determining the mean number of RFC for three or four negative control sera. The percent inhibition for aggregated IgG and Hypertet-toxoid complexes (positive controls), negative control specimens, and patient sera was calculated as a percentage of the baseline value. A represen- tative example of the calculation appears in Table 13. The baseline value, the mean of four negative control sera, for EA rosettes was 102 RFC/200 viable Raji cells. Raji cells incubated with aggregated IgG and Hypertet-toxoid complexes exhibited 45 and 40 EA-RFC, respectively. Serum from patient EX-Ol incubated with Raji cells resulted in 72 EA-RFC or 30% inhibition (1 - 72/102 = 0.3). Ninety-three assays performed using serum from 12 negative con- trols exhibited values ranging from zero percent to 29 percent inhibi- tion. Seventy-eight specimens (84%) exhibited values between zero and nine percent rosette inhibition. Fifteen specimens (16%) resulted in 10 to 29 percent rosette inhibition; however, only four of 93 specimens exceeded 15 percent as shown in Table 14. Serum samples were classified as negative, borderline positive or positive for immune complexes according to the following definitions: Negative: less than 10% inhibition of rosette formation; Borderline Positive: 10 to 15% inhibition; Positive: greater than 15% inhibition. Raji Rosette Inhibition Using Serum from Healthy Blood Donors Normal serum was collected from individuals donating units of blood. An aliquot of the serum drawn for routine blood work was stored at -70° C and evaluated with positive and negative control specimens and 37 TABLE 13. CALCULATION OF EA ROSETTE INHIBITION Treatment of Percent Raji Cells EA-RFC Inhibition NS-Ol 94 8 NS-03 118 0 NS-02 96 6 NS—04 101 0 Baseline value: x = 102 A99 IgG 45 55 Hyp/tox 40 51 Ex-Ol 72 30 38 Ne NNF New Punch mo acmugma v P— NN mm Punch 0 o o o N N Npumz o o o o N N ppimz o.m o .o o o N N opumz o.N w -o o o e e acumz o o o o F _ moumz o o o o N N Neumz o.m N .m o o N N monmz e.m m_-o o F m o moumz m.o N -o o o N w ecumz m.p mplo o F N m moimz m.¢ mpuo o m N_ ON Noumz m.N mNuo v o 0N mm Foumz mmmcm>< magma H-mom Nm_x H-mom Nmp-o_ H-mom No-0 mesa .oz Pocpcou :owuwnwccH afiv pcmocma m>wpwmom mchLoucom m>wammmz Pouch w>mpmmmz >Hpmumcm»_~ocgocmcnsme--az .maocccoemsi-z .mmmmmmu omcoco pmewcweiuouz .m_uwcznocop:LoEopa-uzo a .cm—mnmp mm_zcm:uo mmm~ca xmcu_x mum mw_maowmm m N_co no o .;o .3. x c Npco mu o ccwxm o (.2 o mu.<.z N mu.o.z o o_u.mu.o.z a—u.mu.o.z (DO 5:_xm mo.: 0 eu.mu.a_u.<.u.z N eu.mu.c.u.<.u.z o eu.mu.cpu.u.z _-mom LON .mca Tchwcoa xmac_m Noe ease.wwan .-mcm wELmUOLm—um vmuxm m_moeco_xe< mN-xm azo-ma NN-xm m_om_om>_< mwuc_=omm> _N-xm Pm »0_>Lccmcmmcmaax ow-xm mummmmmumzamemaau No m>wmammz mucw_uma xwmowm me mu.z mom op-xm Aguaaocsam: «a. Pm_ocmmaz m_-xm Po 5.8.mu.u.z mom mo-xm menacsa cwm_=ngom-gomcaz w_-xm _N mu.o.z amnm-zw-z mo-xm Aoacnv mwmocmpom _aocaEamm F6056 --xm __ mucous; 6,8.m8.z meowmoxs»_oa No-xm zu-az o_-xm NP Npco z newcaewmuoca-ouz oo-xm zo-az m_-xm mm m:_xm Az.ov.mu mw3__=omm> mo-xm mom e_-xm mm mu.<.z agoaaocgaac m mmxmnach uzzzz_ ecu emc<:n<>m mc2w_cmao_m “puma: 924 z_¥m .>wzm_x .op wgmec 42 Raji Rosette Inhibition Using Serum from Patients with ColTagen Vascular Disorders Serum was obtained from 11 patients diagnosed with systemic lupus erythematosus (SLE) or mixed connective tissue disease (MCTD). Results of Raji cell ROS—I assay, antinuclear antibody and other pertinent data are recorded in Table 17. ROS-I was positive in five of 11 (45%), with a range of 21 to 82 percent inhibition, borderline in two (18%) and negative in four (36%). There was no apparent correlation between Raji cell ROS-I value and antinuclear antibody titer, percent DNA binding, extractable nuclear antigen titer or complement level. Raji Rosette Inhibition Using Serum from Patients with Rheumatoid Arthritis Serum was obtained from ten rheumatoid arthritis patients having RA latex titers exceeding 1:80. Results of Raji cell ROS-I assay, rheu- matoid factor titer and other pertinent data are recorded in Table 18. ROS-I values were positive in five of 10 (50%) with a range of 22 to 70 percent inhibition, borderline in one (10%) and negative in four (40%). All five CIC positive sera had RF values of 160 or greater, but there was no direct correlation between RF titer and percent inhibition as the four CIC negative sera also had RF values greater than 160. Raji Rosette Inhibition Using Serum from Patients with Rheumatoid Disorders An additional 32 sera specimens were collected from patients with rheumatoid disorders. 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