Clin. exp. Immunol. (1992) 88, 461-466
Low-affinity antibodies against collagen type II produced by lymph node cells are associated with pathology in collagen-induced arthritis in rats J. RAHMAN, J. LOH & N. A. STAINES Immunology Section, King's College London, London, UK
(Acceptedfor publication 16 January 1992)
SUMMARY The relationship between the affinity of antibodies against type II collagen (CII) and arthritis was studied in rats immunized intradermally with bovine CII. Disease was associated with a higher mean titre of serum antibody and a lower mean functional antibody affinity (determined in a chaotropic dissociation assay) against both the immunizing bovine CII and homologous autoantigenic rat CII in comparison with the response in immunized rats that did not develop disease. The functional affinity of the antibodies present in the serum was found to correlate with that of antibodies produced in culture by cells from the lymph nodes draining the site of immunization with collagen. The reduction in mean functional affinity in the anti-collagen response may be the result of the increased production of antibodies of the lowest affinity and a consequent broadening of the affinity heterogeneity. It is proposed that production of low-affinity antibodies in the lymph nodes draining the site of immunization with collagen is important in the pathogenesis of collagen-induced arthritis in rats. Keywords collagen type II antibody affinity lymph nodes arthritis INTRODUCTION Immune sensitization to collagen type II (CII) may lead to the development of polyarthritic disease following injection of CII in either Freund's incomplete adjuvant (FIA) into rats [1] or Freund's complete adjuvant (FCA) into mice, respectively [2]. Several lines of evidence show that T cells are involved in the initiation or maintenance of joint destruction or both; athymic rats are refractory to disease induction by active immunization with CII [3]; passive treatment with anti-thymocyte antiserum decreases the incidence of disease, serum antibody titres and delayed-type hypersensitivity to CII in rats [4] and cells of CIIspecific T cell clones or lines can induce disease in normal or irradiated rats and mice [5]. The involvement of B cells in the disease process is evident by the fact that passive transfer of either complement-fixing IgG antibodies against CII or hybridomas secreting anti-CII MoAbs may induce arthritic symptoms in naive recipients [6-8]. The presence of increased anti-CII antibody levels in the serum of arthritic animals compared with healthy collagen-immunized counterparts [9-11], as well as differences in the levels of antibodies of the IgG2 subclasses in arthritic and non-arthritic JR. present address: Department of Neuropathology, Institute of Psychiatry, Denmark Hill, London, UK. Correspondence: Professor Norman A. Staines, Immunology Section, King's College London, Campden Hill Road, London W8 7AH, UK.
461
rats [12] and mice [11,13] and in mice and rats suppressed for disease induction by gavage with CII [14,15], implies that both quantitative and qualitative differences in the antibody response influence disease susceptibility. In view of the fact that antibody affinity is an important parameter of the immune response, and plays a fundamental role in biological effector functions such as immune complex-dependent pathology [16,17], we have studied the affinity of anti-CII antibodies produced after immunization of rats with CII. There are problems inherent in measuring the affinity of polyclonal antibodies reacting with multi-valent antigens, so for estimating the avidity (which we here refer to as the mean functional affinity) of anti-CII antibodies we used an ELISA that incorporated the chaotropic agent diethylamine (DEA) in the diluent to inhibit the binding of antibodies to the solid-phase antigen. This method for measuring the functional affinity of antibodies depends on the fact that antibodies of relatively high affinity are the more resistant to dissociation by DEA: it has been shown to rank the affinities of MoAbs to DNP and polyclonal antibodies to HSA in the same orders as the ammonium sulphate precipitation method of Steward & Petty [18]. We have previously shown that a high level of antibody production by cells from the lymph nodes draining the injection site was necessary for the subsequent development of disease in animals immunized with CII [19]. We report here that arthritic rats have antibodies of lower mean functional affinity than collagen-immunized rats that remain healthy, and these lowaffinity antibodies are produced in the lymph nodes draining the site of immunization.
462
J. Rahman, J. Loh & N. A. Staines MATERIALS AND METHODS
Animals Inbred female WA/KIR rats aged 3-4 months were obtained from the Kennedy Institute, London, UK, and were maintained in solid-bottomed plastic cages with wood shavings as litter with rat diet 86 (Grain Harvesters, Kent, UK) and water provided ad libitum. Immunization and induction of arthritis Soluble bovine CII (BCII), prepared from nasal septa [10,20], was dissolved in 0 1 M acetic acid, emulsified in FIA and injected intradermally in a single site in the suprascapular region in doses described below. Controls were normal rats matched for sex and age, some of which were injected with FIA. Animals were weighed regularly and the severity of arthritis in each paw was scored on a scale of 0-4 based on increasing peri-articular erythema and swelling of hard and soft tissues [21].
antibody dilutions at an OD of 10 were read off and the percentage of shift values calculated from their reciprocal values (titres): Serum titre without DEA-Serum titre with DEA Serum titre without DEA
100%
Statistical analysis Differences between group means were analysed using Student's two-tailed t-test and regression analysis was performed by calculating Pearson's product moment correlation coefficient.
RESULTS
Parameters of the DEA dissociation ELISA The dose-response curve for a pooled serum standard [19] was shifted progressively to the left in the presence of increasing concentrations of DEA in the CII ELISA (Fig. la). At an OD of 1 0 the interpolated serum dilution in the absence of DEA was defined as the effective total binding of specific antibody. sermanti-C! antiboy levelsInterpolated serum dilution values in the Estimatinof of different the presence perent to propnae e onverted concena ser Blood samples were obtained from the ventral tail artery and DEA concentrations were converted to the appropriate percent0'C Thelevls antibodies ~~~~~~~~age serum atiboies The* levels oof serm stored Ct sera were serawerestoed att - 200C. a negative linear of this.th These a The derived data because ofed ( ati ofshowed crelon reacting with BCII and rat CII (RCII) (prepared in the same way Because a DEA (Fg. with the cer concentration correlation as BCII but from the Swarm rat chondrosarcoma) were concentration of 12-5 mm DEA produced a reduction of about xprsse asthe nd the he results esuts expressed detrmied a LSA [10,20] 10,0] and as the 50% in the binding of the antibody standard to the solid phase, in ann E ELISA determined ftinal ty wee made al sbe eimatesof ofmean reciprocal of the relevant dilutions of serum corresponding to ~all mean functional affinityt were made estimates subsequent the mid point-titration values, in the presence of 12 5 mm DEA. the mid point-titration values.
tIb).
Culture of lymph node cells and estimation of antibody produced in vitro Animals were killed 23 days after immunization, dipped in 70% ethanol and their brachial lymph nodes were removed asceptically. Lymph nodes were teased gently with fine forceps and the released cells suspended and washed three times (1500 g for 10 min each at 40C) in culture medium (RPMI 1640 containing 2% heat-inactivated fetal calf serum (FCS), 12 5 mM HEPES, 100 U/ml penicillin and 100 ,ug/ml streptomycin) (GIBCO, Paisley, UK). Cell cultures of 20 x 106 viable nucleated cells in 10 ml culture medium were maintained in 50 ml tissue culture flasks (Nunc, Roskilde, Denmark) for 7 days at 370C in 5% CO2 in air and 100% relative humidity. After centrifugation (1500 g for 10 min at 40C) the supernatant was lyophilized, reconstituted in 0 4 ml distilled water and then dialysed overnight at 40C against PBS. Anti-CII antibody levels were determined by ELISA in an identical manner to that described for serum
samples. Determination offunctional antibody affinity The functional affinity of anti-CII antibodies was estimated by the influence on antigen-antibody association in the CII-ELISA of the chaotropic agent DEA [22]. Briefly, serial dilutions of anti-CII antibodies were made in PBS containing 1 % casein and 0 05% Tween 20 in the presence and absence of DEA in the wells of ELISA plates coated with BCII or RCII. Plates were incubated for 2 h at 40C and were then processed in the normal way. Results were calculated as follows; the mean optical density (OD) readings obtained for quadruplicate dilutions of anti-CII antisera in the presence and absence of DEA were plotted against the corresponding serum dilution. The interpolated
Mean functional affinity of serum anti-BCII antibodies and its relationship to induced disease Twelve days after immunization with 300 yg of CII in FIA (a dose sufficient to induce disease in only a proportion of the group) some animals started to exhibit clinical symptoms of disease. By 22 days, 64% of the animals were arthritic. Antibody reactive with BCII was observed in the serum of all immunized animals. Antibodies in sera from arthritic rats had a significantly higher mean titre and a significantly lower mean functional affinity compared with those in sera from their immunized but non-arthritic counterparts (Table 1).
High-affinity anti-BCII antibodies are not selectively removed from the circulation of arthritic animals The relationship between the low-affinity anti-CII antibodies in serum and arthritic disease in the experiment above could have been due to a selective adsorption of high-affinity antibodies by autoantigen exposed in damaged tissue. However, study of antibodies made by cells in culture implied otherwise. Rats were immunized with 370 pg CII in FIA (so as to induce disease in only a proportion of the group), and 23 days after immunization animals were terminally bled and cells from lymph nodes draining the sites of immunization were prepared from arthritic and non-arthritic animals. Cells were maintained in culture and the reactivity of the antibodies they secreted were determined against BCII as described. Cultures of lymphoid cells from arthritic rats contained significantly more antibody and of a lower affinity than those antibodies in cultures of cells from non-arthritic rats (Table 2). The mean functional affinity of anti-CIT antibodies made by cells in culture was reflected in the mean functional affinity of antibody in the sera (Fig. 2). In accord with previous observations [19], cells from normal rats or
Low-affinity antibodies against collagen type II (a )
463
Table 1. Serum titres and mean functional affinity of serum antibodies in arthritic and non-arthritic rats immunized with bovine collagen type II
2-5
(CII) 2-0
; I\ \\\\
: 1.5 o
Functional affinity (% shift)
Serum titre
o 0-o 0-5
0.0
0.1
1.0
Non-arthritic rats
Arthritic rats
3-0
2-0
4-0
50
Serum dilution (tog 10)
Serum titre
Functional affinity (% shift)
47
9222
61
6577
5034
62
12656
51
8132
4713
24
13 192 7047 6281 6456 11493
53 56
4669
41
39 47 71 65
5287
27
8032
68
6780+3374
38+ 12
Mean + s.d. 8321 +2617
58+ 10
lb
Fourteen animals were injected intradermally in the suprascapular
2-0 _
region with 300 pg ofbovine CII in Freund's incomplete adjuvant at day 0. Serum titres and antibody affinity were determined by ELISA with
*
3 1-7 >1.4
\
0
bovine CII as the target antigen. The arthritic animals had by day 22 serum antibody titres (P < 005). Sera from arthritic animals exhibited a greater dissociation in the presence of 12 5 mm DEA, i.e. were of lower functional affinity (P
Low-affinity antibodies against collagen type II produced by lymph node cells are associated with pathology in collagen-induced arthritis in rats J. RAHMAN, J. LOH & N. A. STAINES Immunology Section, King's College London, London, UK
(Acceptedfor publication 16 January 1992)
SUMMARY The relationship between the affinity of antibodies against type II collagen (CII) and arthritis was studied in rats immunized intradermally with bovine CII. Disease was associated with a higher mean titre of serum antibody and a lower mean functional antibody affinity (determined in a chaotropic dissociation assay) against both the immunizing bovine CII and homologous autoantigenic rat CII in comparison with the response in immunized rats that did not develop disease. The functional affinity of the antibodies present in the serum was found to correlate with that of antibodies produced in culture by cells from the lymph nodes draining the site of immunization with collagen. The reduction in mean functional affinity in the anti-collagen response may be the result of the increased production of antibodies of the lowest affinity and a consequent broadening of the affinity heterogeneity. It is proposed that production of low-affinity antibodies in the lymph nodes draining the site of immunization with collagen is important in the pathogenesis of collagen-induced arthritis in rats. Keywords collagen type II antibody affinity lymph nodes arthritis INTRODUCTION Immune sensitization to collagen type II (CII) may lead to the development of polyarthritic disease following injection of CII in either Freund's incomplete adjuvant (FIA) into rats [1] or Freund's complete adjuvant (FCA) into mice, respectively [2]. Several lines of evidence show that T cells are involved in the initiation or maintenance of joint destruction or both; athymic rats are refractory to disease induction by active immunization with CII [3]; passive treatment with anti-thymocyte antiserum decreases the incidence of disease, serum antibody titres and delayed-type hypersensitivity to CII in rats [4] and cells of CIIspecific T cell clones or lines can induce disease in normal or irradiated rats and mice [5]. The involvement of B cells in the disease process is evident by the fact that passive transfer of either complement-fixing IgG antibodies against CII or hybridomas secreting anti-CII MoAbs may induce arthritic symptoms in naive recipients [6-8]. The presence of increased anti-CII antibody levels in the serum of arthritic animals compared with healthy collagen-immunized counterparts [9-11], as well as differences in the levels of antibodies of the IgG2 subclasses in arthritic and non-arthritic JR. present address: Department of Neuropathology, Institute of Psychiatry, Denmark Hill, London, UK. Correspondence: Professor Norman A. Staines, Immunology Section, King's College London, Campden Hill Road, London W8 7AH, UK.
461
rats [12] and mice [11,13] and in mice and rats suppressed for disease induction by gavage with CII [14,15], implies that both quantitative and qualitative differences in the antibody response influence disease susceptibility. In view of the fact that antibody affinity is an important parameter of the immune response, and plays a fundamental role in biological effector functions such as immune complex-dependent pathology [16,17], we have studied the affinity of anti-CII antibodies produced after immunization of rats with CII. There are problems inherent in measuring the affinity of polyclonal antibodies reacting with multi-valent antigens, so for estimating the avidity (which we here refer to as the mean functional affinity) of anti-CII antibodies we used an ELISA that incorporated the chaotropic agent diethylamine (DEA) in the diluent to inhibit the binding of antibodies to the solid-phase antigen. This method for measuring the functional affinity of antibodies depends on the fact that antibodies of relatively high affinity are the more resistant to dissociation by DEA: it has been shown to rank the affinities of MoAbs to DNP and polyclonal antibodies to HSA in the same orders as the ammonium sulphate precipitation method of Steward & Petty [18]. We have previously shown that a high level of antibody production by cells from the lymph nodes draining the injection site was necessary for the subsequent development of disease in animals immunized with CII [19]. We report here that arthritic rats have antibodies of lower mean functional affinity than collagen-immunized rats that remain healthy, and these lowaffinity antibodies are produced in the lymph nodes draining the site of immunization.
462
J. Rahman, J. Loh & N. A. Staines MATERIALS AND METHODS
Animals Inbred female WA/KIR rats aged 3-4 months were obtained from the Kennedy Institute, London, UK, and were maintained in solid-bottomed plastic cages with wood shavings as litter with rat diet 86 (Grain Harvesters, Kent, UK) and water provided ad libitum. Immunization and induction of arthritis Soluble bovine CII (BCII), prepared from nasal septa [10,20], was dissolved in 0 1 M acetic acid, emulsified in FIA and injected intradermally in a single site in the suprascapular region in doses described below. Controls were normal rats matched for sex and age, some of which were injected with FIA. Animals were weighed regularly and the severity of arthritis in each paw was scored on a scale of 0-4 based on increasing peri-articular erythema and swelling of hard and soft tissues [21].
antibody dilutions at an OD of 10 were read off and the percentage of shift values calculated from their reciprocal values (titres): Serum titre without DEA-Serum titre with DEA Serum titre without DEA
100%
Statistical analysis Differences between group means were analysed using Student's two-tailed t-test and regression analysis was performed by calculating Pearson's product moment correlation coefficient.
RESULTS
Parameters of the DEA dissociation ELISA The dose-response curve for a pooled serum standard [19] was shifted progressively to the left in the presence of increasing concentrations of DEA in the CII ELISA (Fig. la). At an OD of 1 0 the interpolated serum dilution in the absence of DEA was defined as the effective total binding of specific antibody. sermanti-C! antiboy levelsInterpolated serum dilution values in the Estimatinof of different the presence perent to propnae e onverted concena ser Blood samples were obtained from the ventral tail artery and DEA concentrations were converted to the appropriate percent0'C Thelevls antibodies ~~~~~~~~age serum atiboies The* levels oof serm stored Ct sera were serawerestoed att - 200C. a negative linear of this.th These a The derived data because ofed ( ati ofshowed crelon reacting with BCII and rat CII (RCII) (prepared in the same way Because a DEA (Fg. with the cer concentration correlation as BCII but from the Swarm rat chondrosarcoma) were concentration of 12-5 mm DEA produced a reduction of about xprsse asthe nd the he results esuts expressed detrmied a LSA [10,20] 10,0] and as the 50% in the binding of the antibody standard to the solid phase, in ann E ELISA determined ftinal ty wee made al sbe eimatesof ofmean reciprocal of the relevant dilutions of serum corresponding to ~all mean functional affinityt were made estimates subsequent the mid point-titration values, in the presence of 12 5 mm DEA. the mid point-titration values.
tIb).
Culture of lymph node cells and estimation of antibody produced in vitro Animals were killed 23 days after immunization, dipped in 70% ethanol and their brachial lymph nodes were removed asceptically. Lymph nodes were teased gently with fine forceps and the released cells suspended and washed three times (1500 g for 10 min each at 40C) in culture medium (RPMI 1640 containing 2% heat-inactivated fetal calf serum (FCS), 12 5 mM HEPES, 100 U/ml penicillin and 100 ,ug/ml streptomycin) (GIBCO, Paisley, UK). Cell cultures of 20 x 106 viable nucleated cells in 10 ml culture medium were maintained in 50 ml tissue culture flasks (Nunc, Roskilde, Denmark) for 7 days at 370C in 5% CO2 in air and 100% relative humidity. After centrifugation (1500 g for 10 min at 40C) the supernatant was lyophilized, reconstituted in 0 4 ml distilled water and then dialysed overnight at 40C against PBS. Anti-CII antibody levels were determined by ELISA in an identical manner to that described for serum
samples. Determination offunctional antibody affinity The functional affinity of anti-CII antibodies was estimated by the influence on antigen-antibody association in the CII-ELISA of the chaotropic agent DEA [22]. Briefly, serial dilutions of anti-CII antibodies were made in PBS containing 1 % casein and 0 05% Tween 20 in the presence and absence of DEA in the wells of ELISA plates coated with BCII or RCII. Plates were incubated for 2 h at 40C and were then processed in the normal way. Results were calculated as follows; the mean optical density (OD) readings obtained for quadruplicate dilutions of anti-CII antisera in the presence and absence of DEA were plotted against the corresponding serum dilution. The interpolated
Mean functional affinity of serum anti-BCII antibodies and its relationship to induced disease Twelve days after immunization with 300 yg of CII in FIA (a dose sufficient to induce disease in only a proportion of the group) some animals started to exhibit clinical symptoms of disease. By 22 days, 64% of the animals were arthritic. Antibody reactive with BCII was observed in the serum of all immunized animals. Antibodies in sera from arthritic rats had a significantly higher mean titre and a significantly lower mean functional affinity compared with those in sera from their immunized but non-arthritic counterparts (Table 1).
High-affinity anti-BCII antibodies are not selectively removed from the circulation of arthritic animals The relationship between the low-affinity anti-CII antibodies in serum and arthritic disease in the experiment above could have been due to a selective adsorption of high-affinity antibodies by autoantigen exposed in damaged tissue. However, study of antibodies made by cells in culture implied otherwise. Rats were immunized with 370 pg CII in FIA (so as to induce disease in only a proportion of the group), and 23 days after immunization animals were terminally bled and cells from lymph nodes draining the sites of immunization were prepared from arthritic and non-arthritic animals. Cells were maintained in culture and the reactivity of the antibodies they secreted were determined against BCII as described. Cultures of lymphoid cells from arthritic rats contained significantly more antibody and of a lower affinity than those antibodies in cultures of cells from non-arthritic rats (Table 2). The mean functional affinity of anti-CIT antibodies made by cells in culture was reflected in the mean functional affinity of antibody in the sera (Fig. 2). In accord with previous observations [19], cells from normal rats or
Low-affinity antibodies against collagen type II (a )
463
Table 1. Serum titres and mean functional affinity of serum antibodies in arthritic and non-arthritic rats immunized with bovine collagen type II
2-5
(CII) 2-0
; I\ \\\\
: 1.5 o
Functional affinity (% shift)
Serum titre
o 0-o 0-5
0.0
0.1
1.0
Non-arthritic rats
Arthritic rats
3-0
2-0
4-0
50
Serum dilution (tog 10)
Serum titre
Functional affinity (% shift)
47
9222
61
6577
5034
62
12656
51
8132
4713
24
13 192 7047 6281 6456 11493
53 56
4669
41
39 47 71 65
5287
27
8032
68
6780+3374
38+ 12
Mean + s.d. 8321 +2617
58+ 10
lb
Fourteen animals were injected intradermally in the suprascapular
2-0 _
region with 300 pg ofbovine CII in Freund's incomplete adjuvant at day 0. Serum titres and antibody affinity were determined by ELISA with
*
3 1-7 >1.4
\
0
bovine CII as the target antigen. The arthritic animals had by day 22 serum antibody titres (P < 005). Sera from arthritic animals exhibited a greater dissociation in the presence of 12 5 mm DEA, i.e. were of lower functional affinity (P
