Immunology 1992 76 548-552
Production of rheumatoid factor in adoptively immune guinea-pigs after challenge with Treponema pallidum R. E. BAUGHN, V. WICHER* & K. WICHER* Departments of Microbiology and Immunology and of Dermatology, Baylor College of Medicine and Syphilis Research Laboratory, Veterans Administration Medical Center, Houston, Texas, and * Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York, U.S.A.
Acceptedfor publication 30 March 1992
SUMMARY Guinea-pigs of inbred strains 2 and C4D were infused with various concentrations (I x 108 to 4 x 108) of syngeneic nylon wool-purified Treponema pallidum-immune T lymphocytes (TPI-T) and challenged 24 hr later with virulent T. pallidum (108 organisms). The degree of protection depended on the number of infused T cells and was associated with an accelerated production of IgM rheumatoid factor (RF). Fully protected animals (4 x 108 TPI-T) did not produce treponemal antibodies or circulating immune complexes (CIC) but produced IgM RF detectable 10 days after infection. Partially protected animals ( < 2 x 108 TPI-T) produced, 30 days post-infection, relatively low levels of treponemal antibodies but high levels of CIC and RF. Control animals infused with 2 x 108 TPI-T lymphocytes but not infected with T. pallidum, when monitored for a period of 6 weeks, did not produce treponemal antibodies, CIC, or RF, excluding the possibility that IgM RF could be generated by the donor's B cells contaminating (circa 3%) the TPI-T lymphocytes. Moreover, unprotected syngeneic control animals infused, prior to infection, with T. phagedenis biotype Reiterimmune T cells or with T. pallidum-free testicular inflammatory fluid-immune T cells responded with increasing levels of treponemal antibodies; only a few animals produced RF and CIC 5 months after infection similarly to control guinea-pigs infected only. The production of RF in partially protected animals responding to infection with treponemal antibodies and CIC was apparently associated with the presence of the CIC; but the mechanism of RF production in fully protected animals in which no antibodies or CIC were detected is currently unknown. seems to be more than one pathway of production of these autoantibodies. It has been found that in natural or experimental conditions they may be generated by polyclonal B-cell activators.5 6 They were also present in hyperimmune mice7'8 and rabbits,9 "t where they were most likely elicited by immune complexes. This together with more recent studies'2" 3 provided ground for an assumption that precursors of RF-secreting cells are normal members of the B-cell repertoire.'4
INTRODUCTION The term rheumatoid factor (RF) defines a versatile group of autoantibodies of the IgM, IgG, IgA and even IgE classes reacting specifically with IgG Fc region, CH2 and CH3 domains, or with idiotypic determinants. Although by definition an autoantibody combines only with autologous antigen in its native form, this is not always the case with RF; in a broad sense, RF have been considered as autoantibodies, and it is within this context that they will be discussed in this report. In early studies detection of RF was thought to be limited to patients with rheumatoid arthritis, where they were ascribed a key role in the pathogenesis of joint inflammation.' Later, however, they were found in sera of patients with various nonarthritic disorders, associated with chronic inflammation and infection.2 Further studies revealed that RF can be produced as a consequence of kidney and skin transplantation3 and by lymphocytes from healthy humans or animals.4'5 Indeed, there
The functions ascribed to RF are no less diverse than the nature of the stimuli producing them. They seem to enhance the binding of an antibody to its antigen.'5 IgM RF augments the complement-binding activity of otherwise low-affinity IgG antibodies'6 and, by increasing the size of immune complexes also facilitates their removal from circulation by phagocytic cells.'7 Moreover, while IgM RF-mediated activation of the complement cascade may cause inflammation and destruction ofjoint tissue by binding to B-cell membrane IgG, they may also suppress antibody response.'8 Although RF production has been reported in syphilis for more than three decades'9'20 and seems to be one of the hallmarks of human2"22 and guinea-pig23 newborns congenitally infected with T. pallidum ssp. pallidum (T. pallidum), their role in
Correspondence: Dr K. Wicher, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201-0509, U.S.A.
548
RF in adoptively immune and T. pallidum challenged guinea-pigs the pathogenesis of the disease is still unknown. In both situations, however, circulating immune complexes generated during the course of syphilitic infection have been assumed to be the most plausible triggering mechanism of RF formation. In this report we present evidence of a relatively accelerated production of RF in inbred strain 2 and C4-deficient (C4D) guinea-pigs, after transfer of chancre-immune T cells followed by a challenging infection with T. pallidum. MATERIALS AND METHODS Animals Young (3-4 months old) male inbred strain 2 and homozygous CD4 guinea-pigs were the same as previously reported.24 Manipulations such as infection, injection, trial bleeding and lesion biopsy, were performed with the animals under sedation or general anaesthesia (Ketaset, Bristol Laboratories, Syracuse, NY). At the end of the experimental period the animals were killed by i.v. injection ofeuthanasia agent, Somlethol (American Hoechst, Somerville, NY). Animal procedures have been approved by the Institutional Animal Care and Use Committee of the Wadsworth Center for Laboratories and Research.
Adoptive transfer experiments Immunization of cell donors, purification and examination of T lymphocytes, and the procedure of adoptive transfer has been previously reported.24 The glass and nylon wool-purified lymphocytes were examined by indirect immunofluorescent test using murine anti-guinea-pig monoclonal antibody (mAb) 8BE6 (generously provided by Dr E. Shevach, National Institute of Health, Bethesda, MD)25 recognizing T cells, mAb 31 D2 (p-chain specific),26 reacting with B cells and mAb 342 specific for macrophages.27 The latter two reagents were kindly provided by Dr R. Burger (Robert Koch Institute, Berlin, Germany). The enriched T-cell population consisted of 95-97% T cells, 2-3% B cells and 1-2% macrophages, which is consistent with our previous report24 using polyclonal antiserum. Nylon wool-purified T lymphocytes obtained from T. pallidum-immune (TPI), T. phagedenis biotype Reiter-immune (TRI) and T. pallidum free-inflammatory testicular fluidimmune (ITFI) guinea-pigs, were injected i.v. in various concentrations (1 x 108 to 4 x 108) into 3-5-month-old syngeneic recipients. After 24 hr, experimental and control groups were infected i.d. with 0-1 ml of a suspension of freshly extracted T. pallidum (108 organisms). The guinea-pigs were closely examined for symptoms of infection and bled periodically. The presence of treponemes at the site of infection and lymphoid organs was determined in randomly selected animals by darkfield microscopy, silver staining and rabbit infectivity test (RIT) as described previously.24 Immunological assays Serum antibodies to T. pallidum were examined by the fluorescent treponemal antibody tests (FTA-ABS)28, and enzymelinked immunosorbent assay (ELISA).23 Circulating immune complexes were examined by the Clq solid-phase assay.29 RF was examined by the radioimmunoassay as described in ref. 30 but using reagents appropriate for the guinea-pig serum. Briefly, sera from 10 normal C4D guinea-pigs were pooled and IgG purified by fast protein liquid chromatography (FPLC) using a Protein G Superose HR 10/2 column (Pharmacia LKB Biotech-
549
nology, Uppsala, Sweden). The IgG Fc' fragment (< 25,000 MW) obtained after pepsin digestion was purified by similar procedure. A solution of 5 Mg/ml guinea-pig IgG Fc' fragment or IgG in carbonate buffer, pH 9-6 was added in 100-pl volume to triplicate wells of polyvinyl microtitre plates. Triplicate control wells received 100 p1 of 1% bovine serum albumin (BSA) in carbonate buffer. The plates were incubated overnight at 40, blocked for 1 hr with 1% BSA in phosphate-buffered saline (PBS) and washed three times with PBS containing Tween 20 (PBST). Test guinea-pig sera, diluted 1:100 with PBS containing 0 5% BSA, were added in I00-pl volume into triplicate wells and incubated overnight at room temperature. The plates were then washed three times with PBST and iodinated rabbit anti-guineapig IgM in 0 5% BSA-PBS containing 100,000 c.p.m. activity was then added to each well. The plates were incubated for 90 min at 37° followed by 30 min at 400. After exhaustive washing and drying, the wells were cut and activity counted in the ycounter. Counts > 100 were considered positive ( > 2SD) on the basis of examining 30 sera from healthy guinea-pigs of strains 2 and C4D. In a pilot experiment the RF activity in 10 sera from each strain of guinea-pigs was compared using C4D guinea-pigs IgG Fc' fragment and whole IgG as antigens. Each of the 20 sera was examined quantitatively. The results were almost identical in all sera examined; therefore whole IgG was used as antigen for all experiments. Antibodies to creatine kinase (CK) and fibronectin (Fn) were examined by the same radioimmunoassay as earlier described.29 For detection of antibodies to rabbit proteins by ELISA, a pool (n = 5) of normal rabbit serum (NRS) diluted 1: 1000 in carbonate buffer pH 9-6 was used as antigen for coating microplates (100 pl/well). The test sera were diluted 1:100. Bound antibodies were probed with alkaline phosphataseconjugated goat anti-guinea-pig IgG (Sigma, St Louis, MO) diluted 1: 1000. The reaction was stopped at 45 min by addition of 100 p1 of 3 N NaOH and read in a spectrophotometer. Based on examination of individual and pool of normal guinea-pig sera (Strain 2 and C4) OD values )0 120 (±2SD) were considered positive. RESULTS Clinical course of infection The course of infection in 15 (five strain 2 and 10 C4D) guineapig recipients of various concentrations of TPI-T cells differed significantly from that of 24 control animals receiving either TRI- or ITFI-T cells or no cells at all. In control animals darkfield positive lesions developed between 10 and 15 days post-infection, lasting from 19 to 40 days (strain 2) or from 65 to 86 days (C4D). However, in adoptively immune animals only three of 10 infused with < 2 x 108 TPI-T lymphocytes (two, strain 2 and one C4D) developed an atypical skin reaction, either at 19, 60 or 105 days post-infection containing treponemes detectable only by RIT. The lesions disappeared within 6 days. None of the recipient animals of 4 x 108 TPI-T cells developed lesions for the 5-month observation period.
Immune response In adoptively immune animals the immune response depended on the number of infused T cells. Thus, strain 2 guinea-pig
550
R. E. Baughn, V. Wicher & K. Wicher
recipients of 108 TPI-T cells responded with significantly (P
Production of rheumatoid factor in adoptively immune guinea-pigs after challenge with Treponema pallidum R. E. BAUGHN, V. WICHER* & K. WICHER* Departments of Microbiology and Immunology and of Dermatology, Baylor College of Medicine and Syphilis Research Laboratory, Veterans Administration Medical Center, Houston, Texas, and * Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York, U.S.A.
Acceptedfor publication 30 March 1992
SUMMARY Guinea-pigs of inbred strains 2 and C4D were infused with various concentrations (I x 108 to 4 x 108) of syngeneic nylon wool-purified Treponema pallidum-immune T lymphocytes (TPI-T) and challenged 24 hr later with virulent T. pallidum (108 organisms). The degree of protection depended on the number of infused T cells and was associated with an accelerated production of IgM rheumatoid factor (RF). Fully protected animals (4 x 108 TPI-T) did not produce treponemal antibodies or circulating immune complexes (CIC) but produced IgM RF detectable 10 days after infection. Partially protected animals ( < 2 x 108 TPI-T) produced, 30 days post-infection, relatively low levels of treponemal antibodies but high levels of CIC and RF. Control animals infused with 2 x 108 TPI-T lymphocytes but not infected with T. pallidum, when monitored for a period of 6 weeks, did not produce treponemal antibodies, CIC, or RF, excluding the possibility that IgM RF could be generated by the donor's B cells contaminating (circa 3%) the TPI-T lymphocytes. Moreover, unprotected syngeneic control animals infused, prior to infection, with T. phagedenis biotype Reiterimmune T cells or with T. pallidum-free testicular inflammatory fluid-immune T cells responded with increasing levels of treponemal antibodies; only a few animals produced RF and CIC 5 months after infection similarly to control guinea-pigs infected only. The production of RF in partially protected animals responding to infection with treponemal antibodies and CIC was apparently associated with the presence of the CIC; but the mechanism of RF production in fully protected animals in which no antibodies or CIC were detected is currently unknown. seems to be more than one pathway of production of these autoantibodies. It has been found that in natural or experimental conditions they may be generated by polyclonal B-cell activators.5 6 They were also present in hyperimmune mice7'8 and rabbits,9 "t where they were most likely elicited by immune complexes. This together with more recent studies'2" 3 provided ground for an assumption that precursors of RF-secreting cells are normal members of the B-cell repertoire.'4
INTRODUCTION The term rheumatoid factor (RF) defines a versatile group of autoantibodies of the IgM, IgG, IgA and even IgE classes reacting specifically with IgG Fc region, CH2 and CH3 domains, or with idiotypic determinants. Although by definition an autoantibody combines only with autologous antigen in its native form, this is not always the case with RF; in a broad sense, RF have been considered as autoantibodies, and it is within this context that they will be discussed in this report. In early studies detection of RF was thought to be limited to patients with rheumatoid arthritis, where they were ascribed a key role in the pathogenesis of joint inflammation.' Later, however, they were found in sera of patients with various nonarthritic disorders, associated with chronic inflammation and infection.2 Further studies revealed that RF can be produced as a consequence of kidney and skin transplantation3 and by lymphocytes from healthy humans or animals.4'5 Indeed, there
The functions ascribed to RF are no less diverse than the nature of the stimuli producing them. They seem to enhance the binding of an antibody to its antigen.'5 IgM RF augments the complement-binding activity of otherwise low-affinity IgG antibodies'6 and, by increasing the size of immune complexes also facilitates their removal from circulation by phagocytic cells.'7 Moreover, while IgM RF-mediated activation of the complement cascade may cause inflammation and destruction ofjoint tissue by binding to B-cell membrane IgG, they may also suppress antibody response.'8 Although RF production has been reported in syphilis for more than three decades'9'20 and seems to be one of the hallmarks of human2"22 and guinea-pig23 newborns congenitally infected with T. pallidum ssp. pallidum (T. pallidum), their role in
Correspondence: Dr K. Wicher, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201-0509, U.S.A.
548
RF in adoptively immune and T. pallidum challenged guinea-pigs the pathogenesis of the disease is still unknown. In both situations, however, circulating immune complexes generated during the course of syphilitic infection have been assumed to be the most plausible triggering mechanism of RF formation. In this report we present evidence of a relatively accelerated production of RF in inbred strain 2 and C4-deficient (C4D) guinea-pigs, after transfer of chancre-immune T cells followed by a challenging infection with T. pallidum. MATERIALS AND METHODS Animals Young (3-4 months old) male inbred strain 2 and homozygous CD4 guinea-pigs were the same as previously reported.24 Manipulations such as infection, injection, trial bleeding and lesion biopsy, were performed with the animals under sedation or general anaesthesia (Ketaset, Bristol Laboratories, Syracuse, NY). At the end of the experimental period the animals were killed by i.v. injection ofeuthanasia agent, Somlethol (American Hoechst, Somerville, NY). Animal procedures have been approved by the Institutional Animal Care and Use Committee of the Wadsworth Center for Laboratories and Research.
Adoptive transfer experiments Immunization of cell donors, purification and examination of T lymphocytes, and the procedure of adoptive transfer has been previously reported.24 The glass and nylon wool-purified lymphocytes were examined by indirect immunofluorescent test using murine anti-guinea-pig monoclonal antibody (mAb) 8BE6 (generously provided by Dr E. Shevach, National Institute of Health, Bethesda, MD)25 recognizing T cells, mAb 31 D2 (p-chain specific),26 reacting with B cells and mAb 342 specific for macrophages.27 The latter two reagents were kindly provided by Dr R. Burger (Robert Koch Institute, Berlin, Germany). The enriched T-cell population consisted of 95-97% T cells, 2-3% B cells and 1-2% macrophages, which is consistent with our previous report24 using polyclonal antiserum. Nylon wool-purified T lymphocytes obtained from T. pallidum-immune (TPI), T. phagedenis biotype Reiter-immune (TRI) and T. pallidum free-inflammatory testicular fluidimmune (ITFI) guinea-pigs, were injected i.v. in various concentrations (1 x 108 to 4 x 108) into 3-5-month-old syngeneic recipients. After 24 hr, experimental and control groups were infected i.d. with 0-1 ml of a suspension of freshly extracted T. pallidum (108 organisms). The guinea-pigs were closely examined for symptoms of infection and bled periodically. The presence of treponemes at the site of infection and lymphoid organs was determined in randomly selected animals by darkfield microscopy, silver staining and rabbit infectivity test (RIT) as described previously.24 Immunological assays Serum antibodies to T. pallidum were examined by the fluorescent treponemal antibody tests (FTA-ABS)28, and enzymelinked immunosorbent assay (ELISA).23 Circulating immune complexes were examined by the Clq solid-phase assay.29 RF was examined by the radioimmunoassay as described in ref. 30 but using reagents appropriate for the guinea-pig serum. Briefly, sera from 10 normal C4D guinea-pigs were pooled and IgG purified by fast protein liquid chromatography (FPLC) using a Protein G Superose HR 10/2 column (Pharmacia LKB Biotech-
549
nology, Uppsala, Sweden). The IgG Fc' fragment (< 25,000 MW) obtained after pepsin digestion was purified by similar procedure. A solution of 5 Mg/ml guinea-pig IgG Fc' fragment or IgG in carbonate buffer, pH 9-6 was added in 100-pl volume to triplicate wells of polyvinyl microtitre plates. Triplicate control wells received 100 p1 of 1% bovine serum albumin (BSA) in carbonate buffer. The plates were incubated overnight at 40, blocked for 1 hr with 1% BSA in phosphate-buffered saline (PBS) and washed three times with PBS containing Tween 20 (PBST). Test guinea-pig sera, diluted 1:100 with PBS containing 0 5% BSA, were added in I00-pl volume into triplicate wells and incubated overnight at room temperature. The plates were then washed three times with PBST and iodinated rabbit anti-guineapig IgM in 0 5% BSA-PBS containing 100,000 c.p.m. activity was then added to each well. The plates were incubated for 90 min at 37° followed by 30 min at 400. After exhaustive washing and drying, the wells were cut and activity counted in the ycounter. Counts > 100 were considered positive ( > 2SD) on the basis of examining 30 sera from healthy guinea-pigs of strains 2 and C4D. In a pilot experiment the RF activity in 10 sera from each strain of guinea-pigs was compared using C4D guinea-pigs IgG Fc' fragment and whole IgG as antigens. Each of the 20 sera was examined quantitatively. The results were almost identical in all sera examined; therefore whole IgG was used as antigen for all experiments. Antibodies to creatine kinase (CK) and fibronectin (Fn) were examined by the same radioimmunoassay as earlier described.29 For detection of antibodies to rabbit proteins by ELISA, a pool (n = 5) of normal rabbit serum (NRS) diluted 1: 1000 in carbonate buffer pH 9-6 was used as antigen for coating microplates (100 pl/well). The test sera were diluted 1:100. Bound antibodies were probed with alkaline phosphataseconjugated goat anti-guinea-pig IgG (Sigma, St Louis, MO) diluted 1: 1000. The reaction was stopped at 45 min by addition of 100 p1 of 3 N NaOH and read in a spectrophotometer. Based on examination of individual and pool of normal guinea-pig sera (Strain 2 and C4) OD values )0 120 (±2SD) were considered positive. RESULTS Clinical course of infection The course of infection in 15 (five strain 2 and 10 C4D) guineapig recipients of various concentrations of TPI-T cells differed significantly from that of 24 control animals receiving either TRI- or ITFI-T cells or no cells at all. In control animals darkfield positive lesions developed between 10 and 15 days post-infection, lasting from 19 to 40 days (strain 2) or from 65 to 86 days (C4D). However, in adoptively immune animals only three of 10 infused with < 2 x 108 TPI-T lymphocytes (two, strain 2 and one C4D) developed an atypical skin reaction, either at 19, 60 or 105 days post-infection containing treponemes detectable only by RIT. The lesions disappeared within 6 days. None of the recipient animals of 4 x 108 TPI-T cells developed lesions for the 5-month observation period.
Immune response In adoptively immune animals the immune response depended on the number of infused T cells. Thus, strain 2 guinea-pig
550
R. E. Baughn, V. Wicher & K. Wicher
recipients of 108 TPI-T cells responded with significantly (P
