Experimental arthritis in CC chemokine receptor 2–null mice closely mimics severe human rheumatoid arthritis (original) (raw)
Increased incidence and accelerated joint destruction in Ccr2-deficient mice during CIA. In each of the five separate experiments performed, compared with WT or _Ccr5_–/– mice, _Ccr2_–/– mice consistently exhibited a significantly greater incidence and severity of CIA, whereas the CIA phenotypes of _Ccr5_–/– and WT mice were nearly indistinguishable (Figure 1, A and B). _Ccr2_–/– mice developed extensive swelling and severe ankylosis in multiple joints, whereas the disease in WT and _Ccr5_–/– mice was usually restricted to single digits and rarely involved an entire paw (Figure 1, C and D). These findings were corroborated by radiological examination, which revealed involvement of multiple joints, substantial bone destruction, and decrease in bone density, all features characteristic of severe human RA (Figure 1, E and F). Since the lack of CCR5 expression did not modify significantly the course of CIA in DBA/1J mice, no further analyses were performed on the _Ccr5_–/– animals.
CIA incidence and severity is increased in _Ccr2_–/– mice. The data shown in this figure are representative of one of a minimum of three experiments. (A–F) In the experiment shown, _Ccr2_–/– mice (n = 8), _Ccr5_–/– mice (n = 6), and WT mice (n = 10) were given primary and booster injections of bovine CII and monitored two to three times per week for incidence and severity of arthritis. All mice were backcrossed six generations into the DBA/1J background. Days after immunization in A and B represent days after the second immunization with CII. (A) The cumulative number of arthritic animals in each group is shown as a percentage of the total number immunized with CII. (B) Arthritic score for each group at each time point was divided by the number of arthritic mice to calculate a mean severity score (± SEM). The maximal arthritic score for _Ccr2_–/– mice (11.0 ± 0.8) was significantly higher than the scores for WT (3.9 ± 0.7) and _Ccr5_–/– mice (3.2 ± 1.2) (P < 0.0001). The difference between WT and _Ccr5_–/– mice was not significant (P = 0.5). Photomicrographs (C and D) and radiographs (E and F) from WT and _Ccr2_–/– mice depicting severe arthritis and bone destruction and erosion in _Ccr2_–/– mice. (G) Incidence and (H) severity of arthritis determined 2 weeks after immunization with CII in F8 backcrossed _Ccr2_–/– (n = 3–4 mice per experiment) and WT mice (n = 3–4 mice per experiment).
To exclude any confounding that might occur as a result of the minimal genetic admixture in the F6_Ccr2_–/– DBA/1J mice, we backcrossed these mice an additional two generations. Also, because in these experiments the F6 WT littermates exhibited a benign course compared with _Ccr2_–/– mice, we sought to determine whether further backcrossing of these littermates would alter their susceptibility to CIA relative to commercially available DBA/1J mice. Remarkably, compared with the F6 backcrossed _Ccr2_–/– mice, F8_Ccr2_–/– mice were even more sensitive to the induction of CIA, and all the F8_Ccr2_–/– mice developed arthritis within 14 days of the primary immunization (Figure 1G), whereas the F6_Ccr2_–/– mice developed disease only after the booster injection with CII. Notably, at this early time point the disease severity in the F8_Ccr2_–/– mice was comparable to that observed 50 days after the booster immunization in the F6_Ccr2_–/– mice (Figure 1H). Commercially available DBA/1J mice were only marginally more susceptible than the F8 WT littermates, but the incidence and severity in these commercially obtained control animals remained significantly lower than that observed in the F6 or F8_Ccr2_–/– mice (data not shown).
In addition to the clinical and radiographic changes, by day 30 after the second immunization with CII, the _Ccr2_–/– mice, but not the WT mice, had histopathological features that were highly reminiscent of severe human RA (Table 1 and Supplemental Table 2). The pathology in the joints of these KO mice was characterized by invasion of the pannus tissue into the bone space, destruction of the joint architecture, and severe erosion of cartilage surfaces accompanied by marked infiltration of inflammatory cells, primarily neutrophils and macrophages (Figure 2, A–F). In support of the notion that osteoclasts play an important part in the pathogenesis of focal bone erosion in arthritis (31–34), we observed enhanced osteoclast activity in the joints of _Ccr2_–/– mice (Figure 2, C and D).
Histopathological and cellular responses in _Ccr2_–/– mice after induction of CIA or peritonitis. (A) X-ray of the paw of a _Ccr2_–/– mouse, shown to provide anatomical orientation. The abbreviated names of the bones highlighted in B and C are given in parentheses. Histopathological evaluation of the joints were conducted after (B and C) H&E, (D and E) TRAP (for osteoclasts), and (F) orange G (for enhanced bone visualization) staining. Affected joints derived from WT mice (B and D) show relatively normal articular surfaces, intact joint spaces, and absence of significant periarticular inflammation. _Ccr2_–/– mice (C, E, and F) showed irregular articular surfaces, collapsed joint space, loss of cartilage, and an increase in connective tissue adjacent to the joint space with abundant osteoclasts stained red by TRAP stain. Representative joints collected 30 days after the second immunization are shown. Note that, at this time point, there was little visual evidence of arthritis in the WT mice and hence the relatively normal histopathology in these mice (see Figure 1B). (F and G) To determine the nature of the cell types recruited, FACS analysis was performed on cells derived from the inflamed joints of four mice from each group. (H) Number of macrophages in inflamed joints. (I) Number of peritoneal monocytes/macrophages 72 hours after thioglycollate injection. *P < 0.05; **P < 0.01. Data shown are representative of one of three experiments. In G–I, the white and black histograms refer to WT and _Ccr2_-null mice, respectively.
Comparison of human RA and murine models of autoimmune arthritis
Several leukocyte cell subtypes contribute to human RA pathogenesis (Table 1 and Supplemental Table 2), including neutrophils. Although naive (unmanipulated) WT and _Ccr2_-null mice had similar numbers of circulating neutrophils, monocytes, and lymphocytes (Supplemental Table 3), after induction of CIA the joints of _Ccr2_–/– mice had higher cell numbers than WT mice (5.4 ± 1.2 × 106 versus 2.9 ± 1.0 × 106 cells; mean ± SD). FACS analyses revealed an increase in the percentage and total numbers of neutrophils and monocytes and/or macrophages recruited to the _Ccr2_-null mice (Figure 2, G and H, and data not shown). Mirroring this increased recruitment of monocytes, F4/80 transcripts, a marker characteristic for macrophages, were more abundant in the joints of _Ccr2_-null mice (Table 2). In addition, the abundance of transcripts characteristic for cell types such as B cells (CD19) and T cells (CD3, CD4, and CD8) were also higher in _Ccr2_-null mice than in WT mice (Table 2).
Cellular phenotype in joints derived from CII-immunized mice
The enhanced recruitment of monocyte/macrophages to the joints of _Ccr2_–/– mice was surprising in light of the extensive literature documenting a reduced recruitment of these cell types to inflammatory sites in different disease models tested in these KO mice (Supplemental Table 1). However, concordant with the findings in the literature, after intraperitoneal injection of thioglycollate in the naive DBA/1J mice, lower numbers of monocyte/macrophages were recruited to the peritoneum (Figure 2I). Thus, enhanced or reduced monocyte/macrophage recruitment following genetic inactivation of Ccr2 in DBA/1J mice was clearly highly dependent on the nature of the inflammatory insult. Notably, analogous to our observations here, CCR2-independent recruitment of monocytes and macrophages has also been illustrated in a murine model of idiopathic pulmonary fibrosis (35).
Increased anti-CII Ab, RF, and anti–single-stranded DNA Ab production in Ccr2–/– mice. CII-specific Ab’s are necessary and sufficient to induce CIA (10, 16, 17), and thus enhanced anti-CII-specific Ab production may serve as a major pathogenic mechanisms underlying the severe CIA phenotype of _Ccr2_-null mice. Previous studies supported the notion that inactivation of Ccr2 may be associated with increased Ag-specific Ab production. For example, we have previously shown that after Leishmania major infection, _Ccr2_-null mice in the C57BL/6 genetic background produce higher amounts of _L. major_–specific Ab’s (25). Additionally, enhanced Ab production was also observed in a murine model of allergen hypersensitivity (refs. 23, 24 and Supplemental Table 1). Here, we extended these findings by showing that in the BALB/c background, inactivation of Ccr2 was also associated with increased _L. major_–specific Ab’s after intradermal infection (Supplemental Table 4).
We further extend the notion that inactivation of Ccr2 is associated with aberrant Ab responses to different immunogenic challenges and across different genetic backgrounds, since _Ccr2_–/– DBA/1J mice produced significantly higher levels of anti-CII IgG2a and anti-CII IgG1 compared with WT mice 2 weeks after the primary immunization (Figure 3, A and B). We selected this early time point for analyses of Ab production for two reasons. First, since there is a strong correlation between anti-CII Ab titers before the onset of arthritis and subsequent arthritic severity (36), we surmised that elevated levels of anti-CII Ab at this early time point would support a possible link between aberrant Ab production and the increased disease severity observed in _Ccr2_-null mice. Second, this early time point we posited would potentially limit the confounding that might occur once clinical arthritis is evident, since the disease per se may act as a feedback mechanism to increase autoantibody production, and therefore the finding of abnormal Ab production during the later stages of disease may not necessarily represent a primary immune abnormality. In this light, our findings suggest that CCR2-dependent processes are associated with the magnitude of anti-CII Ab production, and may be associated with the more severe CIA phenotype observed in _Ccr2_-null mice. However, we cannot exclude the possibility that clinically undetectable joint disease during the very early phases of CIA also contributed to the enhanced anti-CII Ab production in _Ccr2_–/– null mice (37).
Elevated levels of autoantibodies 2 weeks after CII immunization in _Ccr2_–/– mice, as shown by OD measurements in serially diluted serum samples. (A) IgG1 and (B) IgG2a against collagen (mean of three WT and four _Ccr2_-deficient mice); *P < 0.05. (C) RF was measured in the sera of three WT and four _Ccr2_–/– mice; **P < 0.0001. Collagen-specific IgG quantification was performed in several experiments, and _Ccr2_–/– mice consistently had higher IgG levels than the WT littermates before arthritic disease was clinically evident, and those levels remained elevated after the arthritic disease started (data not shown).
We next determined whether the production of other autoantibodies was also increased after CII immunization in _Ccr2_–/– mice. Since among all biomarkers currently known for human RA, autoantibodies against IgG (e.g., RF) have consistently been shown to be the best predictor of disease severity (38–40), we measured RF 2 weeks after primary immunization in WT and _Ccr2_–/– mice. After induction of CIA, RF was undetectable in serum from WT mice, a finding concordant with previous studies (10, 17, 39, 41, 42). However, RF was detected in the sera of _Ccr2_–/– mice (Figure 3C), thus reproducing one of the characteristic features of human RA, and notably, a feature of only a handful of other rodent models of RA (Table 1, and refs. 38–40, 42–46). Furthermore, compared with WT mice, higher levels of circulating anti–single-stranded DNA (anti-ssDNA) Ab’s were detected in the _Ccr2_–/– mice (in WT mice, OD = 0.049 versus _Ccr2_-null mice, OD = 0.165; P = 0.001).
Given that an aberrant production of autoantibodies, especially anti-CII Ab’s, appeared to be a major feature of the severe CIA phenotype in _Ccr2_–/– mice, we next tested the hypothesis that this was secondary to an intrinsic defect(s) at the level of _Ccr2_-null B cells that was present in these mice before CII immunization (e.g., enhanced proliferation, increased Ab production, accelerated maturation). Several lines of evidence suggested that _Ccr2_-null B cells before induction of CIA were normal. First, in vitro experiments showed that after stimulation of splenocytes with anti-CD40 (1 μg/ml), anti-CD40 plus IL-4 (10 ng/ml), LPS (20 μg/ml), or LPS (20 μg/ml) plus IL-4 (10 ng/ml), DBA/1J WT and _Ccr2_–/– mice splenocytes produced similar amounts of Ab (data not shown) and had similar proliferation rates (Supplemental Table 5). Second, following B cell receptor stimulation using IgM F(ab′)2 fragments, mature B cells derived from the spleens of WT and _Ccr2_–/– mice appeared to have comparable phosphorylation levels of tyrosine kinases, including that of Syk, a critical proximal kinase that initiates all B cell receptor–dependent signaling pathways (47) (Supplemental Notes and data not shown). Third, the percentage of pro- (B220+CD43+IgM–), pre- (B220+CD43–IgM–), immature (B220+IgM+IgD–), and mature (IgM+IgD+B220hi) B cell populations in the spleens, bone marrow, and peritoneal cavities of naive (nonimmunized) WT and _Ccr2_–/– mice was similar, as were B-1 cells (B220+IgM+CD5+) (Supplemental Table 6). Taken together, these findings suggest that B cell responses in vitro as well as the B cell development in _Ccr2_-null mice do not differ significantly from WT mice.
Since we found no gross defects at the level of _Ccr2_–/– B cells prior to CII immunization to account for the severe CIA phenotype, we next determined the contribution of T cell–mediated immune responses to enhanced Ab production and joint inflammation: cytokine production at the local (joint) and the secondary lymphoid organ level and intrinsic abnormalities in the _Ccr2_-null T cells (i.e., Th cell polarization and AICD).
Cytokine responses in Ccr2–/– mice following CII immunization. There is considerable evidence to suggest that in DBA/1J mice, CIA is a Th1-mediated inflammatory disease (5, 10, 48, 49). Typically, Th1 responses are evaluated by determining cytokine levels from the spleen or DLN of mice with CIA. However, recent studies indicate that the cytokine responses in the peripheral lymphoid organs reflect the immune reactivity against the mycobacterial component of the adjuvant, whereas the cytokine responses in the joints may mirror more accurately the immune response against collagen and not the adjuvant (9, 50, 51). Additionally, we reasoned that although several cytokines could be upregulated systemically, elucidation of those cytokines that drive the inflammatory process at the level of the joints (i.e., local microenvironment) may be especially informative regarding the Th mechanisms underlying the severe joint inflammation that we observed in _Ccr2_-null mice. For these reasons, we first determined the cytokine levels by RPA in the inflamed and noninflamed joints of _Ccr2_–/– mice.
Consistent with the notion that high levels of IFN-γ (9) may be a pathogenic feature of severe joint destruction in CIA, there was abundant expression of this cytokine in the inflamed, but not the noninflamed joints of _Ccr2_–/– mice derived from the same animal, whereas in contrast, Th2 cytokines (i.e., IL-4, IL-5, and IL-13) were undetectable (Table 3). Furthermore, the expression of CCL3, a chemokine associated with Th1 responses (52), as well as other proinflammatory chemokines, were also upregulated in the inflamed joints of _Ccr2_-null mice (Table 3). Notably, in light of the increased recruitment of neutrophils to the _Ccr2_-null joints, the expression of macrophage inflammatory protein 2 (MIP-2), a chemokine that is fairly specific for recruitment of neutrophils, was also elevated (53–55).
Inflammatory mediators in joints derived from CII-immunized _Ccr2_–/– mice
In addition to these Th1/Th2 cytokines, we determined the expression profile of additional inflammatory mediators such as IL-6 and TNF-α and chemokine receptors in the joints of WT and _Ccr2_-null mice (Table 4). Notably, an enhanced expression of IL-6, CCR1, and CCR5 was detected in _Ccr2_-null mice, but there were no differences in the expression levels of TNF-α or its receptors p55 and p75 between KO and WT mice (Table 4). These findings, taken together with the data shown in Table 3, suggest that a biased localized Th1 response and increased IL-6 production in _Ccr2_–/– mice may play a significant role in driving the inflammatory processes in the joint, and that the enhanced expression of several chemokines and their cognate receptors may account for the nature of the cellular infiltrate in the inflamed joints. At least at this early time point, the TNF system did not appear to contribute significantly to joint pathology; however, it is conceivable that at other time points during CIA this gene system may also play a key role.
Inflammatory mediators in joints derived from CII-immunized mice
To extend the aforementioned studies, we also determined the spontaneous as well as Ag (CII)-specific IFN-γ production from the spleens and DLN of WT and _Ccr2_-null mice 25 days after the second immunization, a time point at which joint inflammation was evident in the KO mice. Spontaneous IFN-γ (mean ± SD) production in the DLN (498 ± 284 pg/ml) and spleens (25 ± 33 pg/ml) was evident only in the _Ccr2_-null mice. Additionally, the Ag-induced IFN-γ levels in the DLN were also higher in _Ccr2_–/– (3,500 ± 147 pg/ml) compared with WT (1.7 ± 3 pg/ml; P < 0.001) mice. Ag-induced IFN-γ production in the spleens of _Ccr2_–/– (1,830 ± 803 pg/ml) and WT (1,174 ± 1,890 pg/ml) mice was similar. Interestingly, although IL-4 expression was not detected in the inflamed joints of _Ccr2_-null mice, the Ag-specific IL-4 production from the spleens (WT: 12 ± 12 pg/ml; _Ccr2_–/–: 182 ± 81 pg/ml; P < 0.05) and the DLN (WT: 2.3 ± 1.3 pg/ml; _Ccr2_–/–: 66 ± 64 pg/ml; P < 0.08) was also increased in _Ccr2_-null mice compared with WT mice. Thus, in the inflamed joints a Th1 response may be a pathogenic variable leading to joint destruction, whereas the systemic Th0 responses may reflect the responses to collagen as well as Mycobacterium Ag’s (9).
One possibility to account for this Th1-bias in the relevant local microenvironment was that this Th1 bias was secondary to the genetic background — that is, it was specific to the DBA/1J background in which CIA was elaborated. An alternative possibility is that this Th1 bias is due to the specific nature of the immunogen. To differentiate between these two possibilities, we resorted to the well-characterized model system used to characterize Th1/Th2 polarization in vivo, that is, the infection model with L. major. DBA/1J mice are normally resistant to L. major infection (56); however, we found that after intradermal infection of the ears of WT and _Ccr2_-null mice with L. major, these _Ccr2_-null mice in a DBA/1J background were highly susceptible to infection (increased ear swelling, higher parasite burdens) and increased Th2 (IL-4) levels in the DLN (Supplemental Table 7, and data not shown). Notably, susceptibility to L. major is characteristically associated with an increase in Th2 cytokines. Thus, it would appear that the Th response in DBA/1J mice is secondary to the nature of the immunological challenge: in CIA it is associated with a Th1 response, whereas in L. major infection it is associated with a Th2 response. Further supporting the notion that _Ccr2_-null mice can develop a Th1-biased response, we found that in a graft-versus-host-disease (GVHD) model, _Ccr2-_null mice in a C57BL/6 background develop an enhanced Th1 response associated with a poor GVHD outcome (26).
Normal Th polarization and proliferation but decreased AICD in Ccr2–/– T cells. We next determined whether abnormalities intrinsic to _Ccr2-_null T cells that are present before CII immunization could provide insights into the mechanisms leading to a severe CIA phenotype. In light of the observation that there was increased IFN-γ cytokine production in the inflamed joints as well as the DLN of _Ccr2_-null mice, we determined whether _Ccr2_-null Th cells had a higher intrinsic ability to produce Th1 cytokines after T cell receptor stimulation and under Th1-polarizing conditions. We found that highly purified naive T cells derived from WT and _Ccr2_–/– mice were comparable in their ability to differentiate and produce canonical Th1 or Th2 cytokines under appropriate polarizing conditions (Supplemental Table 8).
In light of the findings of normal Th1 and Th2 skewing in vitro, we hypothesized that either abnormal proliferation or T cell AICD rates in naive (unchallenged) _Ccr2_–/– mice may contribute to both increased cytokine and T cell–dependent Ab production. Although WT and _Ccr2_–/– DBA/1J mice had similar rates of T cell divisions, _Ccr2_-null splenocytes had a significant reduction in the percentage of apoptotic T cells after stimulation with anti-CD3 (Figure 4A). A similar phenotype was recapitulated when _Ccr2_–/– or WT T cells were stimulated with anti-CD3 plus anti-CD28. After 72 hours of stimulation with anti-CD3/CD28, the percentage of CD4+_Ccr2_-null apoptotic cells was significantly lower than in WT T cells (data not shown). These findings are concordant with our recent observation that _Ccr2_-null T cells derived from KO mice on a C57BL/6 background also had impaired AICD (26). This defect in AICD did not appear to be secondary to a gross defect in T cell receptor–dependent signaling, since the tyrosine phosphorylation patterns of critical kinases such as PLC-γ–1, ZAP-70, and MAPK1/2 induced by T cell receptor cross-linking using anti-CD3 Ab in highly purified T cells derived from WT and _Ccr2_–/– mice was similar (Supplemental Notes and data not shown).
Higher accumulation of activated T and B cells in vivo, and decreased AICD in vitro in _Ccr2_–/– mice. (A) Quantitative analysis of proliferation and apoptosis of WT or _Ccr2_-null splenocytes derived from naive mice after stimulation with anti-CD3. carboxy-fluorescein succinimidyl ester–labeled (CFSE-labeled) splenocytes from WT and _Ccr2_–/– mice were stimulated with soluble anti-CD3 antibody (2 μg/ml), and cellular proliferation was assessed by FACS after 72 hours. CFSE-labeled cells were also stained for CD4 and Annexin V, and the percentage of CD4+AnnexinV+ cells in each cell division was determined. The number of generations is noted in the upper left of each panel. Data shown in this panel are representative of at least three separate experiments derived from two animals per group. Single-cell suspensions were prepared from arthritic joints DLN derived from WT or _Ccr2_–/– mice 30 days after the second immunization with CII. Cell numbers (B) and activation markers on T and B cells (C and D) in the DLN of WT and _Ccr2_–/– mice are depicted. The cells were counted and stained with the fluorescent-labeled Ab’s shown in B and C. In D, the percentage of activated T cells (CD4+/CD44+) or (CD4+/RANKL+) or B cells (B220+/CD86+) within the total T or B cell population is shown. The findings shown in A–C are representative of three separate experiments, and the data depicted are from three WT and five _Ccr2_–/– mice.
If the aforementioned in vitro findings of reduced AICD in _Ccr2_-null T cells were potentially contributing to the CIA phenotype in _Ccr2_-null mice, we surmised that we should observe an increase in the accumulation of activated T and B cells after induction of CIA in _Ccr2_–/– mice and be able to transfer the disease phenotype to WT animals by adoptive transfer of _Ccr2_-null whole bone marrow cells and/or purified T cells.
Potential contributions of reduced AICD in Ccr2–/– mice to the CIA phenotype: accumulation of activated T and B cells. CII-immunized _Ccr2_–/– mice exhibited prominent lymphadenopathy. Consistent with this observation, the DLN of _Ccr2_-null mice had a greater cellular content (Figure 4B) that was associated with increased total numbers and percentages of activated T cells (CD4+CD44+ cells; Figure 4, C and D). Notably, we also found that the lymph nodes of _Ccr2_–/– mice had a higher accumulation of T cells that expressed the osteoclastogenic factor RANKL (CD4+RANKL+) that might potentially account for the severe bone loss observed in these mice (Figures 1F and 4, C and D) (32, 57). In addition, the DLN of _Ccr2_–/– mice had nearly three times as many B cells and, more importantly, the number of activated B cells was four times higher in these mice compared with WT mice (Figure 4, C and D, and data not shown). Thus, the increased cellularity in the DLN of _Ccr2_–/– mice was accompanied by an enhanced activation state of the T and B cells. This accumulation in the number of activated T cells was observed only after CII immunization, since prior to CIA induction the percentage of naive as well as activated and memory T cells in _Ccr2_–/– and WT mice was comparable (Supplemental Table 9).
Potential contributions of reduced AICD in Ccr2–/– mice to the CIA phenotype: adoptive-transfer studies. Bone marrow cells from naive WT or _Ccr2_–/– mice were adoptively transferred into lethally irradiated WT mice. At 3 months after transplant, a time point at which the hematopoietic system of the recipients was reconstituted (95% chimerism as described in Methods), we immunized these mice with CII. Wild-type mice reconstituted with WT bone marrow cells did not develop arthritis 3 months after the second immunization with CII (Table 5). In contrast, WT mice transplanted with _Ccr2_–/– bone marrow cells developed frank arthritis within 30 days after the second immunization with clinical arthritic scores similar to those observed in untransplanted _Ccr2_–/– mice immunized with CII (Table 5). In addition, compared with WT mice that were recipients of WT bone marrow cells, higher levels of anti-collagen IgGs were detected in WT recipients of _Ccr2_–/– bone marrow cells (Figure 5). Taken together, these findings indicated that WT DBA/1J mice adoptively transferred with _Ccr2_–/– cells had a CIA phenotype that mimics closely that observed in untransplanted _Ccr2_–/– DBA/1J mice, demonstrating that the expression of CCR2 in the hematopoietic compartment is a critical determinant of CIA.
Transplantation of CCR2-null hematopoietic cells is sufficient to transfer the CIA phenotype to WT animals. Bone marrow cells derived from WT and _Ccr2_–/– mice were transplanted into WT recipients that had been lethally irradiated (1,000 cGy). Three months after transplantation, the protocol to induce CIA was initiated as described in the Methods section. Collagen-specific Ab’s determined in the serum of transplanted animals two weeks after the second immunization.
Transfer of CIA phenotype to WT mice by hematopoietic cell transplant.
Whether the CIA phenotype can be adoptively transferred readily from CII-immunized donor mice into a syngeneic host (not allogeneic SCID) by transplantation of only purified T cells is not clear, since only very few studies have demonstrated that this is possible (58). Most adoptive-transfer studies have used CII-specific T cell clones or whole splenocytes followed by reimmunization of the mice with CII to induce disease (59–63). Nevertheless, we attempted to transfer the CIA phenotype by adoptive transfer of purified T cells using the protocols of Delgado et al. (58). WT or _Ccr2_-null DBA/1J mice were immunized with CII, and 14 days after immunization, T cells purified from their spleens were transferred intravenously into naive DBA/1J mice; the recipient mice were not subsequently challenged with CII. Up to 100 days after transplant we observed no phenotypic changes in the recipient mice.
Ccr2-null mice are highly susceptible to CAIA. The combined injection of anti-CII Ab’s on day 0 and LPS on day 2 induced arthritis in both WT and _Ccr2_–/– mice (Figure 6A). The first sign of slight swelling was detected around day 4, and paw swelling with redness reached a maximum on days 6–7. As demonstrated in previously published reports, we found that the CAIA-affected animals developed swollen, red, and ankylosed joints (12, 20). The swelling and associated other arthritic changes continued until day 10–12 in WT and _Ccr2_–/– mice. However, after this time point we observed a differential response in the resolution phase of the disease: signs of arthritis gradually decreased after day 12 and were minimal by day 23 in WT mice, whereas in _Ccr2_–/– mice signs of severe arthritis persisted, suggesting a delay in the processes that lead to a resolution in inflammation. Histological analysis confirmed these findings, since 23 days after injection of the arthritogenic Ab’s the joints of the WT mice had minimal signs of disease, whereas the joints of _Ccr2_–/– mice had prominent signs of chronic arthritis with pannus formation, and destructive bone and cartilage erosion, predominantly of the distal joints (Figure 6, B and C). Collectively, these findings indicated a possible requirement of CCR2 expression for the resolution of the ongoing inflammatory processes during CAIA.
Enhanced CAIA phenotype in _Ccr2_–/– mice. (A) Mean arthritic score after induction of CAIA was determined in WT and _Ccr2_–/– mice. Histopathological analyses (H&E staining) of joints from (B) WT and (C) Ccr2–/– mice. Significant joint destruction was evident in Ccr2–/– mice compared with WT mice. Letters indicate (a) chronic inflammatory infiltrate and extensive synoviocyte proliferation; (b) loss of interarticular space; (c) cyst formation. *P < 0.001.