GPCR-mediated leukocyte trafficking is critical in inflammatory disease states, and the GRK regulatory system has been implicated in animal models of autoimmune disease. Recently, GRK6-deficient animals have been shown to have increased neutrophil migration to the gut and impaired regulatory T cell responses in a DSS-induced colitis model of inflammatory bowel disease [15
]. In the EAE model of multiple sclerosis, GRK2 heterozygote animals exhibit earlier inflammation that correlates with T cell and macrophage infiltration into the CNS, but then subsequently have less disease and inflammatory cell infiltrate than wildtype controls at later time points [17
]. Given that GRK6 and -2 levels are reduced in disease flares of adjuvant induced arthritis [12
] and in the PBMC of rheumatoid arthritis patients [11
], we investigated how and which GRK subtypes play a role in leukocyte migration to the joint in an acute model of inflammatory arthritis, the K/BxN serum transfer model. This model system closely mirrors the acute flares of rheumatoid arthritis and allowed us to evaluate mononuclear cell contribution toward disease independent of the lymphocytic response.
The in vitro
studies further support a role for GRK6 and -2 in granulocyte-mediated chemotaxis to LTB4 and C5a (). LTB4 is a potent neutrophil chemoattractant [24
] and critical for disease in the K/BxN model [22
]. One of its high affinity receptors (BLT1) is a GPCR that has been shown to have GRK6 and -2 mediated receptor internalization [25
] and desensitization [26
] respectively. Previous work by Kavelaars et al. has demonstrated enhanced chemotaxis in GRK6 deficient granulocytes in vitro
to LTB4 [14
], and our data here support this finding. We also show that GRK6 deficient granulocytes have enhanced chemotaxis to C5a compared to controls. The C5a anaphylatoxin receptor (C5aR) is a GPCR important in the chemotaxis and activation of myeloid cells [27
] and necessary for K/BxN disease expression [23
]. Over expression of wildtype or catalytically inactive forms of GRK6 and -2 in COS-7 cells does not change agonist-mediated phosphorylation of C5aR [28
]. However, Milcent et. al. noted that transfection of COS-7 cells with wildtype GRK6 consistently increased expression of C5aR by two fold and that GRK6 appeared to be autophosphorylated [28
], suggesting a biologic relevance not apparent in this in vitro
model system. Our data suggests that GRK6 may have receptor-kinase interactions mediated through the BLT and C5aR receptors on granulocytes that are physiologically relevant as it pertains to inflammatory disease.
Interleukin-8 (IL-8 or CXCL8) is also a known chemoattractant of granulocytes and is thought to have pathophysiologic significance in rheumatoid arthritis through its chemoattractant [29
] and angiogenic [30
] properties. However, we did not observe differences in granulocyte migration to IL-8 in the animals with gene defects in GRK2, -5, or -6 compared to controls (). The role of IL-8 in the K/BxN model in specific is largely unknown, whereas LTB4 and C5a in this model have been found to be of critical importance to disease pathogenesis [22
Our data also suggest that the enhanced inflammatory disease seen in GRK2 heterozygote mice may be mediated through one or both of the BLT GPCRs. GRK2+/− granulocytes have slightly increased chemotaxis to LTB4 in vitro
, albeit our results did not achieve statistical significance. This could be partially explained by the heterozygous expression of GRK2 that may have reduced this phenotype. Further support that GRK2 plays an important role in BLT1 signaling has been suggested in transfection model systems [25
]. LTB4-induced internalization of BLT1 can be blocked by dominant negative GRK2 co-expression, and GRK2 co-localizes with the BLT1 receptor through the C terminus [25
We also show that in addition to increased organ-specific inflammation, GRK6-deficient animals have enhanced systemic effects in the K/BxN model. Weight loss is observed in inflammatory disease states such as rheumatoid arthritis and has been linked to elevated cytokine production, particularly TNF-α [31
]. We were unable to reliably detect TNF-α in the serum of our arthritic animals to investigate its role in the cachexia, but we do show that GRK6−/− animals have elevated systemic levels of IL-6 that coincide with the observed weight loss. IL-6 can be elevated in rheumatoid arthritis patients and may correlate with disease activity [32
]. In addition, IL-6 has been associated with profound cachectic states such as malignancy [33
]. Importantly, IL-6 is not elevated chronically in the K/BxN model in either wildtypes or GRK6-deficient animals ( and unpublished observations), and the presence of malignancy has not been observed in the K/BxN model in either controls or GRK-deficient mice.
Unlike the DSS-colitis model studied by Kavelaars et al., we did not observe significant differences in systemic production of IL-1β between groups [15
]. This could be explained by the different types of inflammatory responses being observed (i.e. DSS, chronic v. K/BxN, acute) or by different pathophysiologic mechanisms regulating the two disease models. Although we did observe increased weight loss in the GRK2+/− mice, we did not detect any significant differences in systemic cytokine production, possibly because their overall weight loss and disease was less severe.
In conclusion, the GRK system is an important regulatory pathway in the K/BxN serum transfer model of arthritis, particularly as it pertains to the early infiltration of granulocyte-mediated acute inflammatory responses. Granulocytes, which are critical to disease expression in the K/BxN model, have increased chemotaxis to LTB4 and C5a, but not IL-8, and GRK6−/− mice have more arthritis, weight loss, and IL-6 production. These data highlight the unique and subtype-specific effects of the GRKs on leukocyte trafficking and inflammatory disease and underscore the important regulatory role that GRK molecules may play in the acute versus chronic phases of autoimmunity. Although specific gene polymorphisms in the GRKs have not been described in the human rheumatoid arthritis population, an increased understanding of these regulators as they pertain to acute versus chronic inflammatory cell trafficking may lead to targeted therapies or diagnostics for patients with rheumatoid arthritis or other autoinflammatory diseases.