Autoimmunity. 1992, Vol. 13, pp. 285-290 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
DOES MITOGEN-INDUCED ANTIBODY PRODUCTION BY NORMAL BLOOD CELLS MIMIC SPONTANEOUS PRODUCTION IN LUPUS? ORA DAR", MYER R. SALAMAN, MARTIN H. SEIFERT' and DAVID A. ISENBERG2
Autoimmunity Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/20/14 For personal use only.
Department aflmmunoloRy, St Mary's Hospital Mediml School, 'Department of Rheumatology, St. Mary's Hospital and 'Bloomshury Rheuniatology Unit, Department of Rheumatology Research, University College and Middlesex School of Medicine, London (ReceivedMarch 6,1992;infinal form June 6,1992) Blood cells from patients with systemic lupus erythematosus (SLE) showed a raised level of spontaneous IgG production that included antibodies to DNA and to common environmental antigens (influenza virus haemagglutinin, adenovirus hexon and mannan from Candida alhicnns). In contrast, no IgG antibody was produced against an antigen not normally encountered in the UK (egg antigen from Schistosoma mansoni) or a selfantigen not generally associated with SLE (thyroglobulin). IgM production was raised to a lesser extent and only antibodies to DNA were detected. When normal cells were stimulated with pokeweed mitogen or S. aureus organisms, the specificity pattern of IgG production was similar to that described above for SLE with the major exception of the absence of IgG anti-DNA. IgM antibodies to DNA and all the other antigens were detected, but the specificity of the IgM ELISA assays for the protein antigens needs further clarification. The activity of IgM anti-DNA relative to total IgM was far greater in the SLE system. These results provide further evidence that a response to self-antigen is required for production of pathogenic IgG autoantibodies in SLE. KEY WORDS: Systemic lupus erythematosus, immunoglobulin production, polyclonal activation, antibodies to DNA, mitogen stimulation.
INTRODUCTION We have previously demonstrated that blood cells from patients with systemic lupus erythematosus (SLE) produce spontaneously not only IgG autoantibodies characteristic of the disease-antibodies to DNA-but also increased levels of IgG antibodies to microbial antigens that are commonly present in the environment'. The numbers of cells making antibodies to DNA were no greater than for the environmental antigens'. These findings are consistent with the view that both a self-antigen response and a non-specific polyclonal stimulus are involved in the production of IgG autoantibodies in SLE3. Further information on the relative contribution of these processes has now been obtained by comparing the specificities and levels of IgG and IgM antibodies produced spontaneously in SLE with those produced by normal cells stimulated with mitogens. In addition to DNA and common microbiological antigens, we have included in the study an autoantigen not generally associated with
*Dr Dar's current address is Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel. Correspondence to: Myer R. Salaman, Department of Immunology, St. Mary's Hospital Medical School, Norfolk Place, London W2 IPG.
285
SLE (thyroglobulin) and an antigen preparation from Schistosoma mansoni, an organism to which most people in the UK have not been exposed.
METHODS
Patients and controls Peripheral blood samples were obtained from female SLE patients attending clinics at St Mary's Hospital and the Bloomsbury Rheumatology Unit. All patients fulfilled the American Rheumatism Association's revised criteria for this disease4. The group of thirteen in Table 1 included one patient of clinical grade 1 (inactive), seven of grade 2 (mild), and five of grade 3 (moderate)'. Ten of the patients were being treated with prednisolone, in three cases in conjunction with azathioprine. The group in Table 2 comprised ten additional patients, one at grade 1, eight of grade 2 and one of grade 4 (severe). Six of these were receiving prednisolone, in four cases with azathioprine. A female control group came from staff at St Mary's Hospital Medical School.
Spontaneous production of antibodies Mononuclear leucocytes (PBMC) were obtained from
286
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heparinised blood of SLE patients and normal donors and cultured as previously described'. In brief, one ml cultures containing 2x10' PBMC in RPMI 1640 with 10% heat-inactivated fetal calf serum (RPMIPCS) were incubated for 2 days at 37°C. Cell-free supernatants pooled from triplicate cultures were then collected for assay of antibodies and total Ig. Differences between SLE and normal groups were assessed by the Mann-Whitney U-test.
Autoimmunity Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/20/14 For personal use only.
Mitogen-induced production of antibodies PBMC from normal donors were set up in culture as described above in the presence and absence of pokeweed mitogen (PWM) or Staphylococcus aureus Cowan strain 1 (SAC). Both mitogens were tested on each donor except in a few instances where there were only sufficient cells for one. After 6 days the cells were washed twice by centrifugation at 200 g for 7 min in RPMI/FCS and were re-cultured for a further 6 days in the absence of mitogen. Cell-free supernatants were then pooled from triplicate cultures as above. PWM (Gibco, Paisley, UK) was reconstituted as directed in 10 ml water and was used at a final concentration of 1 % (v/v). SAC organisms were kindly provided by Dr C. Ison, Dept of Medical Microbiology, St. Mary's Hospital Medical School. Bacteria were fixed by treatment in 1% formol saline for 2 days at 4°C. After extensive washing in saline they were used at a final concentration of 10' organisms/ml. These mitogen levels were shown to be optimal for Ig production. In time course experiments with PWM it was found that Ig production in the first week of culture was at its greatest on day 6 and in the second week it reached a plateau on days 1 1 and 12. The level attained in the second week was often at least 2-fold greater than that in the first week. Viability of the cells by trypan blue exclusion on day 12 was about 70%.
Determination of IgG and I g M antibodies The ELISA method used for determination of IgG antibodies to native (n) and denatured (d) DNA, influenza virus haemagglutinin (HA), adenovirus hexon (HX) and mannan from Candida alkicans (MN) has been described'. In brief, the microtiter plates (M29AR; Sterilin, Feltham, UK) were coated with the appropriate antigen and the plates were then exposed to culture supernatant. Antibody binding to the plates was detected with alkaline phosphatase conjugates specific for IgG (A-3 150; Sigma, Poole, UK) or IgM (A-3275). For determination of antibodies to thyroglobulin (TG), plates were coated overnight at 4°C with antigen (2.5 ,ug/ml) in carbonate coating buffer (Don Whitley, Shipley, UK). Human thyroglobulin was
kindly supplied by Dr P. Byfield, Clinical Research Centre, Harrow. Plates pre-coated with Schistosoma mansoni egg fraction (CEF-6) were kindly supplied by Dr J. Lilleywhite (London School of Hygiene and Tropical Medicine). Undiluted samples of supernatants were tested on each plate with the corresponding standards. These were plasma from a SLE patient (anti-DNA), a normal plasma (anti-HA, HX and MN), serum from a patient who had been infected with S. mansoni (anti-CEF-6) and, for anti-TG, MRC Research Standard A (65/93) from the National Institute for Biological Standards and Control, London. The standards were diluted in phosphate buffered saline containing 0.05% (v/v) Tween 20 (Sigma) as follows: IgG assays, 1 in 5000 (nDNA, MN, CEF-6, TG), 10,000 (dDNA, HX) and 20,000 (HA); IgM assays, 1 in 1000 for all except nDNA (5000) and dDNA (10,000). Mean optical density at 405 nm for three replicate determinations was corrected for background obtained in the absence of antigen on the plate (
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
DOES MITOGEN-INDUCED ANTIBODY PRODUCTION BY NORMAL BLOOD CELLS MIMIC SPONTANEOUS PRODUCTION IN LUPUS? ORA DAR", MYER R. SALAMAN, MARTIN H. SEIFERT' and DAVID A. ISENBERG2
Autoimmunity Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/20/14 For personal use only.
Department aflmmunoloRy, St Mary's Hospital Mediml School, 'Department of Rheumatology, St. Mary's Hospital and 'Bloomshury Rheuniatology Unit, Department of Rheumatology Research, University College and Middlesex School of Medicine, London (ReceivedMarch 6,1992;infinal form June 6,1992) Blood cells from patients with systemic lupus erythematosus (SLE) showed a raised level of spontaneous IgG production that included antibodies to DNA and to common environmental antigens (influenza virus haemagglutinin, adenovirus hexon and mannan from Candida alhicnns). In contrast, no IgG antibody was produced against an antigen not normally encountered in the UK (egg antigen from Schistosoma mansoni) or a selfantigen not generally associated with SLE (thyroglobulin). IgM production was raised to a lesser extent and only antibodies to DNA were detected. When normal cells were stimulated with pokeweed mitogen or S. aureus organisms, the specificity pattern of IgG production was similar to that described above for SLE with the major exception of the absence of IgG anti-DNA. IgM antibodies to DNA and all the other antigens were detected, but the specificity of the IgM ELISA assays for the protein antigens needs further clarification. The activity of IgM anti-DNA relative to total IgM was far greater in the SLE system. These results provide further evidence that a response to self-antigen is required for production of pathogenic IgG autoantibodies in SLE. KEY WORDS: Systemic lupus erythematosus, immunoglobulin production, polyclonal activation, antibodies to DNA, mitogen stimulation.
INTRODUCTION We have previously demonstrated that blood cells from patients with systemic lupus erythematosus (SLE) produce spontaneously not only IgG autoantibodies characteristic of the disease-antibodies to DNA-but also increased levels of IgG antibodies to microbial antigens that are commonly present in the environment'. The numbers of cells making antibodies to DNA were no greater than for the environmental antigens'. These findings are consistent with the view that both a self-antigen response and a non-specific polyclonal stimulus are involved in the production of IgG autoantibodies in SLE3. Further information on the relative contribution of these processes has now been obtained by comparing the specificities and levels of IgG and IgM antibodies produced spontaneously in SLE with those produced by normal cells stimulated with mitogens. In addition to DNA and common microbiological antigens, we have included in the study an autoantigen not generally associated with
*Dr Dar's current address is Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel. Correspondence to: Myer R. Salaman, Department of Immunology, St. Mary's Hospital Medical School, Norfolk Place, London W2 IPG.
285
SLE (thyroglobulin) and an antigen preparation from Schistosoma mansoni, an organism to which most people in the UK have not been exposed.
METHODS
Patients and controls Peripheral blood samples were obtained from female SLE patients attending clinics at St Mary's Hospital and the Bloomsbury Rheumatology Unit. All patients fulfilled the American Rheumatism Association's revised criteria for this disease4. The group of thirteen in Table 1 included one patient of clinical grade 1 (inactive), seven of grade 2 (mild), and five of grade 3 (moderate)'. Ten of the patients were being treated with prednisolone, in three cases in conjunction with azathioprine. The group in Table 2 comprised ten additional patients, one at grade 1, eight of grade 2 and one of grade 4 (severe). Six of these were receiving prednisolone, in four cases with azathioprine. A female control group came from staff at St Mary's Hospital Medical School.
Spontaneous production of antibodies Mononuclear leucocytes (PBMC) were obtained from
286
0.DAR ET AL.
heparinised blood of SLE patients and normal donors and cultured as previously described'. In brief, one ml cultures containing 2x10' PBMC in RPMI 1640 with 10% heat-inactivated fetal calf serum (RPMIPCS) were incubated for 2 days at 37°C. Cell-free supernatants pooled from triplicate cultures were then collected for assay of antibodies and total Ig. Differences between SLE and normal groups were assessed by the Mann-Whitney U-test.
Autoimmunity Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/20/14 For personal use only.
Mitogen-induced production of antibodies PBMC from normal donors were set up in culture as described above in the presence and absence of pokeweed mitogen (PWM) or Staphylococcus aureus Cowan strain 1 (SAC). Both mitogens were tested on each donor except in a few instances where there were only sufficient cells for one. After 6 days the cells were washed twice by centrifugation at 200 g for 7 min in RPMI/FCS and were re-cultured for a further 6 days in the absence of mitogen. Cell-free supernatants were then pooled from triplicate cultures as above. PWM (Gibco, Paisley, UK) was reconstituted as directed in 10 ml water and was used at a final concentration of 1 % (v/v). SAC organisms were kindly provided by Dr C. Ison, Dept of Medical Microbiology, St. Mary's Hospital Medical School. Bacteria were fixed by treatment in 1% formol saline for 2 days at 4°C. After extensive washing in saline they were used at a final concentration of 10' organisms/ml. These mitogen levels were shown to be optimal for Ig production. In time course experiments with PWM it was found that Ig production in the first week of culture was at its greatest on day 6 and in the second week it reached a plateau on days 1 1 and 12. The level attained in the second week was often at least 2-fold greater than that in the first week. Viability of the cells by trypan blue exclusion on day 12 was about 70%.
Determination of IgG and I g M antibodies The ELISA method used for determination of IgG antibodies to native (n) and denatured (d) DNA, influenza virus haemagglutinin (HA), adenovirus hexon (HX) and mannan from Candida alkicans (MN) has been described'. In brief, the microtiter plates (M29AR; Sterilin, Feltham, UK) were coated with the appropriate antigen and the plates were then exposed to culture supernatant. Antibody binding to the plates was detected with alkaline phosphatase conjugates specific for IgG (A-3 150; Sigma, Poole, UK) or IgM (A-3275). For determination of antibodies to thyroglobulin (TG), plates were coated overnight at 4°C with antigen (2.5 ,ug/ml) in carbonate coating buffer (Don Whitley, Shipley, UK). Human thyroglobulin was
kindly supplied by Dr P. Byfield, Clinical Research Centre, Harrow. Plates pre-coated with Schistosoma mansoni egg fraction (CEF-6) were kindly supplied by Dr J. Lilleywhite (London School of Hygiene and Tropical Medicine). Undiluted samples of supernatants were tested on each plate with the corresponding standards. These were plasma from a SLE patient (anti-DNA), a normal plasma (anti-HA, HX and MN), serum from a patient who had been infected with S. mansoni (anti-CEF-6) and, for anti-TG, MRC Research Standard A (65/93) from the National Institute for Biological Standards and Control, London. The standards were diluted in phosphate buffered saline containing 0.05% (v/v) Tween 20 (Sigma) as follows: IgG assays, 1 in 5000 (nDNA, MN, CEF-6, TG), 10,000 (dDNA, HX) and 20,000 (HA); IgM assays, 1 in 1000 for all except nDNA (5000) and dDNA (10,000). Mean optical density at 405 nm for three replicate determinations was corrected for background obtained in the absence of antigen on the plate (
