Alan M. Fong

Defective lymphocyte chemotaxis in β-arrestin2- and GRK6-deficient mice

Lymphocyte chemotaxis is a complex process by which cells move within tissues and across barriers such as vascular endothelium and is usually stimulated by chemokines such as stromal cell-derived factor-1 (CXCL12) acting via G protein-coupled receptors. Because members of this receptor family are regulated (“desensitized”) by G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and β-arrestin binding, we examined signaling and chemotactic responses in splenocytes derived from knockout mice deficient in various β-arrestins and GRKs, with the expectation that these responses might be enhanced. Knockouts of β-arrestin2, GRK5, and GRK6 were examined because all three proteins are expressed at high levels in purified mouse CD3+ T and B220+ B splenocytes. CXCL12 stimulation of membrane GTPase activity was unaffected in splenocytes derived from GRK5-deficient mice but was increased in splenocytes from the β-arrestin2- and GRK6-deficient animals. Surprisingly, however, both T and B cells from β-arrestin2-deficient animals and T cells from GRK6-deficient animals were strikingly impaired in their ability to respond to CXCL12 both in transwell migration assays and in transendothelial migration assays. Chemotactic responses of lymphocytes from GRK5-deficient mice were unaffected. Thus, these results indicate that β-arrestin2 and GRK6 actually play positive regulatory roles in mediating the chemotactic responses of T and B lymphocytes to CXCL12.

Chemokine receptor mutant CX3CR1-M280 has impaired adhesive function and correlates with protection from cardiovascular disease in humans

The chemokine receptor CX3CR1 is a proinflammatory leukocyte receptor specific for the chemokine fractalkine (FKN or CX3CL1). In two retrospective studies, CX3CR1 has been implicated in the pathogenesis of atherosclerotic cardiovascular disease (CVD) based on statistical association of a common receptor variant named CX3CR1-M280 with lower prevalence of atherosclerosis, coronary endothelial dysfunction, and acute coronary syndromes. However, the general significance of CX3CR1-M280 and its putative mechanism of action have not previously been defined. Here we show that FKN-dependent cell-cell adhesion under conditions of physiologic shear is severely reduced in cells expressing CX3CR1-M280. This was associated with marked reduction in the kinetics of FKN binding as well as reduced FKN-induced chemotaxis of primary leukocytes from donors homozygous for CX3CR1-M280. We also show that CX3CR1-M280 is independently associated with a lower risk of CVD (adjusted odds ratio, 0.60, P = 0.008) in the Offspring Cohort of the Framingham Heart Study, a long-term prospective study of the risks and natural history of this disease. These data provide mechanism-based and consistent epidemiologic evidence that CX3CR1 may be involved in the pathogenesis of CVD in humans, possibly by supporting leukocyte entry into the coronary artery wall. Moreover, they suggest that CX3CR1-M280 is a genetic risk factor for CVD.

β-Arrestin-2 regulates the development of allergic asthma

Asthma is a chronic inflammatory disorder of the airways that is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. Migration of Th2 cells to the lung is key to their inflammatory function and is regulated in large part by chemokine receptors, members of the seven-membrane-spanning receptor family. It has been reported recently that T cells lacking beta-arrestin-2, a G protein-coupled receptor regulatory protein, demonstrate impaired migration in vitro. Here we show that allergen-sensitized mice having a targeted deletion of the beta-arrestin-2 gene do not accumulate T lymphocytes in their airways, nor do they demonstrate other physiological and inflammatory features characteristic of asthma. In contrast, the airway inflammatory response to LPS, an event not coordinated by Th2 cells, is fully functional in mice lacking beta-arrestin-2. beta-arrestin-2-deficient mice demonstrate OVA-specific IgE responses, but have defective macrophage-derived chemokine-mediated CD4+ T cell migration to the lung. This report provides the first evidence that beta-arrestin-2 is required for the manifestation of allergic asthma. Because beta-arrestin-2 regulates the development of allergic inflammation at a proximal step in the inflammatory cascade, novel therapies focused on this protein may prove useful in the treatment of asthma.

CX3CR1 Deficiency Impairs Dendritic Cell Accumulation in Arterial Intima and Reduces Atherosclerotic Burden

Objective—Dendritic cells (DCs) have recently been found in atherosclerosis-predisposed regions of arteries and have been proposed to be causal in atherosclerosis. The chemokine receptor CX3CR1 is associated with arterial injury and atherosclerosis. We sought to determine whether a link exists between arterial DC accumulation, CX3CR1, and atherosclerosis. Methods and Results—Mouse aortas were isolated and subjected to en face immunofluorescence analysis. We found that DCs were located predominantly in the intimal regions of arterial branch points and curvatures. Consistent with the increased accumulation of intimal DCs in aged and ApoE−/− aortas compared with young WT aortas (P=0.004 and 0.05, respectively), the incidence of atherosclerosis was 88.9% for aged WT and 100% for ApoE−/− mice compared with 0% for young WT mice. CX3CR1 was expressed on intimal DCs and DC numbers were decreased in CX3CR1-deficient aortas of young, aged, and ApoE−/− mice (P=0.0008, 0.013, and 0.0099). The reduced DC accumulation in CX3CR1-deficiency was also correlated with decreased atherosclerosis in these animals. Conclusions—The accumulation of intimal DC increases in aged and ApoE−/− aortas and correlates with the generation of atherosclerosis. CX3CR1-deficiency impairs the accumulation of DC in the aortic wall and markedly reduces the atherosclerotic burden.

Ultrastructure and Function of the Fractalkine Mucin Domain in CX3C Chemokine Domain Presentation

Fractalkine (FKN), a CX3C chemokine/mucin hybrid molecule on endothelium, functions as an adhesion molecule to capture and induce firm adhesion of a subset of leukocytes in a selectin- and integrin-independent manner. We hypothesized that the FKN mucin domain may be important for its function in adhesion, and tested the ability of secreted alkaline phosphatase (SEAP) fusion proteins containing the entire extracellular region (FKN-SEAP), the chemokine domain (CX3C-SEAP), or the mucin domain (mucin-SEAP) to support firm adhesion under flow. CX3C-SEAP induced suboptimal firm adhesion of resting peripheral blood mononuclear cells, compared with FKN-SEAP, and mucin-SEAP induced no firm adhesion. CX3C-SEAP and FKN-SEAP bound to CX3CR1 with similar affinities. By electron microscopy, fractalkine was 29 nm in length with a long stalk (mucin domain), and a globular head (CX3C). To test the function of the mucin domain, a chimeric protein replacing the mucin domain with a rod-like segment of E-selectin was constructed. This chimeric protein gave the same adhesion of peripheral blood mononuclear cells as intact FKN, both when immobilized on glass and when expressed on the cell surface. This implies that the function of the mucin domain is to provide a stalk, extending the chemokine domain away from the endothelial cell surface to present it to flowing leukocytes.

Defective antitumor responses in CX3CR1-deficient mice

Innate immunity is critically important for tumor surveillance and regulating tumor metastasis. Fractalkine (FKN, CX3CL1), operating through the receptor CX3CR1, is an effective chemoattractant and adhesion receptor for NK cells and monocytes, important constituents of the innate immune response. Previous studies have shown that over‐expression of CX3CL1 by tumor cells enhances antitumor responses. However, since most tumors do not express CX3CL1, it remains unclear if CX3CL1/CX3CR1 has a role in tumor immunity in the absence of ligand over‐expression. To determine the role of CX3CL1 and CX3CR1 in regulating antitumor immune responses, we tested the response of wildtype and CX3CR1‐deficient animals to unmanipulated B16 melanoma that does not express CX3CL1. We studied the distribution and trafficking of mononuclear cells (MNC) under homeostatic conditions and in the presence of B16 metastatic melanoma, cytotoxic activity, and cytokine production in wild‐type and CX3CR1‐deficient animals. We found that B16‐treated CX3CR1−/− mice had increased lung tumor burden and cachexia. There was a selective reduction of monocytes and NK cells in the lungs of CX3CR1‐deficient animals under homeostatic conditions and in response to B16. CX3CR1‐deficient NK cells effectively killed B16 cells in cytotoxicity assays. However, CX3CR1‐deficient NK cells exhibited a tumorigenic cytokine production profile with defective IFN‐γ expression and enhanced IL‐6 production in response to TLR3 activation with polyIC. Our studies indicate that CX3CR1 is an important contributor to innate immunity at multiple levels. Its role in tumor immunity is not limited by expression of CX3CL1 by tumor cells.

Increased Acute Inflammation, Leukotriene B4-Induced Chemotaxis, and Signaling in Mice Deficient for G Protein-Coupled Receptor Kinase 6

Directed migration of polymorphonuclear neutrophils (PMN) is required for adequate host defense against invading organisms and leukotriene B4 (LTB4) is one of the most potent PMN chemoattractants. LTB4 exerts its action via binding to BLT1, a G protein-coupled receptor. G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases (GRK) in an agonist-dependent manner, resulting in receptor desensitization. Recently, it has been shown that the human BLT1 is a substrate for GRK6. To investigate the physiological importance of GRK6 for inflammation and LTB4 signaling in PMN, we used GRK6-deficient mice. The acute inflammatory response (ear swelling and influx of PMN into the ear) after topical application of arachidonic acid was significantly increased in GRK6−/− mice. In vitro, GRK6−/− PMN showed increased chemokinetic and chemotactic responses to LTB4. GRK6−/− PMN respond to LTB4 with a prolonged increase in intracellular calcium and prolonged actin polymerization, suggesting impaired LTB4 receptor desensitization in the absence of GRK6. However, pre-exposure to LTB4 renders both GRK6−/− as well as wild-type PMN refractory to restimulation with LTB4, indicating that the presence of GRK6 is not required for this process to occur. In conclusion, GRK6 deficiency leads to prolonged BLT1 signaling and increased neutrophil migration.

CX3CR1 Deficiency Confers Protection From Intimal Hyperplasia After Arterial Injury

Objective—A functional polymorphism in the chemokine receptor CX3CR1 is associated with protection from vascular diseases including coronary artery disease and internal carotid artery occlusive disease. We investigated the mechanisms by which CX3CR1 may be involved by evaluating the inflammatory response to arterial injury in CX3CR1-deficient animals. Methods and Results—Femoral arteries of CX3CR1−/− and wild-type (WT) mice were injured with an angioplasty guide wire. After 1, 5, 14, and 28 days, arteries were harvested and evaluated by histology, morphometry, and immunohistochemistry. Arterial injury upregulated the CX3CR1 ligand CX3CL1. In CX3CR1−/− compared with WT animals, the incidence of neointima formation was 58% lower (P=0.0017), accompanied by no difference in the area of platelet accumulation at day 1 (P=0.48) but a significant decrease in intimal monocyte infiltration at day 5 (P=0.006), vascular smooth muscle cell (VSMC) proliferation at days 5 and 14, and intimal area at day 28 (P=0.009). Conclusions—In an endothelial denudation injury model, CX3CR1 deficiency protects animals from developing intimal hyperplasia as a result of decreased monocyte trafficking to the lesion. CX3CR1 deficiency decreases VSMC proliferation and intimal accumulation either directly or indirectly as a result of defective monocyte infiltration.

Enhanced Th17-Cell Responses Render CCR2-Deficient Mice More Susceptible for Autoimmune Arthritis

CCR2 is considered a proinflammatory mediator in many inflammatory diseases such as rheumatoid arthritis. However, mice lacking CCR2 develop exacerbated collagen-induced arthritis. To explore the underlying mechanism, we investigated whether autoimmune-associated Th17 cells were involved in the pathogenesis of the severe phenotype of autoimmune arthritis. We found that Th17 cells were expanded approximately 3-fold in the draining lymph nodes of immunized CCR2−/− mice compared to WT controls (p = 0.017), whereas the number of Th1 cells and regulatory T cells are similar between these two groups of mice. Consistently, levels of the Th17 cell cytokine IL-17A and Th17 cell-associated cytokines, IL-6 and IL-1β were approximately 2–6-fold elevated in the serum and 22–28-fold increased in the arthritic joints in CCR2−/− mice compared to WT mice (p = 0.04, 0.0004, and 0.01 for IL-17, IL-6, and IL-1β, respectively, in the serum and p = 0.009, 0.02, and 0.02 in the joints). Furthermore, type II collagen-specific antibodies were significantly increased, which was accompanied by B cell and neutrophil expansion in CCR2−/− mice. Finally, treatment with an anti-IL-17A antibody modestly reduced the disease severity in CCR2−/− mice. Therefore, we conclude that while we detect markedly enhanced Th17-cell responses in collagen-induced arthritis in CCR2-deficient mice and IL-17A blockade does have an ameliorating effect, factors additional to Th17 cells and IL-17A also contribute to the severe autoimmune arthritis seen in CCR2 deficiency. CCR2 may have a protective role in the pathogenesis of autoimmune arthritis. Our data that monocytes were missing from the spleen while remained abundant in the bone marrow and joints of immunized CCR2−/− mice suggest that there is a potential link between CCR2-expressing monocytes and Th17 cells during autoimmunity.

Decreased Th17 and antigen‐specific humoral responses in CX3CR1‐deficient mice in the collagen‐induced arthritis model

OBJECTIVE CX(3) CR1 is a chemokine receptor that uniquely binds to its ligand fractalkine (CX(3) CL1) and has been shown to be important in inflammatory arthritis responses, largely due to its effects on cellular migration. This study was undertaken to test the hypothesis that genetic deficiency of CX(3) CR1 is protective in the chronic inflammatory arthritis model collagen-induced arthritis (CIA). Because CX(3) CR1 is expressed on T cells and antigen-presenting cells, we also examined adaptive immune functions in this model. METHODS Autoantibody formation, clinical, histologic, T cell proliferative, and cytokine responses were evaluated in wild-type and CX(3) CR1-deficient DBA/1J mice after immunization with heterologous type II collagen (CII). RESULTS CX(3) CR1(-/-) mice had an ∼30% reduction in arthritis severity compared to wild-type mice, as determined by 2 independent measures, paw swelling (P < 0.01) and clinical disease score (P < 0.0001). Additionally, compared to wild-type mice, CX(3) CR1(-/-) mice had an ∼50% decrease in anti-CII autoantibody formation (P < 0.05), decreased Th17 intraarticular cytokine expression (P < 0.01 for interleukin-17 [IL-17] and P < 0.001 for IL-23), and decreased total numbers of Th17 cells in inflamed joints (P < 0.05). CONCLUSION Our findings indicate that CX(3) CR1 deficiency is protective in inflammatory arthritis and may have effects that extend beyond migration that involve adaptive immune responses in autoimmune disease.