We have utilized the sykf/f MRP8-cre+ mice, which lack Syk in neutrophils, to examine the role of these cells in the innate immune-mediated effector stage of inflammatory arthritis. Since Syk kinase is required for FcγR-induced signaling, the neutrophils in these mice fail to respond to immune complexes. Loss of Syk signaling in neutrophils is sufficient to protect mice from K/BxN serum induced arthritis. Clinical swelling is greatly reduced or absent in the sykf/f MRP8-cre+ mice; the synovium of these animals is undisturbed, with no evidence of bone and cartilage damage. Sykf/f MRP8-cre+ mice also demonstrate decreased antibody accumulation along the cartilage and lower serum cytokine levels. Further, Syk signaling pathways in neutrophils are required at several stages of immune response, including early induction of vascular permeability. However, using mixed chimeras, it is clear that Syk is not required for neutrophil migration into the joint if inflammation is already established. Neither mast cells nor basophils are required for arthritis development following K/BxN serum challenge. These observations help re-define the pathogenesis of inflammatory arthritis in this model and emphasize the neutrophil dependence of this disease.
These conclusions are based on the neutrophil specificity of the MRP8-cre
gene. Although monocytes/macrophages may express MRP8
), we found no significant deletion of Syk or upregulation of Cre expression in these cell types. Therefore, while we cannot completely rule out a contribution from syk
deletion in monocytes/macrophages, it would be minor compared to neutrophil deletion.
The original model of disease progression in K/BxN arthritis postulates that tissue resident mast cells are required for the initial recognition of anti-GPI immune complexes, promoting vascular permeability and antibody deposition, and elaborating proinflammatory mediators leading to neutrophil recruitment, activation and tissue injury (8
). Indeed, mast cell-deficient c-kitW/Wv
mice are protected from K/BxN serum induced arthritis, and disease can be restored by engraftment of wild type mast cells. However, we and others have found that the mast cell-deficient strain c-kitSh/Sh
is susceptible to K/BxN serum induced arthritis and the similar anti-collagen mAb/LPS injection model (12
). The difference between the c-kitSh/Sh
strains likely reflects the hematopoietic defect in the W/Wv mouse, which includes neutropenia (48
). Together, these results show that mast cell Fc/Syk signaling is not required for the disease process as previously thought. In contrast, recent studies suggest a critical role for neutrophil FcγRs. Arthritis progresses normally in human-FcγRIIA transgenic mice that express FcγRs only on neutrophils (20
), and in FcγRn/I/IIA/IIB/III/-deficient mice that express FcγRIV only on macrophages and neutrophils (12
). In both these models FcγR expression is absent in all other cell lineages, and it is not known whether forcing Fc receptor expression in other cell types would have a compensatory effect on disease development. In contrast, FcγR signaling is intact in all cells except neutrophils in sykf/f MRP8-cre
mice. In concert with the above studies, one can generally conclude that neutrophil recognition of immune complexes is the major driver of K/BxN serum induced arthritis.
By generating mixed bone marrow chimeras, we were able to directly compare the behavior of Syk-deficient and wild type neutrophils during inflammation. Combined with data from Sykf/f MRP8-cre+
mice, our results show that Syk deficiency has effects on specific pathways and mechanisms. Despite their expected inability to respond to immune complexes in vitro
, in an in vivo
inflammatory setting Syk-deficient neutrophils mobilize secretory granules normally, possibly in response to factors produced by wild type neutrophils (2
), and migrate normally. Syk deficiency does impede the ability of neutrophils to promote vascular permeability and produce cytokines at the inflammatory site. Syk-deficient neutrophils also show enhanced/accelerated cell death.
Systemic immune complexes induce both acute (5-10 minutes after administration) and long term (hours to days) changes to the vascular endothelium that increase permeability and extravasation (19
). Arthritis can occur in the absence of acute vascular leak in the joint, though disease is slightly attenuated (41
). Notably, Fc receptor expression by neutrophils can rescue arthritis on an FcRγ−/−
background, but does not rescue the vascular leak defect observable at 45 minutes post serum injection (20
). This observation appears to be at odds with our finding that deletion of Syk in neutrophils causes a decrease in antibody deposition in the joint. However, our results in the cutaneous Arthus reaction show that edema in response to immune complexes is reduced in sykf/f MRP8-cre+
mice after several hours. We suggest that very early acute vascular changes occur through a separate mechanism than the more long-term process of edema, where the latter at least partially depends on IgG recognition by neutrophils.
Syk is also responsible for signaling through other neutrophil receptors that utilize ITAM-dependent signaling pathways, such as integrins, some C-type lectin receptors and cytokine receptors (52
). Reduced integrin signaling may contribute to impaired neutrophil migration, as Syk-deficient neutrophils do show reduced recruitment in the cremaster muscle model following superfusion with chemoattractant peptides (53
). However, we have found no defect in Syk-deficient neutrophil recruitment in thioglycollate induced sterile peritonitis (54
), hemorrhagic vasculitis in the skin (55
) or as shown here in the K/BxN model of arthritis. We conclude that the loss of integrin signaling in Syk-deficient neutrophils has variable effects on cellular migration dependent on the inflammation model. Whether Syk deficiency would alter cellular recruitment in other tissue sites remains to be tested.
Since sykf/f MRP8-cre+
mice are protected from arthritis despite normal migration in Syk-deficient neutrophils, the block in inflammation likely occurs upstream of substantial neutrophil recruitment. Similar findings were reported with mice lacking BLT1, the LTB4 receptor, in which reconstitution with wild type neutrophils induced arthritis and the recruitment of BLT1−/−
neutrophils to the joint (19
). We propose that Syk-dependent signaling in neutrophils is required for the elaboration of chemokines and cytokines, such as TNFα and LTB4, which induce further recruitment of additional monocytes and neutrophils, leading to more cytokine production and tissue damage, in the fashion of a self-amplifying loop. Activation of tissue resident cells alone is not sufficient to induce significant neutrophil recruitment in sykf/f MRP8-cre+
mice, even though Syk-deficient neutrophils could otherwise migrate into the joint if a sufficient inflammatory signal was present. Undoubtedly, the majority of neutrophil activation occurs in response to tissue-deposited immune complexes. However, the decreased antibody deposition in joints of sykf/f MRP8-cre+
mice suggests that at least part of the neutrophil activation occurs in the peripheral blood.
The role of Fc receptors in human autoimmune disease is complex, since these molecules mediate both activating and inhibitory signaling. Hypomorphic alleles of the human FcγRIIA, RIIIA, and RIIIB are associated with increased disease severity and nephritis in patients with SLE (56
). These hypoactive FcγRs could result in decreased immune complex clearance, paradoxically leading to accumulation of IgGs in tissues that would mediate chronic immune cell activation (58
). Syk is therefore at attractive therapeutic target, as it is required for signaling through all FcγRs. Indeed, Syk inhibitors are efficacious in multiple animal models of autoimmune arthritis and SLE; phase II clinical trials with rheumatoid arthritis patients show promise (59
). Our data suggests that part of the efficacy of Syk inhibitors could stem from the inhibition of immune-complex induced activating signals in innate immune cells (7
Overall, these results suggest that signaling through Syk in neutrophils is the major mediator of arthritis in the K/BxN model, while immune complex recognition by other cells, in particular mast cells and basophils, plays a less important role in disease development. In combination with similar findings in immune complex nephritis (7
), this suggests that neutrophils are the dominant pathogenic cell in most immune complex-mediated diseases. Obviously, this hypothesis will require further testing, but it does significantly alter the pathogenic models of immune complex inflammation, particularly diminishing the role of mast cells. This would have direct implications in the development of cell-targeted therapeutics for treatment of immune complex disease.