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. 2002 Apr;128(1):44–51. doi: 10.1046/j.1365-2249.2002.01794.x

Vascular cell adhesion molecule-1 (VCAM-1) blockade in collagen-induced arthritis reduces joint involvement and alters B cell trafficking

R A CARTER 1, I K CAMPBELL 1, K L O’DONNEL 1, I P WICKS 1
PMCID: PMC1906373  PMID: 11982589

Abstract

Vascular cell adhesion molecule-1 (VCAM-1 or CD106) is important in leucocyte trafficking and its increased expression is associated with a number of chronic inflammatory diseases, including rheumatoid arthritis (RA). We used a neutralizing monoclonal antibody (M/K-2·7) to investigate the role of VCAM-1 in collagen-induced arthritis (CIA), an autoimmune model of RA. A single injection of M/K-2·7 (0·5 mg) into naive mice caused leucocytosis within 20 h, due to increased numbers of circulating B cells and macrophages, as well as neutrophils. The most marked effect was on the numbers of immature B cells (B220loIgM+) which were increased approximately fourfold. CIA was elicited in DBA/1 mice by immunization with chick type II collagen (CII) in Freund’s complete adjuvant, followed by a repeat injection 21 days later. Repeated M/K-2·7 administration from the time of primary CII immunization reduced the clinical severity, but not the incidence, of CIA compared to isotype-control monoclonal antibody-treated mice. Histological assessment showed fewer arthritic joints in M/K-2·7-treated mice; however, affected joints showed the same range of severity as those of control mice. Anti-CII IgG1 levels were reduced in anti-VCAM-1-treated mice but the cellular immune response to CII was unaffected. In contrast, VCAM-1 blockade from the onset of clinical features of CIA did not prevent disease progression. These results establish a role for VCAM-1 in promoting polyarticular involvement in CIA, most probably via an effect on B cells.

Keywords: animal model, inflammation, lymphocyte trafficking, monoclonal antibody, rheumatoid arthritis

INTRODUCTION

Leucocyte trafficking to sites of injury and infection is required for the initiation of an effective immune response and is controlled by a highly regulated sequence of adhesive interactions between leucocytes and vascular endothelium [1]. Vascular cell adhesion molecule-1 (VCAM-1 or CD106) was originally identified as an inducible molecule on endothelium capable of binding lymphoid and tumour cell lines [2,3]. VCAM-1 binds predominantly the integrin α4β1 (CD29/CD49d), which is expressed by most circulating leucocytes [4]. Interactions between endothelial VCAM-1 and α4β1 integrin-bearing leucocytes contribute to the recruitment of these cells to sites of inflammation [5]. VCAM-1 is not expressed on resting vascular endothelium, but is induced rapidly in response to inflammatory stimuli, such as the cytokines tumour necrosis factor and interleukin-1 [2,5].

Gene knockout and in vitro studies suggest VCAM-1 is critical during early embryogenesis [6,7] and may be involved in haemopoiesis [8,9], B cell development [10,11] and T cell activation [12–14]. More information on the function of VCAM-1 in normal physiology has been obtained from recent reports describing conditional VCAM-1 mutant mice [15,16]. Induced deficiency of VCAM-1 interfered with lymphoid trafficking between the vascular compartment and the bone marrow, causing peripheral blood leucocytosis. In particular, there was retention of immature B cells in peripheral blood and a reduction in mature B cells in the bone marrow. Using interferon-induced Cre-loxP-mediated deletion of VCAM-1, there was also a reduced IgG1 response to a model T cell-dependent antigen, although the germinal centre reaction in lymph nodes (LN) was apparently unaffected. These results suggest that VCAM-1 expression in the bone marrow is important for B cell development and activation.

Elevated VCAM-1 expression on both vascular and nonvascular cell types is associated with many chronic inflammatory diseases, including rheumatoid arthritis (RA). VCAM-1 is strongly expressed on several cell types in RA synovium including fibroblast-like synovial lining cells [17], chondrocytes [18] and endothelial cells [17,19,20]. In vitro, peripheral blood T lymphocytes bind to type B synovial lining cells through an α4β1 integrin-VCAM-1-dependent pathway [19]. VCAM-1 may therefore facilitate the recruitment and retention of leucocytes in the inflamed synovium. VCAM-1 may also enhance B lymphocyte survival in the synovium [21–23], promote angiogenesis [24] and may mediate synovial attachment to cartilage and bone [25] (see [26] for review).

Monoclonal antibodies (MoAb) have proved useful tools to investigate collagen-induced arthritis (CIA), which is a widely used experimental model of RA. This approach allows the widespread neutralization of a target molecule without concern about abnormal physiological development of a gene knockout mouse. For example, anti-ICAM-1 MoAb reduced markedly the incidence and severity of CIA [27] and the administration of anti-ICAM-1 MoAb to RA patients has shown therapeutic benefit [28,29]. In this study, we investigated the role of VCAM-1 in murine CIA and we present the first report showing an effect of VCAM-1 blockade in autoimmune arthritis in vivo. Surprisingly, systemic administration of a neutralizing anti-VCAM-1 MoAb from the time of immunization with type II collagen (CII) attenuated disease, whereas VCAM-1-blockade from the onset of clinical arthritis did not affect disease progression. These results help to evaluate the role of VCAM-1 at different stages of autoimmune inflammatory arthritis.

MATERIALS AND METHODS

MoAbs

The rat hybridoma, M/K-2·7 [8], produces a neutralizing IgG1 MoAb specific for mouse VCAM-1 and was purchased from ATCC (Rockville, MD, USA). An isotype-matched control, the rat hybridoma, GL113, was obtained from DNAX (Palo Alto, CA, USA). Hybridomas were grown in specific serum-free media in a MiniPERM Bioreactor (Heraeus Equipment) and MoAbs were purified by protein G-sepharose (Pharmacia Biotech, Uppsala, Sweden) affinity chromatography.

Mice

Male DBA/1 mice were purchased from the Animal Resources Centre (Canning Vale, WA, Australia) and were used at 8–12 weeks of age. The mice were fed standard rodent chow and water ad libitum and housed in sawdust-lined cages in groups of up to five. All experiments were approved by the Royal Melbourne Hospital Animal Ethics Committee.

Peripheral blood leucocyte counts

Peripheral blood was obtained from mice by retro-orbital plexus venesection and was anticoagulated with sodium-EDTA. Erythrocytes were removed using red cell removal buffer (0·16m NH4Cl, 0·13 mm EDTA, 12 mm NaHCO3). Total leucocyte counts were performed using an Advia 120 Hematology System (Bayer Diagnostics). To assess leucocyte subclasses, cells were stained with antibodies to the following molecules: biotinylated anti-B220 (clone RA3·6B2, ATCC, Manassas, VA, USA), followed by streptavidin-Tricolour (Caltag Laboratories, Burlingame, CA, USA); PE conjugated MoAbs to CD4 (clone CT-CD4, Caltag), CD8 (clone 53–6·7, Sigma Chemical Co., St Louis, MO, USA) and CD11b (clone M1/70·15, Caltag); and FITC-conjugated antibodies to IgM (polyclonal, Southern Biotechnology Associates, Birmingham, AL, USA), neutrophil marker (clone 7/4, Caltag) and I-Ab (clone M5/114, ATCC). The level of non-specific staining was determined using isotype matched antibodies of irrelevant specificity and propidium iodide staining was used to assess cell viability (Calbiochem, La Jolla, CA, USA). Cells were analysed by flow cytometry on a FACScan using Cell Quest software (Becton Dickinson). The number of cells in the respective leucocyte subclasses was evaluated by multiplying the total leucocyte counts by the percentage of each cell type determined by flow cytometry.

CIA

An emulsion was formed by dissolving 2 mg/ml chick CII (Sigma) overnight at 4°C in 10 mm acetic acid and combined with an equal volume of Freund's complete adjuvant, prepared by adding heat-killed Mycobacterium tuberculosis (strain H37 Ra; Difco Laboratories, Detroit, MI, USA) to Freund's incomplete adjuvant (Sigma) at 5 mg/ml. All mice were injected intradermally (i.d.) at several sites into the base of the tail with a total of 100 μl emulsion containing 100 μg CII and 250 μg M. tuberculosis. Mice were immunized for maximum CIA response by administration of a repeat of the primary CII injection as a boost at day 21 [30].

MoAb treatment

To examine the effect of VCAM-1 blockade on CIA development, neutralizing anti-VCAM-1 (M/K-2·7) or isotype-matched control (GL113) MoAbs (0·5 mg/injection) were administered intraperitoneally (i.p.) the day before primary CII immunization and repeated every 2–3 days thereafter until CII challenge. To assess the therapeutic effect of VCAM-1 blockade on established disease, M/K-2·7 or GL113 (0·5 mg daily/10 days) were administered i.p. from the onset of clinical disease.

Clinical assessment of arthritis

Animals were assessed for redness and swelling of limbs and a clinical score allocated for each mouse daily for up to 42 days. The scoring system was as described previously [31] where 0 = normal, 1 = slight swelling, 2 = extensive swelling and 3 = joint distortion and/or rigidity. The maximum score per mouse was 12. Clinical and histological evaluations were assessed by two independent investigators blinded to the experimental groups.

Histological assessment of arthritis

At sacrifice, the limbs of the mice were removed, fixed, decalcified and processed for histological assessment of haematoxylin and eosin (H&E)-stained sections [31]. Arthritic features of metacarpophalangeal (MCP) and metatarsophalangeal (MTP) joints of all mice were scored histologically by a blinded assessor as described previously for an acute arthritis model [32]. The features of joint exudate (presence of inflammatory cells or fibrin-like material in the joint space), synovitis (thickening of the synovium and periosteum but excluding pannus), pannus (encroachment of hyperplastic synovium over the articular surface or bone at the cartilage–bone junction), and the extent and depth of cartilage and bone degradation were each scored on a scale from 0 (normal) to 5 (severe). Differential cell counts were performed by examining four high power fields of H&E-stained joint sections (n = four mice) to determine the relative numbers of neutrophils, eosinophils, lymphocytes and monocyte/macrophages.

Detection of serum anti-CII antibodies

Enzyme-linked immunosorbent assays (ELISA) for antibody to CII were performed as described previously [33]. Horseradish peroxidase-conjugated goat antimouse IgG (Sigma), IgG1, IgG2a or IgM (Southern Biotechnology Associates) detection antibodies were used. Standard curves were constructed from sera of CII-hyperimmunized mice using arbitrary units.

Lymphocyte proliferation assays

To examine the in vitro effect of VCAM-1 blockade on antigen-specific T cells, mice were sacrificed 10 days after primary CII immunization and single cell suspensions were prepared from the inguinal LNs in RPMI supplemented with 2-mercaptoethanol and 1% (v/v) autologous mouse serum. LN cells (4 × 105 cells in 0·2 ml) were plated in 96-well plates and stimulated in triplicate with 50 μg/ml chick CII in the presence of 1, 10 or 100 μg/ml M/K-2·7 or GL113. Cells were incubated at 37°C in 5% CO2 for 4 days and pulsed with 1 μCi [3H]thymidine for the final 18 h. Cells were harvested and [3H]thymidine uptake was measured using a microplate scintillation counter (Canberra Packard, Victoria, Australia).

To examine the effect of in vivo VCAM-1 blockade on the proliferative response of CII-sensitized inguinal LN cells in vitro, mice received i.p. injections of either M/K-2·7 or GL113 (0·5 mg/ injection) beginning the day before primary CII immunization and thereafter every second day until inguinal LN harvest on day 10. LN cell proliferation assays were set up as described above, except the cells were stimulated with CII without the in vitro addition of anti-VCAM-1 or isotype-matched control MoAbs.

Statistics

The Mann–Whitney two-sample rank test was used to determine the level of statistical significance between means of groups for clinical scores. The χ2 test was used to analyse histological data. Student’s t-test for the difference of two means was used to compare anti-CII antibody levels, LN cell proliferation and differential leucocyte counts between groups. For all tests P < 0·05 was considered statistically significant.

RESULTS

Peripheral blood leucocytosis following in vivo blockade of VCAM-1

In view of its proposed role as an adhesion molecule involved in cell trafficking and the phenotype of conditional VCAM-1 mutants, the effect of VCAM-1 blockade on circulating peripheral blood leucocyte levels was assessed. Blood was obtained from mice that had been injected 20h earlier with either anti-VCAM-1 neutralizing MoAb (M/K-2·7) or isotype-matched control MoAb (GL113). Table 1 shows there was a 1·5-fold increase in the number of total blood leucocytes of mice treated with M/K-2·7 compared to control mice. M/K-2·7-treated mice showed increases in the number of circulating cells for all leucocyte subclasses examined, although only certain cells of the B cell lineage, macrophages and neutrophils showed statistical significance (P < 0·05). Based on B220 and IgM staining, two groups could be identified within the B cell lineage (Fig. 1): B220hiIgM+− R1 and B220loIgM+− R2. R1 and R2 appear to correspond to mature and immature B cell populations, respectively. The most marked effect of VCAM-1 blockade was on the number of circulating immature B cells which were increased 3·8-fold in the peripheral blood (Table 1). A single injection of 0·5 mg M/K-2·7 was therefore sufficient to inhibit VCAM-1 function in vivo.

Table 1.

Effect of VCAM-1 blockade on circulating peripheral blood leucocytes in naive DBA/1 mice

MoAb treatment*
GL113 M/K-2·7 Fold increase†
Total leucocytes 9·35 ± 0·66 13·79 ± 1·23§ 1·5
Leucocyte subclass‡
 CD4+ T cells 2·16 ± 0·26 2·31 ± 0·24 1·1
 CD8+ T cells 0·68 ± 0·09 0·81 ± 0·08 1·2
 Mature B cells 1·06 ± 0·19 1·63 ± 0·39 1·5
 Immature B cells 0·32 ± 0·05 1·23 ± 0·23§ 3·8
 Macrophages 0·20 ± 0·04 0·47 ± 0·10 2·4
 Neutrophils 4·43 ± 0·17 5·83 ± 0·48 1·3
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Mice were injected i.p. with either anti-VCAM-1 (M/K-2·7) or isotype control (GL113) MoAbs (0·5 mg/mouse) and blood was removed 20h later, cell counts performed and cell populations analysed by flow cytometry (see Materials and methods). Data are representative of two experiments.

*

Data show the mean (± s.e.m.) number of cells/μl × 10−3 for six mice/group.

†

Ratio of cell counts for M/K-2·7-treated/GL113-treated mice.

‡

Mature B cells: B220hiIgM+ (R1, see Fig. 1); immature B cells: B220loIgM+ (R2); macrophages: CD11b+, I-Ab+.

§

P < 0·01;

P < 0·05, compared to GL113-treated group.

Fig. 1.

Fig. 1

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FACS analysis of peripheral blood B cell subpopulations follow-ing VCAM-1 blockade in vivo. Mice were injected i.p. with either anti-VCAM-1 (M/K-2·7) or isotype control (GL113) MoAbs (0·5 mg/mouse), blood was removed 20 h later and B cell subpopulations were analysed by flow cytometry (see Materials and methods). Two populations were identified and gated as R1 (B220hiIgM+– mature B cells) and R2 (B220loIgM+– immature B cells).

VCAM-1 blockade during CIA development results in reduced clinical severity of disease

To investigate the role of VCAM-1 in CIA development, M/K-2·7 or GL113 were administered over 3 weeks beginning the day before primary CII immunization. The treatment schedule was based on a previous study where administration of a neutralizing anti-ICAM-1 MoAb over a similar time-course reduced the incidence and severity of CIA in DBA/1 mice /27/. M/K-2·7 treatment did not significantly affect either the incidence of arthritis or the mean day of disease onset (Table 2). However, there was a statistically significant (P < 0·01) reduction in overall clinical severity of disease as compared to animals treated with control MoAb. Less severe disease in M/K-2·7-treated mice was clinically apparent from day 28 postprimary CII immunization and continued through until termination on day 42 (Fig. 2). As summarized in Table 2, this decrease was seen as both a reduction in the number of affected paws and a decrease in the severity of arthritis in the affected paws. The results show pooled data from two experiments; analysis of data from each experiment revealed similar findings.

Table 2.

Effect of VCAM-1 blockade on clinical arthritis during the induction phase of disease

MoAb treatment
GL113 M/K-2·7
Incidence of CIA (%) 18/19 (95) 18/18 (100)
Day of arthritis onset* 26·7 ± 0·2 26·3 ± 0·3
Clinical score*† 7·2 ± 0·5 4·4 ± 0·7‡
Number of affected paws* 3·3 ± 0·2 2·3 ± 0·3§
Severity of affected paws* 2·1 ± 0·1 1·7 ± 0·2§
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Mice were immunized with CII as described in Materials and methods. Anti-VCAM-1 (M/K-2·7) or isotype control (GL113) MoAbs (0·5 mg) were injected i.p. the day before primary immunization and repeated every 2–3 days thereafter until CII challenge.

*

Results are expressed as mean ± s.e.m.

†

Results represent the average clinical score per mouse across the time course of the experiment (days 26–42).

‡

P < 0·01;

§

P < 0·05, compared to GL113-treated group.

Fig. 2.

Fig. 2

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Effect of VCAM-1 blockade during the induction phase of CIA. CII-immunized mice were treated with anti-VCAM-1 (M/K-2·7) or isotype control (GL113) MoAbs as described in Table 2. Results show the mean (± s.e.m.) clinical scores (maximum of 12 per mouse) for 18–19 mice/group (pooled data from two experiments). See Table 2 for statistical assessment. •, GL113; ○, M/K-2·7.

VCAM-1 blockade during CIA development reduces the number of arthritic joints but not the histopathological progression of arthritis

To evaluate further the effect of VCAM-1 blockade on CIA, histological analyses were performed on MCP and MTP joints which were assessed for five features of inflammatory arthritis [32]. Mice treated with M/K-2·7 showed a significantly lower percentage of arthritic joints compared to the GL113-treated mice (36%versus 69%, respectively; P < 0·0005) (Table 3). Of the joints affected with CIA, the majority for each treatment group were severely damaged, there being no difference in the level of severity of the histological features of joint exudate, synovitis, pannus formation or cartilage or bone degradation between the two treatment groups (Table 3 and Fig. 3). Differential cell counts of H&E-stained joint sections showed similar proportions of cellular subclasses within the joint exudates (data not shown). In short, the level of inflammation and tissue destruction proceeded to a similar extent for each treatment group once a given joint was involved. Therefore, reduced numbers of arthritic joints within a paw most probably accounts for the lower clinical scores observed in arthritic paws of M/K-2·7-treated mice (Table 2).

Table 3.

Histological evaluation of the effect of VCAM-1 blockade on CIA during the induction phase of disease

MoAb treatment
GL113 M/K-2·7
Arthritis incidence* 114/165 (69) 48/133 (36)†
Joint features‡
 Exudate 3·0 ± 0·3 3·5 ± 0·4
 Synovitis 4·4 ± 0·2 4·4 ± 0·2
 Pannus 4·4 ± 0·2 3·8 ± 0·3
 Cartilage degradation 4·0 ± 0·2 3·6 ± 0·3
 Bone degradation 4·4 ± 0·2 4·1 ± 0·3
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Histological features of arthritis were assessed on H&E stained paraffin sections of MCP and MTP joints from CII-immunized mice treated with anti-VCAM-1 (M/K-2·7) or isotype control (GL113) MoAbs as described in Table 2.

*

Results show the number of arthritic joints/total joints with percentages in parentheses.

†

P < 0·0005 compared to GL113-treated mice.

‡

Arthritic MTP joints of the rear right paws were assessed for the designated histopathological features, each on a scale from 0 (normal) to 5 (severe). Values show the mean ± s.e.m. histological scores for mice treated with M/K-2·7 (n = 16 joints) or GL113 (n = 18).

Fig. 3.

Fig. 3

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Severe histopathology in arthritic joints of mice treated with anti-VCAM-1 MoAb. CII-immunized mice were treated with anti-VCAM-1 (M/K-2·7) (a) or isotype control (GL113) (b) MoAbs as described in Table 2. Mice were sacrificed 42 days after primary immunization and the MCP and MTP joints processed for histological evaluation. Severe histopathology was observed in the arthritic joints of both groups of mice with features of joint exudate, inflammatory cell infiltration and invasive pannus formation resulting in cartilage and bone destruction (arrows). Similar cell types were noted in the exudates of each treatment group, with a predominance of polymorphonuclear leucocytes (arrowheads). H&E staining, original magnification × 400.

Altered anti-CII antibody response following VCAM-1 blockade

CIA induction requires both humoral and cell-mediated immune responses to CII [30,34], and in view of the effect of VCAM-1 blockade on circulating leucocyte numbers (Table 1), it is possible that either or both of these could be affected in M/K-2·7-treated mice. Serum levels of anti-CII antibody were therefore analysed to investigate whether less severe disease in anti-VCAM-1-treated mice was due to impaired humoral immunity to CII. As shown in Fig. 4a, the anti-CII IgG1 levels of M/K-2·7-treated mice were significantly lower than those of GL113-treated animals (P < 0·05). There were no differences in the total IgG, IgG2a or IgM anti-CII levels.

Fig. 4.

Fig. 4

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Effect of VCAM-1 blockade during the induction phase of disease on the humoral and cellular immune responses to CII. (a) Humoral response to CII. Mice were immunized with CII and treated with anti-VCAM-1 (M/K-2·7) or isotype control (GL113) MoAbs as described in Table 2. CII-specific total IgG, IgG1, IgG2a or IgM antibody levels were determined on sera from day 42 by ELISA as described in Materials and methods. Results show the mean ± s.e.m. values for M/K-2·7-treated (n = 18) or GL113-treated (n = 19) mice. *P < 0·05, compared to GL113-treated mice. (b,c) Cellular response to CII. Mice were immunized with CII and lymphocyte proliferation assays established in the presence and absence of CII (see Materials and methods) from cells of the inguinal LNs removed at day 10. To assess in vitro VCAM-1 blockade (b), LN cells were cultured in the presence of different concentrations of either M/K-2·7 or GL113. For in vivo VCAM-1 blockade (c), mice were injected with either M/K-2·7 or GL113 beginning the day before CII immunization and every second day thereafter until day 10. Results show the mean (± s.e.m.) [3H]thymidine uptake (in cpm) above background levels in the absence of CII stimulation. No statistically significant differences were observed between the groups. ■, GL113; □, M/K-2·7.

VCAM-1 blockade does not affect the cellular response to CII

To determine the effect of VCAM-1 blockade on cell-mediated immunity against CII in vitro, we examined the effect of M/K-2·7 on the proliferative response of CII-sensitized inguinal LN cells. Figure 4b shows that at concentrations of 1, 10 and 100 μg/ml, the anti-VCAM-1 MoAb had no suppressive effect on LN proliferation as compared to the controls. The effect of in vivo VCAM-1 blockade on cell-mediated immunity against CII in vitro was also examined following the administration of M/K-2·7 to mice before primary CII immunization and thereafter until LN harvest on day 10. Figure 4c shows that the in vivo administration of M/K-2·7 had no suppressive effect on LN proliferation as compared to the controls.

VCAM-1 blockade in established CIA

As M/K-2·7 treatment was beneficial when administered during CIA development, the therapeutic effect of VCAM-1 blockade on established disease was examined. The MoAb treatment schedule was based on previous studies where neutralizing antibodies against M-CSF [35] and CD4 [36] attenuated established CIA in DBA/1 mice. Mice were immunized with CII and assessed for clinical signs of arthritis, as above. As clinical signs of arthritis developed, mice were paired according to the arthritic score and assigned to either the GL113 or M/K-2·7 treatment groups, which ensured mice of similar initial arthritic scores were evenly distributed. Mice were then treated with 10 daily i.p. injections of MoAb. There was no significant difference in the disease severity up to day 14 of arthritis for M/K-2·7-treated mice compared to GL113-treated animals (data not shown).

DISCUSSION

The intercellular adhesion molecule VCAM-1 is expressed strongly by vascular and non-vascular cells in synovial tissue from patients with RA [17,20,37]. To explore the function of VCAM-1 in inflammatory joint disease and to evaluate VCAM-1 as a potential therapeutic target in RA, MoAb blockade of VCAM-1 in a murine model of RA was investigated. Systemic administration of neutralizing anti-VCAM-1 MoAbs from the time of CII immunization attenuated the severity of CIA – an effect not reported previously. Clinical observations showed reduced severity of CIA in anti-VCAM-1-treated mice compared to animals treated with control MoAb. However, administration of anti-VCAM-1 MoAbs did not affect either the incidence of arthritis or the mean day of onset relative to control mice.

Attenuation of disease development with VCAM-1 blockade focused our attention on early immunological mechanisms in CIA. Interestingly, a quantitative trait locus containing the VCAM-1 gene has been implicated as controlling disease severity in CIA in rats [38]. CIA induction requires both humoral and cell-mediated immune responses to CII [30,34]. A role for VCAM-1 in migration/recirculation of B cells to the bone marrow was revealed by recent conditional VCAM-1 knockout studies [15,16]. One conditional VCAM-1 knockout also had an impaired T cell-dependent IgG1 humoral response [15]. Our observation that the peripheral blood lymphocyte count was increased (in percentage and absolute terms) in M/K-2·7-treated mice compared to GL113-treated mice is consistent with the knockout phenotype and demonstrates the in vivo effectiveness of the anti-VCAM-1 MoAb. Consistent with results from the conditional VCAM-1 mutants, we also found reduced IgG1 anti-CII antibodies. Reduced anti-CII antibodies provides a potential explanation for reduced CIA, because anti-CII antibodies are essential for the development of CIA. Whilst previous work has suggested that CIA in DBA/1 mice is not correlated with IgG1 anti-CII antibodies [39], data using our immunization protocol are in fact suggestive of an association [30]. Our results, together with the impaired B cell migration demonstrated in conditional VCAM-1 mutants, raise the possibility that maturation of the B cell response within the bone marrow is important for the development of CIA. Several studies suggest VCAM-1 may play an important role in B lymphocyte survival in the synovium [21–23]. It is also possible that survival of synovial B cells, or local production of anti-CII antibodies within the joints [40], is impaired by anti-VCAM-1 treatment.

Several studies have demonstrated the importance of antigen presenting cell–T cell interactions in the pathogenesis of CIA. For example, MoAb blockade of CD40 ligand (CD40L) interactions or CD80/CD86-mediated pathways suppressed disease induction and progression [41,42]. The reduced incidence and severity of CIA in mice following the administration of a neutralizing anti-ICAM-1 MoAb may be due in part to less efficient antigen presentation [27]. VCAM-1 is expressed on both lymphoid and myeloid dendritic cell subsets in spleen [16] and follicular dendritic cells [3,43]. In vitro data suggest VCAM-1 may function as a co-stimulatory molecule for T cell activation [12–14] and VCAM-1 blockade could act by disrupting the interaction between VCAM-1 on antigen presenting cells and α4β1 integrin on T lymphocytes. However, M/K-2·7 administered in vivo or added in vitro did not affect CII-specific LN cell proliferation. It is therefore less likely that VCAM-1 has a major influence on cellular immunity to CII. In keeping with this, VCAM-1 blockade did not prevent the development of mBSA/IL-1 induced acute arthritis (data not shown), a model which we have shown to be CD4+ T cell-dependent, but B cell-independent [32].

The lack of complete disease suppression following anti-VCAM-1 treatment suggests other adhesion pathways are also involved in leucocyte recruitment to inflamed joints. For example, ICAM-1 and hyaluronic acid mediate leucocyte trafficking and are up-regulated in RA synovium [20,44] Respective counter-receptors for these molecules include LFA-1 and CD44, which are both expressed at increased levels on synovial T cells and other infiltrating mononuclear cells [45]. α4β1 integrin also binds a cell attachment domain (CS-1) in an alternatively spliced region of fibronectin [46]. mRNA for the CS-1 fibronectin isoform is present in RA synovial endothelium [47] and therefore some α4β1 integrin+ leucocytes may infiltrate the synovium through adhesion to fibronectin. Moreover, studies of adjuvant arthritis in rats demonstrated that administration of anti-α4β1 integrin MoAbs or CS-1 fibronectin peptides suppressed synovial leucocyte infiltration [48]. A recent study of α4β1 dependent leucocyte adhesion in rats also raises the possibility of a novel (non-VCAM-1, non-CS-1) α4β1 ligand [49].

Somewhat surprisingly, in view of its role as an inducible endothelial adhesion molecule, VCAM-1 blockade from the onset of clinical disease did not significantly alter disease progression. This suggests that by the stage of clinical presentation, VCAM-1-independent pathways may predominate in CIA. It is also possible that a higher dose of anti-VCAM-1 MoAb may be required at this stage, given the up-regulation of VCAM-1 associated with established disease [17,37,50]. During CIA development, administration of anti-VCAM-1 MoAb may also prevent soluble VCAM-1-mediated chemotaxis of monocytes and T cells to the joints [51,52]. As an increase in soluble VCAM-1 is associated with advanced inflammatory joint disease [53], it is again possible that the concentrations of anti-VCAM-1 MoAb used in the present study may be insufficient to neutralize the levels of soluble VCAM-1 that may occur during established CIA. Alternatively, the administration of anti-VCAM-1 MoAb at the time of disease onset (when soluble VCAM-1 levels are likely to be elevated) could result in immune complex formation within joints, exacerbating arthritis and so counteracting any benefit of VCAM-1 neutralization. It is unlikely that the lack of response to M/K-2·7 in the established disease protocol was due to the development of neutralizing anti-M/K-2·7 antibodies as the period of treatment with MoAb was short (10 days) and an effect of M/K-2·7 on CIA was observed in the longer treatment protocol.

In summary, these results establish a significant role for VCAM-1 in the immunopathogenesis of inflammatory arthritis and indicate that VCAM-1 is important in the development of CIA, most likely due to altered B cell trafficking. In contrast, the importance of VCAM-1 seems to decline with progression of CIA. Our results highlight the need to carefully dissect the function of adhesion molecules at different stages of disease and suggest that in human RA, inhibition of multiple adhesion pathways may be superior to blockade of a single adhesion molecule.

Acknowledgments

We are grateful to K. Lawlor for technical assistance, A. Milligan for animal care, S. Mihajlovic for histology, Natalie Thorne for statistical advice and D. Tarlinton for critical review of the manuscript. Supported by the National Health and Medical Research Council of Australia, the Arthritis Foundation of Australia and the Reid Charitable Trusts. R.A.C. was supported by a postgraduate scholarship from the University of Melbourne.

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