CELLULAR
IMMUNOLOGY
142,326-337 (1992)
The Effect of Anti-adhesion Molecule Antibody on the Development of Collagen-Induced Arthritis KIICHI KAKIMOTO, TAKASHI NAKAMURA,* KOJI ISHII, TOHRU TAKASHI,~ HIROSHI IIGou,t HIDEO YAGITA,~ Kou OKUMURA,$ AND KAORU ONOUE Department of Biochemistry, Institute for Medical Immunology, Kumamoto University Medical School; Honjo 2-2-1, Kumamoto 860, Japan; *Department of Radiology, Nagasaki University School of Dentistry; f Tokyo R & D Center, Daiichi Pharmaceutical Company Ltd.; and *Department cf Immunology, Juntendo University School of Medicine Received January 13, 1992; accepted March 6, 1992 In order to study how inflammatory cells including autoimmune lymphocytes interact with each other to develop collagen-induced arthritis (CIA), we injected monoclonal antibodies against mouse LFA-1 and ICAM- into DBA/l mice immunized with type II collagen (CII). Both antibodies suppressedthe development of CIA. Theseantibodies showedno effecton anti-C11antibody response,although they both significantly suppressedDTH response.It was suggestedthat antiadhesionmolecule antibodies suppressCIA mainly through their effect on cell-mediated immunity, without affecting humoral immunity under the conditions used. o 1992 Academic PKSS, IIK.
INTRODUCTION Recently, progress has been made in defining the molecules involved in the interactions of various cells including lymphoid and nonlymphoid cells (1,2). It was shown that cell-adhesion molecules play important roles in immune response and inflammation (3, 4). It is known that adhesion molecules are expressed on various cells composing a synovial microenvironment in the resting state. These molecules are upregulated in responseto the cytokines released(5). Among them, lymphocyte function-associated antigen- 1 (LFA- 1)’ which is a member of integrin family and its ligand, intercellular adhesion molecule- 1 (ICAM- 1) of the immunoglobulin superfamily, work as one of the main pairs of molecules to mediate the leucocyte adhesion process (6). While the events that lead to the initiation of the cascade of immune responses resulting in chronic proliferative synovitis in rheumatoid arthritis (RA) are not known, it seemsthat autoreactive T cells play an important role in the pathogenesis of this disease(7,8). Although the specific antigen that triggers the autoreactive T cell response in RA remains unknown, several animal models have been described in which defined antigens such as bacterial cell wall peptidoglycan which induces adjuvant arthritis (9) and connective tissue protein which produces type II collagen (CII)-induced arthritis i Abbreviations used: CIA, type II collagen-induced arthritis; CII, type II collagen; DTH, delayed type hypersensitivity; ICAM- 1, intercellular adhesion molecule-1; LFA- 1, lymphocyte function-associatedantigen1; MDP, muramyl dipeptide; REI, radiometric ear index. 326 0008-8749/92 $5.00 Copyright 0 1992 by Academic Press., Inc. All rights of reproduction in any form reserved.
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(CIA) (10, 11) and proteoglycan-induced arthritis (12) were used. CIA, in particular, is well known for its clinical and pathological similarity to RA ( 10). A cell-mediated autoimmune reaction, as well as a humoral one, appearsto play a critical role in CIA. As demonstrated by us ( 13) and others ( 14) CIA can be induced by passive transfer of the CII-specific T cell line. In this study, we investigated the role of the adhesion molecules in murine CIA by using monoclonal antibodies to mouse LFA- 1 and ICAM- 1. It was found that injection of anti-adhesion molecule antibodies suppressedCIA possibly by affecting cell-mediated immunity against CII. MATERIALS AND METHODS Animals. Female DBA/l mice, 7-10 weeks of age, were purchased from the Seiwa Institute for Experimental Animals (Fukuoka, Japan). CZZ.Human CII was purified from costal cartilage according to Miller’s method ( 15). Briefly, native CII was solubilized by limited pepsin digestion of costal cartilage and purified by DEAE cellulose chromatography and fractional salt precipitation. The purity of CII wasassessedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( 16) and contamination-free preparations were used. Monoclonaf antibodies. Rat monoclonal antibodies (MoAb) against LFA- 1 (KBA; IgGz,) ( 17) and ICAM- 1 (YN l/ 1.7;IgGZb)( 18)were purified by the useof caprylic acid (19) from the ascitesof ICR nu/nu mice transplanted intraperitoneally with each hybridoma cell. Normal rat IgG wasprepared from rat serum using DEAE ion exchange chromatography and used as control immunoglobulin (Ig) throughout the experiment. Induction ofarthritis. The CIA was induced by the intracutaneous immunization of 200 pg per mouse of human CII emulsified in Freund’s complete adjuvant followed by the booster injection of the sameamount of CII emulsified into Freund’s incomplete adjuvant 3 weeks later. Peptidoglycan/muramyl dipeptide (MDP)-induced arthritis was induced in the mice by the single intravenous injection of 100 pg MDP per mouse as we reported previously (20, 2 1). MDP was kindly donated by Dr. Atsuro Inoue of Daiichi Pharmaceutical Co., Ltd. This arthritis is induced by acute inflammatory cell infiltration (polymorphonuclear leukocytes and mononuclear cells) which seemsto be induced by the chemotactic activity of degraded components of the complements activated by peptidoglycan through an alternate pathway (20, 2 1). Administration of antibodies. One hundred micrograms per mouse of MoAb or normal control Ig was injected intraperitoneally starting on the day of immunization with CII (Day 0) and thereafter injected every other day for 2 weeks as well as Days 2 I, 24, and 28 (10 times for a total of 1 mg Ig/mouse). In the case of MDP-induced arthritis, a single injection of Ig was done only on the day of MDP immunization. Titration of anti-CII antibody. Antibody against CII was measured by enzymelinked immunosorbent assay(ELISA) (22). Alkaline phosphatase-conjugatedgoat antimouse IgG antibody (Tago Inc., Burlingame, CA) was used as the second antibody, Optimally diluted sera collected on Day 35 were assayed and the absorbance was determined by an ELISA microreader (Dynatech Laboratories Inc., Alexandria, VA), Measurement of DTH responseto CII. Delayed type hypersensitivity (DTH) was measured by ear skin testing on Day 21 using a 50-~1Hamilton syringe as described by Farmer et al. (23) and the responsewas read at 48 hr. The DTH responsewas also quantified by a modified method of radiometric ear assay(24). Briefly, CII wasdissolved
328
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ET AL.
at a concentration of one mg/ml in 0.15 M NaCI, 0.02 M Tris-HCI buffer, pH 8.0, by stirring for 24 hr. To radiolabel the dividing cells that accumulate at the site of DTH, mice were injected subcutaneously with [3H]thymidine at a dose of 0.4 Ci/body wt (g). Twenty-four hours later, 5 ~1of the collagen solution was injected intradermally into the right ear, using a Hamilton microsyringe with a 27-gaugeneedle. As a control, the buffer solvent alone was injected into the left ear in the same manner. After an additional 24 hr, the mice were killed and both ears dissected, and digested in NCS tissue solubilizer (Amersham Corp., Arlington Heights, IL). The radioactivity present in each ear was counted. The radiometric ear index (REI) was expressedas the ratio of cpm in the challenged ear vs the control ear. In vitro eflect of MoAb on the proliferative responseof lymphocytes to CII. The effect of MoAb was assessedon the sensitized lymph node cells collected on Day 14 from DBA/ 1 mice after immunization with human CII. Lymphocytes that were suspended at 2 X 1O4cells/ml in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Grand Island, NY) supplemented with 1% autologous mouse serum, 50 units/ml penicillin, 50 pg/ml streptomycin, and 5 X lop5 M 2-mercaptoethanol (2-ME) were incubated in flat-bottomed microtiter plates (Falcon 3072, Becton-Dickinson, Mountain View, CA) in triplicate wells. Each well was stimulated with 40 pg/ml of CII together with various concentrations of MoAb and cultivated for 4 days in 10%C02-air. Each well was pulsed with 0.2 &i of [3H]thymidine (sp act 5 &i/nmol, RCC, Amersham, England) for the last 9 hr. The cells were then harvested on fiberglass filters and the incorporation of thymidine was measured in a liquid scintillation counter. The proliferative response was expressed as the mean cpm. The effect of MoAb on the CIIinduced proliferative response of the CII-specific T cell line (Thy-l+, Lyt-l+, Lyt-2-, L3T4+) was also studied in the same way except for addition of 1 X lo6 syngeneic spleen cells (3000-R irradiated) as accessorycells (ACs) to the culture. As for the CIIspecific T cell line capable of inducing arthritis by passive transfer in DBA/l mice, we have reported elsewhere (23). Clinical evaluation of arthritis. The animals were graded for severity of arthritis according to the method reported by Wood et al. (25). The lesions of the extremities distal to the elbow or knee were graded on a scale of 0 to 4 based on the number of joints involved and the degreeof erythema and swelling. The maximal score was 12 because lesions on the injected foot were not included in the numerical score. The soft x-ray examination was carried out using Sofron (type SRO M-50) (Sohken, Tokyo, Japan). Histological examination of arthritis. Mice were killed and exanguinated. Joints (knees, ankles, feet) were removed and fixed in formalin, decalcified with ethylenediamine tetraacetic acid (EDTA) (Sigma Chemical Co., St. Louis, MO), and stained with hematoxylin and eosin. Statistics. Statistical significance was analyzed by the Student’s t test. RESULTS The e&ct of MoAbs on incidence and severity of CIA. As shown in Fig. 1a, the mice which were treated with control Ig developed CIA with 100% incidence. In contrast, the mice which were injected with KBA (anti-LFA-1) or YN1/1.7 (anti-ICAM-1) developed the arthritis with an incidence of 50 and 75%, respectively. Injection of MoAbs affected not only the incidence but also the severity of arthritis. Thus, Fig. 1b reveals that treatment with KBA or YN l/ 1.7 suppressedthe severity of arthritis significantly
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FIG. I The effect of MoAbs on incidence and severity of CLA. DBA/I mice (10 mice per group) were boosted with human CII on Day 2 I. Control Ig. anli-LFA- I (KBA), or anti-ICAM- 1 (YN l/ 1.7, abbreviated as YN-I) MoAb was injected ip every other day, amounting to a total of 1 mg per mouse. Details are described under Materials and Methods. (a) The effect on incidence. Differences between control Ig and KBA, and between control Ig and YN l/ I .7 are statistically significant (P < 0.0 1). (b) The effect on severity. Differencesbetween control Ig (normal rat IgG) and KBA and between control Ig and YNlil.7 are statistically significant (P i 0.01 and P < 0.05, respectively).
with the control Ig injection. Figure 2 depicts the soft x-ray findings of these animals. Destructive changes of joints and bone cortex are seen in untreated mice (Fig. 2a) and mice treated with control Ig (Fig. 2b). On the other hand, the joints and bones of mice treated with KBA (Fig. 2c) or YNl/ 1.7 (Fig. 2d) showed slight or only marginal changes. Figure 3 shows the histological findings of these joints. The mice injected with control Ig showed inflammatory cell infiltration into the joint space, synovium, and surrounding tissues, the typical findings of chronic proliferative synovitis (Fig. 3b), compared with the untreated normal mice (Fig. 3a). In contrast, mice treated with KBA or YN1/1.7 which showed suppressed development of arthritis revealed only slight, if any, infiltration of inflammatory cells (Figs. 3c and 3d). The effect of MoAbs on anti-CII humoral and cell-mediated immunity. Since the immune response against CII is involved in the pathogenesis of CIA, we assessedthe level of anti-C11 antibody in the serum of these mice and measured the cell-mediated immunity against CII by means of DTH reaction in the ear. As seen in Fig. 4a, no significant difference of antibody production was observed among control Ig-, KBA-, and YN I / 1.7-injected animals, whereas a significant difference in the DTH response to CII was found between the control Ig- and MoAb-injected groups (Fig. 4b). The DTH response of mice treated with KBA or YN1/1.7 was suppressed to 0.39 -t 0.02 and 0.42 -+ 0.01 mm, respectively, compared with 0.60 -t 0.02 mm in control mice. This result was reproducible and confirmed by radiometric ear assay. Thus, REI was 1.56 -+ 0.2 for the control Ig group, 0.89 t 0.17 for the KBA group, and 0.93 t 0.19 for the YN l/1.7 group. as compared
330
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FIG. 2. The effect of MoAbs on CIA (soft x-ray findings). Note that the destructive change seen in mice injected with control Ig (b) was largely suppressed in both KBA-treated (c) and YN1/1.7-treated (d) mice. The same paws which appear in Fig. 3 are shown. (a) This shows the findings of a normal untreated mouse.
The efict of MoAbs on T cell response to CII in vitro. In order to study the effect of MoAbs on cell-mediated immunity against CII in more detail, we examined the effect of MoAbs on the in vitro proliferative response of CII-sensitized lymph node cells as well as the CII-specific T cell line stimulated with CII. As shown in Fig. 5, both of the antibodies suppressed the proliferative response of lymph node cells by
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FIG. 2-Continued
about 50% at 10 rig/ml of MoAb (Fig. 5a). The proliferative response of the T cell line was also suppressed strongly by KBA, and to a lesser extent by YN l/ 1.7 (Fig. 5b) at 10 pug/ml of MoAbs. The efict of MoAbs on MDP-induced arthritis. Next, we studied the effect of the MoAbs on MDP-induced acute arthritis. As shown in Fig. 6, all mice treated with
332
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FIG. 3. The effect of MoAbs on CIA (histological findings). The finding of chronic proliferative synovitis characteristic of CIA is seen in mice treated with control Ig (b). Only slight inflammatory cell infiltration was seen in mice which showed arthritis suppressed by the KBA (c) and YN1/1.7 (d). (a) This shows the findings of a normal untreated mouse. Hematoxylin-eosine staining; X90 (a, b) and X I80 (c, d). Mice were sacrificed on Day 60.
control Ig developed this acute arthritis within 12 hr after MDP injection and reached maximum severity at 24-36 hr. The arthritis then subsided in 3-5 days after injection as reported previously in untreated mice (2 1). The injection of KBA or YNl/l.7
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3-Contimred
suppressed development of the arthritis dramatically in terms of the incidence as well as the severity: KBA suppressed the arthritis completely and YN1/1.7 lowered the incidence by 80% (Fig. 6). DISCUSSION CIA is a useful and unique experimental model of human RA (10). It has been established that autoimmunity to CII is involved in the development of CIA (10, 26),
334
KAKIMOTO
ET AL.
x
0 l
I
1
-rI p
IMMUNOLOGY
142,326-337 (1992)
The Effect of Anti-adhesion Molecule Antibody on the Development of Collagen-Induced Arthritis KIICHI KAKIMOTO, TAKASHI NAKAMURA,* KOJI ISHII, TOHRU TAKASHI,~ HIROSHI IIGou,t HIDEO YAGITA,~ Kou OKUMURA,$ AND KAORU ONOUE Department of Biochemistry, Institute for Medical Immunology, Kumamoto University Medical School; Honjo 2-2-1, Kumamoto 860, Japan; *Department of Radiology, Nagasaki University School of Dentistry; f Tokyo R & D Center, Daiichi Pharmaceutical Company Ltd.; and *Department cf Immunology, Juntendo University School of Medicine Received January 13, 1992; accepted March 6, 1992 In order to study how inflammatory cells including autoimmune lymphocytes interact with each other to develop collagen-induced arthritis (CIA), we injected monoclonal antibodies against mouse LFA-1 and ICAM- into DBA/l mice immunized with type II collagen (CII). Both antibodies suppressedthe development of CIA. Theseantibodies showedno effecton anti-C11antibody response,although they both significantly suppressedDTH response.It was suggestedthat antiadhesionmolecule antibodies suppressCIA mainly through their effect on cell-mediated immunity, without affecting humoral immunity under the conditions used. o 1992 Academic PKSS, IIK.
INTRODUCTION Recently, progress has been made in defining the molecules involved in the interactions of various cells including lymphoid and nonlymphoid cells (1,2). It was shown that cell-adhesion molecules play important roles in immune response and inflammation (3, 4). It is known that adhesion molecules are expressed on various cells composing a synovial microenvironment in the resting state. These molecules are upregulated in responseto the cytokines released(5). Among them, lymphocyte function-associated antigen- 1 (LFA- 1)’ which is a member of integrin family and its ligand, intercellular adhesion molecule- 1 (ICAM- 1) of the immunoglobulin superfamily, work as one of the main pairs of molecules to mediate the leucocyte adhesion process (6). While the events that lead to the initiation of the cascade of immune responses resulting in chronic proliferative synovitis in rheumatoid arthritis (RA) are not known, it seemsthat autoreactive T cells play an important role in the pathogenesis of this disease(7,8). Although the specific antigen that triggers the autoreactive T cell response in RA remains unknown, several animal models have been described in which defined antigens such as bacterial cell wall peptidoglycan which induces adjuvant arthritis (9) and connective tissue protein which produces type II collagen (CII)-induced arthritis i Abbreviations used: CIA, type II collagen-induced arthritis; CII, type II collagen; DTH, delayed type hypersensitivity; ICAM- 1, intercellular adhesion molecule-1; LFA- 1, lymphocyte function-associatedantigen1; MDP, muramyl dipeptide; REI, radiometric ear index. 326 0008-8749/92 $5.00 Copyright 0 1992 by Academic Press., Inc. All rights of reproduction in any form reserved.
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(CIA) (10, 11) and proteoglycan-induced arthritis (12) were used. CIA, in particular, is well known for its clinical and pathological similarity to RA ( 10). A cell-mediated autoimmune reaction, as well as a humoral one, appearsto play a critical role in CIA. As demonstrated by us ( 13) and others ( 14) CIA can be induced by passive transfer of the CII-specific T cell line. In this study, we investigated the role of the adhesion molecules in murine CIA by using monoclonal antibodies to mouse LFA- 1 and ICAM- 1. It was found that injection of anti-adhesion molecule antibodies suppressedCIA possibly by affecting cell-mediated immunity against CII. MATERIALS AND METHODS Animals. Female DBA/l mice, 7-10 weeks of age, were purchased from the Seiwa Institute for Experimental Animals (Fukuoka, Japan). CZZ.Human CII was purified from costal cartilage according to Miller’s method ( 15). Briefly, native CII was solubilized by limited pepsin digestion of costal cartilage and purified by DEAE cellulose chromatography and fractional salt precipitation. The purity of CII wasassessedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( 16) and contamination-free preparations were used. Monoclonaf antibodies. Rat monoclonal antibodies (MoAb) against LFA- 1 (KBA; IgGz,) ( 17) and ICAM- 1 (YN l/ 1.7;IgGZb)( 18)were purified by the useof caprylic acid (19) from the ascitesof ICR nu/nu mice transplanted intraperitoneally with each hybridoma cell. Normal rat IgG wasprepared from rat serum using DEAE ion exchange chromatography and used as control immunoglobulin (Ig) throughout the experiment. Induction ofarthritis. The CIA was induced by the intracutaneous immunization of 200 pg per mouse of human CII emulsified in Freund’s complete adjuvant followed by the booster injection of the sameamount of CII emulsified into Freund’s incomplete adjuvant 3 weeks later. Peptidoglycan/muramyl dipeptide (MDP)-induced arthritis was induced in the mice by the single intravenous injection of 100 pg MDP per mouse as we reported previously (20, 2 1). MDP was kindly donated by Dr. Atsuro Inoue of Daiichi Pharmaceutical Co., Ltd. This arthritis is induced by acute inflammatory cell infiltration (polymorphonuclear leukocytes and mononuclear cells) which seemsto be induced by the chemotactic activity of degraded components of the complements activated by peptidoglycan through an alternate pathway (20, 2 1). Administration of antibodies. One hundred micrograms per mouse of MoAb or normal control Ig was injected intraperitoneally starting on the day of immunization with CII (Day 0) and thereafter injected every other day for 2 weeks as well as Days 2 I, 24, and 28 (10 times for a total of 1 mg Ig/mouse). In the case of MDP-induced arthritis, a single injection of Ig was done only on the day of MDP immunization. Titration of anti-CII antibody. Antibody against CII was measured by enzymelinked immunosorbent assay(ELISA) (22). Alkaline phosphatase-conjugatedgoat antimouse IgG antibody (Tago Inc., Burlingame, CA) was used as the second antibody, Optimally diluted sera collected on Day 35 were assayed and the absorbance was determined by an ELISA microreader (Dynatech Laboratories Inc., Alexandria, VA), Measurement of DTH responseto CII. Delayed type hypersensitivity (DTH) was measured by ear skin testing on Day 21 using a 50-~1Hamilton syringe as described by Farmer et al. (23) and the responsewas read at 48 hr. The DTH responsewas also quantified by a modified method of radiometric ear assay(24). Briefly, CII wasdissolved
328
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ET AL.
at a concentration of one mg/ml in 0.15 M NaCI, 0.02 M Tris-HCI buffer, pH 8.0, by stirring for 24 hr. To radiolabel the dividing cells that accumulate at the site of DTH, mice were injected subcutaneously with [3H]thymidine at a dose of 0.4 Ci/body wt (g). Twenty-four hours later, 5 ~1of the collagen solution was injected intradermally into the right ear, using a Hamilton microsyringe with a 27-gaugeneedle. As a control, the buffer solvent alone was injected into the left ear in the same manner. After an additional 24 hr, the mice were killed and both ears dissected, and digested in NCS tissue solubilizer (Amersham Corp., Arlington Heights, IL). The radioactivity present in each ear was counted. The radiometric ear index (REI) was expressedas the ratio of cpm in the challenged ear vs the control ear. In vitro eflect of MoAb on the proliferative responseof lymphocytes to CII. The effect of MoAb was assessedon the sensitized lymph node cells collected on Day 14 from DBA/ 1 mice after immunization with human CII. Lymphocytes that were suspended at 2 X 1O4cells/ml in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Grand Island, NY) supplemented with 1% autologous mouse serum, 50 units/ml penicillin, 50 pg/ml streptomycin, and 5 X lop5 M 2-mercaptoethanol (2-ME) were incubated in flat-bottomed microtiter plates (Falcon 3072, Becton-Dickinson, Mountain View, CA) in triplicate wells. Each well was stimulated with 40 pg/ml of CII together with various concentrations of MoAb and cultivated for 4 days in 10%C02-air. Each well was pulsed with 0.2 &i of [3H]thymidine (sp act 5 &i/nmol, RCC, Amersham, England) for the last 9 hr. The cells were then harvested on fiberglass filters and the incorporation of thymidine was measured in a liquid scintillation counter. The proliferative response was expressed as the mean cpm. The effect of MoAb on the CIIinduced proliferative response of the CII-specific T cell line (Thy-l+, Lyt-l+, Lyt-2-, L3T4+) was also studied in the same way except for addition of 1 X lo6 syngeneic spleen cells (3000-R irradiated) as accessorycells (ACs) to the culture. As for the CIIspecific T cell line capable of inducing arthritis by passive transfer in DBA/l mice, we have reported elsewhere (23). Clinical evaluation of arthritis. The animals were graded for severity of arthritis according to the method reported by Wood et al. (25). The lesions of the extremities distal to the elbow or knee were graded on a scale of 0 to 4 based on the number of joints involved and the degreeof erythema and swelling. The maximal score was 12 because lesions on the injected foot were not included in the numerical score. The soft x-ray examination was carried out using Sofron (type SRO M-50) (Sohken, Tokyo, Japan). Histological examination of arthritis. Mice were killed and exanguinated. Joints (knees, ankles, feet) were removed and fixed in formalin, decalcified with ethylenediamine tetraacetic acid (EDTA) (Sigma Chemical Co., St. Louis, MO), and stained with hematoxylin and eosin. Statistics. Statistical significance was analyzed by the Student’s t test. RESULTS The e&ct of MoAbs on incidence and severity of CIA. As shown in Fig. 1a, the mice which were treated with control Ig developed CIA with 100% incidence. In contrast, the mice which were injected with KBA (anti-LFA-1) or YN1/1.7 (anti-ICAM-1) developed the arthritis with an incidence of 50 and 75%, respectively. Injection of MoAbs affected not only the incidence but also the severity of arthritis. Thus, Fig. 1b reveals that treatment with KBA or YN l/ 1.7 suppressedthe severity of arthritis significantly
ADHESION MOLECULES AND AUTOIMMUNE
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ARTHRITIS
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0 3
4
5
6
7
8
9
The
after
10
ilunization
(decks)
FIG. I The effect of MoAbs on incidence and severity of CLA. DBA/I mice (10 mice per group) were boosted with human CII on Day 2 I. Control Ig. anli-LFA- I (KBA), or anti-ICAM- 1 (YN l/ 1.7, abbreviated as YN-I) MoAb was injected ip every other day, amounting to a total of 1 mg per mouse. Details are described under Materials and Methods. (a) The effect on incidence. Differences between control Ig and KBA, and between control Ig and YN l/ I .7 are statistically significant (P < 0.0 1). (b) The effect on severity. Differencesbetween control Ig (normal rat IgG) and KBA and between control Ig and YNlil.7 are statistically significant (P i 0.01 and P < 0.05, respectively).
with the control Ig injection. Figure 2 depicts the soft x-ray findings of these animals. Destructive changes of joints and bone cortex are seen in untreated mice (Fig. 2a) and mice treated with control Ig (Fig. 2b). On the other hand, the joints and bones of mice treated with KBA (Fig. 2c) or YNl/ 1.7 (Fig. 2d) showed slight or only marginal changes. Figure 3 shows the histological findings of these joints. The mice injected with control Ig showed inflammatory cell infiltration into the joint space, synovium, and surrounding tissues, the typical findings of chronic proliferative synovitis (Fig. 3b), compared with the untreated normal mice (Fig. 3a). In contrast, mice treated with KBA or YN1/1.7 which showed suppressed development of arthritis revealed only slight, if any, infiltration of inflammatory cells (Figs. 3c and 3d). The effect of MoAbs on anti-CII humoral and cell-mediated immunity. Since the immune response against CII is involved in the pathogenesis of CIA, we assessedthe level of anti-C11 antibody in the serum of these mice and measured the cell-mediated immunity against CII by means of DTH reaction in the ear. As seen in Fig. 4a, no significant difference of antibody production was observed among control Ig-, KBA-, and YN I / 1.7-injected animals, whereas a significant difference in the DTH response to CII was found between the control Ig- and MoAb-injected groups (Fig. 4b). The DTH response of mice treated with KBA or YN1/1.7 was suppressed to 0.39 -t 0.02 and 0.42 -+ 0.01 mm, respectively, compared with 0.60 -t 0.02 mm in control mice. This result was reproducible and confirmed by radiometric ear assay. Thus, REI was 1.56 -+ 0.2 for the control Ig group, 0.89 t 0.17 for the KBA group, and 0.93 t 0.19 for the YN l/1.7 group. as compared
330
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FIG. 2. The effect of MoAbs on CIA (soft x-ray findings). Note that the destructive change seen in mice injected with control Ig (b) was largely suppressed in both KBA-treated (c) and YN1/1.7-treated (d) mice. The same paws which appear in Fig. 3 are shown. (a) This shows the findings of a normal untreated mouse.
The efict of MoAbs on T cell response to CII in vitro. In order to study the effect of MoAbs on cell-mediated immunity against CII in more detail, we examined the effect of MoAbs on the in vitro proliferative response of CII-sensitized lymph node cells as well as the CII-specific T cell line stimulated with CII. As shown in Fig. 5, both of the antibodies suppressed the proliferative response of lymph node cells by
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FIG. 2-Continued
about 50% at 10 rig/ml of MoAb (Fig. 5a). The proliferative response of the T cell line was also suppressed strongly by KBA, and to a lesser extent by YN l/ 1.7 (Fig. 5b) at 10 pug/ml of MoAbs. The efict of MoAbs on MDP-induced arthritis. Next, we studied the effect of the MoAbs on MDP-induced acute arthritis. As shown in Fig. 6, all mice treated with
332
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ET AL.
FIG. 3. The effect of MoAbs on CIA (histological findings). The finding of chronic proliferative synovitis characteristic of CIA is seen in mice treated with control Ig (b). Only slight inflammatory cell infiltration was seen in mice which showed arthritis suppressed by the KBA (c) and YN1/1.7 (d). (a) This shows the findings of a normal untreated mouse. Hematoxylin-eosine staining; X90 (a, b) and X I80 (c, d). Mice were sacrificed on Day 60.
control Ig developed this acute arthritis within 12 hr after MDP injection and reached maximum severity at 24-36 hr. The arthritis then subsided in 3-5 days after injection as reported previously in untreated mice (2 1). The injection of KBA or YNl/l.7
ADHESION
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FIG.
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3-Contimred
suppressed development of the arthritis dramatically in terms of the incidence as well as the severity: KBA suppressed the arthritis completely and YN1/1.7 lowered the incidence by 80% (Fig. 6). DISCUSSION CIA is a useful and unique experimental model of human RA (10). It has been established that autoimmunity to CII is involved in the development of CIA (10, 26),
334
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ET AL.
x
0 l
I
1
-rI p
