Plasma from a patient with RA (RA1) and a normal donor N1 were injected i.p. into groups of 8–12-wk-old B6.FcγRIIB−/−
mice. At this age, there is minimal evidence for lupus-like autoimmune disease reported in older B6.FcγRIIB−/−
). Mice were injected with human samples on days 0, 2, and 7 for a total of 2.5 ml plasma per mouse. Plasma from RA1 caused mild soft tissue swelling indicated by ankle thickness and an overall arthritis score in all (6/6) mice tested (, top). Both hind ankles were affected; however, the involvement of other joints was difficult to detect by simple visual inspection. The joint swelling was transient and resolved by day 20. In contrast, none (0/4) of the mice injected with control plasma from healthy individual N1 developed measurable ankle inflammation or swelling (, B and C, bottom). The transient nature of the inflammation caused by RA1 plasma is consistent with mouse serum–induced models of arthritis that generally result in more severe lesions. One explanation for the transient nature of the lesions is accelerated clearance of human Abs in recipient FcγRIIB−/−
mice. To test this possibility, we monitored the clearance of human IgG Abs in mice using mouse sera collected at different time points. Levels of circulating human IgG Abs in mice injected with human plasma samples from both RA1 and N1 were maximal on day 3, followed by a second peak on day 8 (). The level of human IgG in the mouse circulation gradually decreased and disappeared by day 20. A similar pattern of IgG degradation was observed in mice injected with plasma from healthy control N1; however, the level of circulating human IgGs was significantly lower at all time points compared with that from the RA patient. We then tested whether FcγRIIB−/−
mice developed a mouse anti–human Ab (MAHA) response, which could potentially neutralize circulating pathogenic human Abs. Plasma from both RA patient RA1 and healthy individual N1 resulted in a gradual accumulation of MAHAs after the onset of inflammation (). This temporal pattern suggests that MAHAs do not contribute to the lesions and may diminish the pathogenic activity of the human plasma. Collectively, these results provide direct evidence that plasma from a patient with RA is able to initiate acute inflammatory arthritis when injected into genetically susceptible FcγRIIB−/−
Figure 1. Induction of inflammatory arthritis in FcγRIIB−/− with plasma from an RA patient. 8–12-wk-old B6.FcγRIIB−/− mice were injected with plasma from a patient with RA (RA1) or a healthy blood donor (N1) (more ...)
To test whether the absence of the inhibitory receptor FcγRIIB is necessary for arthritis initiation, age-matched B6 mice (n = 3) were injected with the same dose of plasma from RA1 (2.5 ml/mouse). As illustrated in , no mice (0/3) showed joint inflammation. This result demonstrates that FcγRIIB deficiency is required for mice to become susceptible to arthritis induced by human RA plasma.
Histological analysis was performed to determine whether lesions could be detected in the joints of FcγRIIB−/− mice injected with arthritogenic human plasma (). We observed a normal synovial lining, acellular joint space, and smooth articular cartilage in the ankle joints of mice (n = 2) injected with normal plasma N1 and killed on day 11 after initial injection (). In contrast, the joints of mice injected with plasma from RA1 and subsequently killed on days 11, 14, and 15 (total n = 3) had arthritic lesions. The mouse killed on day 11 had marked inflammation within the joint space and subsynovial tissues (). Mild synovial hyperthrophy and hyperplasia were present; however, the bone and articular cartilage were unaffected. The arthritic lesions were characterized by large numbers of inflammatory cells, predominantly neutrophils with some macrophages and a few lymphocytes (). The mice analyzed on days 14 and 15 (n = 2) had edema of the periarticular soft tissue as well as mild synovial hyperplasia and hypertrophy with diffuse inflammatory cell infiltrations (representative images shown in ). Within the joint space there was a small amount of fibrin mixed predominately with plasma cells, a lesser number of neutrophils, and a few fibroblasts (). The inflammatory infiltrates present in the synovium contained predominantly lymphocytic and plasmacytic cells with a moderate number of neutrophils and a few macrophages (). In addition, there was multifocal perivascular infiltration within perivascular soft tissue that contained neutrophils, macrophages, and a few multinuclear giant cells (). These results provide direct evidence that plasma from an individual with active RA contains pathogenic components able to initiate acute inflammatory arthritis.
Figure 2. Histopathology of ankle joints. (A) Hematoxylin and eosinstained tissue sections from the ankle joint obtained from a representative B6.FcγRIIB−/− mouse injected with plasma collected from healthy individual N1 and killed on day (more ...)
To determine whether the ability to promote inflammatory lesions is consistent among RA sera, serum samples obtained from three additional RA patients were tested (Table S1, available at http://www.jem.org/cgi/content/full/jem.20051951/DC1
). Patients were classified as having RA on the basis of the American College of Rheumatology criteria (16
). Groups of FcγRIIB−/−
mice were injected with sera collected from three additional RA patients (RA2, RA3, and RA4) and three healthy blood donors (N2, N3, and N4) using the same injection protocol. Once again, we observed moderate severity and onset of arthritis in mice injected with the pathogenic sera from RA patients (, and Table S2, available at http://www.jem.org/cgi/content/full/jem.20051951/DC1
), but not with sera from three additional normal blood donors ( and Table S2).
Figure 3. Passive transfer of arthritis with sera obtained from three additional RA patients. FcγRIIB−/− mice were injected with sera from three additional arthritic patients (RA2, RA3, and RA4; A, B, and C) and three normal blood donors (more ...)
Patients with RA demonstrate hypergammaglobulinemia with a wide range of RA-associated autoAbs. Therefore, we wanted to determine whether RA-related autoAbs were present in human blood samples collected from the RA patients and healthy controls. The total concentrations of IgG were variable in the samples obtained from the RA patients as well as healthy controls (). The IgG isotype ratios of sera obtained from both RA patient and healthy control groups were similar (Table S3, available at http://www.jem.org/cgi/content/full/jem.20051951/DC1
). Of the RA-associated Abs tested, anti-GPI Ab was more prevalent in plasma from RA1, yet there was no difference in the level of this autoAb in the other three RA patients in the cohort as compared with healthy individuals (). Anti-CCP Abs were elevated in three out of four samples from RA patients () and was not detected in sera obtained from healthy donors. All RA patients and one normal donor (N2) were positive for IgM RF (), and all four arthritic patients and two of the normal blood donors (N1 and N4) were IgG RF+
(). Although there was considerable variation in autoAb levels among the RA and normal donor sera, the anti-CCP and IgM RF correlated best with the potential to induce inflammatory arthritis in mice.
Figure 4. Levels of human IgG Abs. (A) Total IgG (mg/ml). (B) Anti-GPI Abs (OD, 405 nm). (C) Anti-CCP IgG Abs (relative units, RU/ml). Values >5 RU/ml were considered positive. Mean ± SD is indicated. (D) IgM RF (values ≥20 RU/ml are considered (more ...)
Plasma from RA patient RA1 and healthy blood donor N1 was fractionated into IgG+
components (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20051951/DC1
), and their disease-inducing capacity was tested in FcγRIIB−/−
mice (). The IgG+
fraction obtained from RA patient RA1, but not the IgG−
fraction, was capable of provoking arthritis in FcγRIIB−/−
recipient mice (). At this concentration (total of 47.4 mg IgG/mouse), the potency of the IgG+
fraction was similar to whole plasma with the same relative IgG concentration in the starting volume ( and ). Furthermore, when IgG−
fractions were combined and injected (), mice developed arthritis with the same arthritic lesions as IgG+
() and the whole plasma group (). In contrast, mice injected with IgG-bound (14.7 mg IgG/mouse) or unbound IgG fractions (<0.0001 mg IgG/mouse) from nonarthritic donor N1 did not show any inflammation even when IgG concentrations three times (44.1 mg IgG/mouse) above that obtained from normal N1 plasma were used (). IgG−
fractions from one additional patient with established RA were purified and injected into FcγRIIB−/−
mice (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20051951/DC1
). Once again the IgG+
but not IgG−
fraction was able to in duce mild and transient arthritic lesions in mice. Collectively, these results suggest that the pathogenic component of the human plasma leading to induction of arthritis in mice resides in the IgG fraction.
Figure 5. Induction of arthritis with purified human IgG Abs from patient RA1. Groups of FcγRIIB−/− mice (n = 3 each group) were injected with protein G column-separated IgG− and IgG+ fractions of human blood samples. (more ...)
To date, few studies have documented the successful passive transfer of arthritis into mice by injection of purified human IgG Abs. Transfusion of peripheral leukocytes, lymph node cells, and plasma from patients with RA into nonrheumatoid human recipients failed to provoke any evidence of rheumatic disease (17
). Other experiments included injections of a patient's own IgG Abs into one knee and an identical dose of rheumatoid IgG into the other (18
), resulting in RA serum–induced inflammatory reactions persisting for several days. In 1984, Wooley et al. (19
) reported that purified anti–type II collagen IgGs from a patient with seronegative rheumatoid-like arthritis induced mild, transient arthritis in 20–25% of the injected collagen type II–susceptible mice. To our knowledge, there has been no further documentation of the reliable induction of arthritic lesions in mice after transfer of human Abs. Therefore, despite the presence of autoAbs in many patients with RA and related arthropathies, their importance in autoimmune pathogenesis remains uncertain.
We used a mouse model sensitized by a deficiency in Fc receptor FcγRIIB to exaggerate humoral immunity as a bioassay to investigate the pathogenic activity of human RA–associated Abs. Our data show that (a) plasma or serum collected from individuals with active RA was able to initiate inflammatory arthritis in B6.FcγRIIB−/− mice with pathological features of human RA; (b) the inflammatory lesions in recipient animals are mild and transient in nature but highly reproducible; (c) the pathogenic component of the human plasma from RA1 leading to induction of arthritis in mice resides in the Ig fraction.
These studies suggest that human Abs from RA patients can inflict pathological lesions consistent with the underlying RA syndrome. Although too limited to establish an actual link between human-specific autoAbs and RA, they raise the intriguing possibility that the FcγRIIB−/−
mouse model can be used as a bioassay to thoroughly evaluate the pathogenic involvement of humoral autoimmunity in RA. In contrast to mouse anti-GPI and anti–collagen type II Abs, the specificities of human Abs playing a role in the pathogenesis of RA are poorly defined. In particular, Abs that react with one or more ubiquitous normal protein (e.g., GPI) show poor association with human RA, whereas Abs reactive with citrullinated proteins show better correlation (20
). It remains to be demonstrated whether anti-CCP, or other undefined autoAbs, are responsible for the inflammatory lesions we observed. Our studies suggest the FcγRIIB−/−
mouse system can be used productively for evaluating the pathological potential of currently known and novel RA-associated human Abs. Conceivably, the same model also could be used to evaluate Abs associated with other human autoimmune disorders. Finally, this model has a potential application as a disease model for examining novel anti-autoimmune therapies.