Research ArticleINFLAMMATION

Atypical complement receptor C5aR2 transports C5a to initiate neutrophil adhesion and inflammation

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Science Immunology  10 May 2019:
Vol. 4, Issue 35, eaav5951
DOI: 10.1126/sciimmunol.aav5951

Atypical attraction

Neutrophils contribute to inflammatory responses by moving from the circulation into target tissues through a series of chemoattractant-driven steps involving arrest and transmigration. Here, Miyabe et al. use an immune complex–induced arthritis murine model to show that neutrophil arrest requires endothelial expression of the atypical complement C5a receptor 2 (C5aR2) to drive transport of C5a into the vessel lumen, which is required for initiation of C5aR1-driven neutrophil arrest. Expression of the atypical chemokine receptor 1 (ACKR1) on endothelial cells mediates transport of CXCR2 chemokine ligands into the lumen and CXCR2-dependent transendothelial migration of neutrophils. These results clarify a role for “atypical” chemoattractant receptors working in conjunction with classical chemoattractant receptors to drive neutrophil arrest and diapedesis.

Abstract

Chemoattractant-induced arrest of circulating leukocytes and their subsequent diapedesis is a fundamental component of inflammation. However, how tissue-derived chemoattractants are transported into the blood vessel lumen to induce leukocyte entry into tissue is not well understood. Here, intravital microscopy in live mice has shown that the “atypical” complement C5a receptor 2 (C5aR2) and the atypical chemokine receptor 1 (ACKR1) expressed on endothelial cells were required for the transport of C5a and CXCR2 chemokine ligands, respectively, into the vessel lumen in a murine model of immune complex–induced arthritis. Transported C5a was required to initiate C5aR1-mediated neutrophil arrest, whereas transported chemokines were required to initiate CXCR2-dependent neutrophil transdendothelial migration. These findings provide new insights into how atypical chemoattractant receptors collaborate with “classical” signaling chemoattractant receptors to control distinct steps in the recruitment of neutrophils into tissue sites of inflammation.

INTRODUCTION

Chemoattractant-induced egress of leukocytes from the circulation into the extravascular space is a fundamental component of inflammation. Leukocyte recruitment from blood into tissues follows a well-established paradigm: (i) tethering and rolling on the vessel wall in the direction of flow; (ii) firm arrest on the endothelium; (iii) spreading out and crawling in all directions on the vessel to locate a receptive location for (iv) transendothelial migration (TEM) to extravasate into tissue. This process of leukocyte recruitment across post-capillary blood vessels into tissue is tightly controlled by adhesion molecules and chemoattractants (1).

Chemoattractants that induce integrin-mediated leukocyte firm arrest and subsequent diapedesis, such as chemokines, are thought in large part to be produced by activated endothelial cells (ECs) in an inflammatory site (1). These endothelially produced chemokines are then retained by proteoglycans on the surface of the endothelium where they guide localized leukocyte recruitment (2). However, the mechanism by which leukocytes enter sites of inflammation where the endothelium has not been activated to produce chemokines is not entirely clear. This would likely be most relevant at early times in an inflammatory reaction when chemoattractants might be produced in the tissue before the activation of the overlying endothelium. Leukocyte recruitment induced by some chemoattractants, such as C5a, fMLP, and LTB4, was found to be independent of protein synthesis and EC activation (3). The role of these tissue-derived chemoattractants in signaling to circulating leukocytes to enter sites of inflammation is also unclear.

Atypical chemokine receptor 1 (ACKR1) has been postulated to play a role in this process by transporting chemokines produced in tissue across the endothelium into the vessel lumen (4). ACKRs are seven transmembrane spanning proteins structurally related to conventional chemokine receptors but are not coupled to G proteins and therefore do not activate the signaling pathways necessary to induce cell migration (57). To date, four ACKRs (ACKR1-4) have been described. ACKRs act as chemokine sinks helping to maintain chemokine gradients in vivo across cells. In addition, ACKR1 (previously called DARC for Duffy antigen receptor) expressed on ECs has been shown to transcytose multiple inflammatory chemokines, including CXCR2 ligands, across ECs in vitro (4). Mice deficient in Ackr1 have decreased neutrophil recruitment into sites of inflammation in several disease models, suggesting that ACKR1 may play this role in vivo (4, 812). In addition, a recent study demonstrated a role for endothelial ACKR1 in facilitating neutrophil TEM (13). However, in lipopolysaccharide-induced models of peritoneal and lung inflammation, Ackr1 deficiency resulted in increased neutrophil recruitment into the peritoneum and alveolar space, respectively, suggesting that ACKR1 might also modulate inflammation as a sink for chemokines (14, 15). Furthermore, a variant of ACKR1 that selectively abolishes its expression on erythroid cells, including nucleated erythroid cells in the bone marrow (BM), results in neutropenia in healthy individuals of African ancestry (16, 17). Thus, the role of ACKR1 in neutrophil biology is complex and is context and cell type dependent.

In the K/BxN arthritogenic serum transfer (AST) model of immune complex (IC)–induced arthritis, neutrophils are the main effector cells required for the development of arthritis (18, 19). In contrast, mast cells are only required in certain strains of mice that have a relative neutrophil deficiency (20, 21), and macrophages can modulate the anti-inflammatory role of intravenous immunoglobulin (IVIG) in the model but are not required effector cells (22). Recently, we adapted the technique of multiphoton intravital microscopy (MP-IVM) to image neutrophil recruitment into the joint in the K/BxN AST model of IC-induced arthritis (23). We found that the complement C5a receptor 1 (C5aR1) expressed on neutrophils was critical for initial integrin-dependent neutrophil arrest and spreading on the endothelium of the joint in the AST model of arthritis (24). In this model, C5a is generated by the alternative pathway on the cartilage surface of the joint (25) and accumulates in the joint fluid before other chemoattractants (24). Furthermore, C5a is retained on the surface of ECs by proteoglycans where it can be presented to rolling neutrophils (24). However, it is not known how tissue-derived C5a is transported into the blood vessel lumen to induce C5aR1-mediated neutrophil arrest on the endothelium.

In addition to C5aR1, C5a binds a second seven-transmembrane spanning receptor called C5L2 or C5aR2 (2628). C5aR2 is widely expressed on myeloid cells (29), ECs (30), and epithelial cells (31). Unlike C5aR1, C5aR2 does not bind G proteins and therefore does not initiate G protein–dependent signaling (27). Thus, C5aR2 has been considered an “atypical” receptor that scavenges C5a. More recently, C5aR2 has been shown to elicit cellular signaling via β-arrestin (32) and can alter the course of tissue inflammation in a pro- or anti-inflammatory manner depending on the model (3337). Thus, the function of C5aR2 and its role in neutrophil recruitment in vivo are not well understood.

Here, we use MP-IVM of the arthritic joint to define a new role for C5aR2 expressed on the joint endothelium. We observe C5aR2 playing a role in transporting tissue-derived C5a into the blood vessel lumen to induce the initial arrest of neutrophils mediated by C5aR1 expressed on circulating neutrophils. In addition, we found that ACKR1 transports CXCR2 chemokine ligands across the joint endothelium to induce neutrophil TEM and entry into the joint that is mediated by CXCR2 expressed on arrested neutrophils. Thus, we describe the sequential roles of two atypical chemoattractant receptors, C5aR2 and ACKR1, that collaborate with “classical” chemoattractant receptors, C5aR1 and CXCR2, to control distinct steps in the entry of neutrophils from the circulation into the tissue in a model of IC-induced inflammation.

RESULTS

C5aR2 on nonhematopoietic cells is required for IC-induced arthritis

We first studied the role of C5aR2 in the K/BxN AST model of arthritis. C5ar2-deficient knockout (KO) and wild-type (WT) littermate control mice were intraperitoneally treated with AST on days 0 and 2 to induce arthritis. C5ar2-KO mice had markedly reduced clinical arthritis scores and change in ankle thickness (~2-fold less) compared with WT littermate control mice (fig. S1, A and B). To analyze neutrophil infiltration into the joint space, we isolated synovial fluid (SF) cells from C5ar2-KO and WT littermate control mice on day 7 after AST and analyzed them by flow cytometry. Fewer neutrophils (~3-fold less) were recovered from the SF of C5ar2-KO mice on day 7 after AST compared with WT littermate controls (fig. S1, C and D). In addition, the histopathological joint score, which quantitates inflammatory cell infiltration into synovial tissue (ST), was lower (~3-fold) in C5ar2-KO mice compared with WT littermate controls (fig. S1, E and F). These data demonstrate that C5ar2 deficiency attenuated IC-induced arthritis via impairment of neutrophil recruitment into the joint.

Next, to determine the role of C5aR2 on hematopoietic versus nonhematopoietic cells in the pathogenesis of IC-induced arthritis, we generated BM chimeric (BMC) mice and subjected them to the AST model of arthritis (Fig. 1A). These BMC mice showed no differences in circulating neutrophil numbers (Fig. 1B). Clinical arthritis scores and changes in ankle thickness in C5ar2-KO host BMC mice were markedly lower (~4-fold) compared with WT host BMC mice (Fig. 1, C and D). The number of SF neutrophils recovered on day 7 after AST in C5ar2-KO host BMC mice was also markedly decreased (~10-fold) compared with WT host BMC mice (Fig. 1E). In addition, the histopathological scores of C5ar2-KO host BMC mice were less severe (~4-fold) than WT host BMC mice (Fig. 1, F and G). In contrast, WT host BMC mice with C5ar2-KO BM had no attenuation of clinical or histopathological scores or synovial neutrophil numbers. These data demonstrate that C5aR2 expressed on nonhematopoietic cells regulates neutrophil recruitment into the joints in IC-induced arthritis.

Fig. 1 Role for C5aR2 on nonhematopoietic cells in IC-induced arthritis.

(A) Schematic of C5aR2 BMC mice generation. WT or C5ar2-KO BM was transferred into lethally irradiated WT or C5ar2-KO mice. (B) Polymorphonuclear neutrophil (PMN) counts in the blood of BMC mice. (C to G) After reconstitutions, BMC mice were intraperitoneally treated with AST on days 0 and 2. n.s., not significant. (C) Clinical arthritis score. (D) Change in ankle thickness. (E) Number of neutrophils recovered from the SF on day 7 after AST. (F and G) Representative H&E staining and histopathological score on day 7 after AST. Arrowheads indicate inflammatory cell infiltration. Scale bars, 200 μm. Data indicate means ± SEM. n = 4 to 6 mice per group and P value calculated using ordinary one-way ANOVA with a post hoc Tukey’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

ACKR1 on nonhematopoietic cells is required for IC-induced arthritis

ACKR1 has been shown to transcytose inflammatory chemokines in vitro, such as CXCR2 ligands, and has been suggested to play this role in vivo (4, 811). Because CXCR2 ligands are required for neutrophil recruitment and the development of arthritis in the K/BxN model (38), we also studied whether ACKR1 contributed to the pathogenesis of IC-induced arthritis. Ackr1-KO and WT littermate control mice were injected with AST on days 0 and 2. Ackr1-KO mice had markedly less disease (~6-fold) as measured by clinical arthritis scores and changes in ankle thickness compared with WT littermate controls (fig. S2, A and B). We also isolated cells from the joints of Ackr1-KO or WT littermate control on day 7 after AST to analyze SF neutrophil numbers. There were no neutrophils recovered from the joints of Ackr1-KO mice on day 7 after AST, and the histopathological scores in these mice were markedly reduced (~10-fold) compared with WT littermate controls (fig. S2, C to E). Thus, Ackr1 deficiency also ameliorated arthritis by inhibiting neutrophil recruitment into joints.

ACKR1 is mainly expressed on ECs and erythrocytes (39, 40). We therefore analyzed the role of ACKR1 on hematopoietic versus nonhematopoietic cells in the pathogenesis of IC-induced arthritis (Fig. 2A). The number of circulating neutrophils in BMC mice with Ackr1-KO BM was decreased compared with BMC mice with WT BM as has been reported previously (Fig. 2B) (17). However, Ackr1-KO host BMC mice had markedly reduced clinical arthritis scores and changes in ankle thickness (~4-fold) compared with WT host BMC mice (Fig. 2, C and D). There were no neutrophils recovered from the joints of Ackr1-KO host BMC mice on day 7 after AST, and the histopathological scores were reduced (~2- to 4-fold) in these mice compared with WT host BMC mice (Fig. 2, E to G). Thus, ACKR1 expressed on nonhematopoietic cells regulates neutrophil recruitment into the joints in IC-induced arthritis.

Fig. 2 Role for ACKR1 on nonhematopoietic cells in IC-induced arthritis.

(A) Schematic generation of ACKR1 BMC mice. WT or Ackr1-KO BM was transferred into lethally irradiated WT or Ackr1-KO mice. (B) Neutrophil counts in the blood of BMC mice. (C to G) BMC mice were intraperitoneally treated with AST on days 0 and 2. (B) Clinical arthritis score. (C) Change in ankle thickness. (D) Number of neutrophils in SF derived from each BMC mice on day 7 after AST. (E and F) Representative H&E staining and histopathological score of BMC mice on day 7 after AST. Arrowheads indicate inflammatory cell infiltration. Scale bars, 200 μm. Data indicate means ± SEM. n = 5 mice per group and P value calculated using ordinary one-way ANOVA with a post hoc Tukey’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001.

C5aR2 is expressed on ECs in the joint

C5aR2 has been shown to be expressed on CD31+ ECs from human carotid arteries (30). To determine the pattern of C5aR2 expression in the joint, we harvested ST from untreated and day 7 AST-treated WT mice for quantitative polymerase chain reaction (qPCR), flow cytometry, and immunofluorescence analysis. C5ar2 mRNA was detected in the ST of WT mice at baseline and increased during the development of inflammatory arthritis (Fig. 3A). About 80% of CD45CD31+ ECs expressed C5aR2 on the cell surface in the ST from WT mice with AST on day 7, whereas C5aR2 protein was detected on the surface of about 20% of CD45CD31+ ECs isolated from the ST of untreated WT mice as determined by flow cytometry (Fig. 3, B and C). In contrast, C5aR2 protein was detected on the surface of <10% of CD45+ cells and <20% of CD31CD45 cells recovered from both untreated and AST-treated mice (Fig. 3, B and C), suggesting that C5aR2 was expressed predominantly on ECs in the arthritic joint. Immunohistochemistry also revealed C5aR2 immunoreactivity on von Willebrand factor–positive (VWF+) blood vessels within the ST of WT mice with arthritis but not in C5ar2-KO mice similarly treated with AST as negative controls (fig. S3). C5aR2 immunoreactivity was detected on arterial, venular, and capillary vessels in the joints of WT mice after AST on day 7 (fig. S3). These data demonstrate that C5aR2 is primarily expressed on ECs in the joint and that its expression is increased after joint inflammation.

Fig. 3 C5aR2 expression on joint endothelium.

(A) C5ar2 mRNA levels in the ST of untreated WT mice and WT mice on day 7 after AST. Data indicate means ± SEM. n = 3 mice per condition and P value calculated using unpaired two-tailed Student’s t test. **P < 0.01. (B) Cell surface protein expression of C5aR2 on CD45CD31+ ECs, CD45CD31 cells, and CD45+ cells was determined by flow cytometry on live cells recovered from ST of untreated or day 7 AST-treated WT and C5ar2-KO mice. SSC-A, side scatter area; FSC-A, forward scatter area; FSC-H, forward scatter height. (C) Populations of C5aR2-expressing cells isolated from ST of untreated or day 7 AST-treated WT littermate controls and C5ar2-KO mice. n = 4 mice per group.

C5aR2 is required for initial neutrophil adhesion, whereas ACKR1 is required for neutrophil extravasation in IC-induced arthritis

Our BMC mice data demonstrated that C5aR2 and ACKR1 on nonhematopoietic cells are required for neutrophil entry into the joint in IC-induced arthritis. To define how these two atypical chemoattractant receptors expressed on joint ECs collaborate to control neutrophil recruitment into the joint, we turned to MP-IVM (23). We generated LysM-GFP BMC mice to visualize the migratory behavior of green fluorescent protein (GFP)–expressing WT neutrophils in WT (control), C5ar2-KO, and Ackr1-KO host mice. The joints of these BMC mice were imaged in live mice on day 3 (early phase of arthritis) and day 7 (full-blown arthritis) after AST (Fig. 4A). On both days, we observed similar numbers of neutrophils adhering to the joint endothelium in WT and Ackr1-KO host BMC mice (Fig. 4, B to D). In contrast, there were markedly fewer (~5-fold) neutrophils adhering to endothelium in the joints of C5ar2-KO host BMC mice on both days 3 and 7 (Fig. 4, B to D, and movies S1 and S2). The number of newly extravasated neutrophils in the joints of both Ackr1-KO and C5ar2-KO host BMC mice was markedly less than that in WT host BMC mice on both days 3 and 7 (Fig. 4, B, C, and E, and movies S1 and S2). Further, the percentage of adherent cells that extravasated was markedly reduced in Ackr1-KO host BMC mice compared with both WT and C5ar2-KO host BMC mice (Fig. 4F). Together, this resulted in fewer neutrophils found in the joint tissue of C5ar2-KO and Ackr1-KO host BMC mice compared with WT host BMC mice on day 7 after AST (Fig. 4, B, C, and G, and movies S1 and S2). These data demonstrate that C5aR2 expressed on nonhematopoietic cells was required for initial neutrophil adhesion, whereas ACKR1 was required for subsequent neutrophil extravasation within the joint in IC-induced arthritis.

Fig. 4 Imaging joints of live WT, Ackr1-KO, and C5aR2-KO BMC mice on days 3 and 7 after AST.

BMC (A) Schematic BMC mice generation. WT-LysM-GFP BM was transferred into lethally irradiated WT, Ackr1-KO, or C5ar2-KO mice. After reconstitution, joints were imaged on days 3 and 7 after AST. (B and C) In vivo joint imaging of WT, Ackr1-KO, or C5ar2-KO BMC mice for 35 min on day 3 (B) and day 7 (C) after AST. Scale bars, 50 μm. (D) Number of newly adherent neutrophils on joint endothelium of WT, Ackr1-KO, or C5ar2-KO BMC mice. (E) Number of newly extravasated neutrophils in joint tissue of WT, Ackr1-KO, or C5ar2-KO BMC mice. (F) Percentage of adherent neutrophils that extravasated within joints of WT, Ackr1-KO, or C5ar2-KO BMC mice. (G) Number of neutrophils observed in ST of WT, Ackr1-KO, or C5ar2-KO BMC. Data indicate means ± SEM. n = 3 to 4 mice per group and P value calculated using ordinary one-way ANOVA with a post hoc Tukey’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

C5aR2 is required for C5a-induced neutrophil adhesion, whereas ACKR1 is required for CXCL1-induced adhesion and extravasation

To directly study whether C5aR2 transports C5a and ACKR1 transports CXCR2 ligands into the blood vessel lumen in vivo to initiate neutrophil adhesion and extravasation, we used a superfusion assay and MP-IVM. We again made use of BMC mice using WT-LysM-GFP BM transferred into lethally irradiated WT, Ackr1-KO, or C5ar2-KO mice. In this assay, chemoattractants are directly added to the surface of surgically exposed joints, which are then imaged for 70 min (fig. S4A) (24). Using this assay, we found that C5a induced robust neutrophil adhesion to the endothelium of WT host BMC mice but not to the endothelium of C5ar2-KO host BMC mice (Fig. 5A and movie S3). Although C5a potently induced adhesion, it did not induce neutrophil extravasation in WT host BMC mice (Fig. 5B and movie S3). In contrast, the CXCR2 ligands CXCL1 and CXCL2 induced both neutrophil adhesion and extravasation in the joints of WT host BMC mice but induced neither in Ackr1-KO host BMC mice (Fig. 5, C to F, and movies S4 and S5).

Fig. 5 In vivo superfusion assays.

WT-LysM-GFP BM was transferred into lethally irradiated WT, Ackr1-KO, or C5ar2-KO mice as in Fig. 4. After reconstitution, the indicated chemoattractants (0.1 pmol) dissolved in sterile PBS (100 μl) were superfused onto surgically exposed joints, which were then imaged for 70 min. Cumulative graph of neutrophil adhesion (A, C, and E) and extravasation (B, D, and F) after (A and B) C5a application onto joints of WT and C5ar2-KO BMC mice, (C and D) CXCL1 application onto joints of WT and Ackr1-KO BMC, and (E and F) CXCL2 application onto joints of WT and Ackr1-KO BMC. Data indicate means ± SEM. n = 3 mice per group and P value calculated using ordinary one-way ANOVA with a post hoc Tukey’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

To determine the specificity of C5aR2 and ACKR1 to transport C5a and CXCR2 ligands, respectively, and to demonstrate that there was no intrinsic defect in the ability of C5ar2-KO and Ackr1-KO mice to support neutrophil arrest and TEM, we performed sequential in vivo superfusion assays using the same LysM-GFP BMC mice described above (fig. S4B). Although C5a could not initiate neutrophil adhesion in C5ar2-KO host BMC mice, CXCL1, CXCL2, and LTB4 could induce neutrophil adhesion and extravasation into the joints of these mice similar to what we observed in WT host BMC control mice (Fig. 6, A and B, and movie S6). CXCL1 was a more potent inducer of neutrophil adhesion and extravasation compared with CXCL2 and LTB4. Likewise, whereas CXCL1 could not induce neutrophil adhesion or extravasation when applied onto the joints of Ackr1-KO host BMC mice, the subsequent application of either C5a or LTB4 was able to induce neutrophil arrest, and LTB4 was also able to induce neutrophil extravasation (Fig. 6, C and D, and movie S7). However, the subsequent application of CXCL2 was unable to induce neutrophil arrest or transmigration in these mice (Fig. 6, C and D, and movie S7). These data demonstrated that C5aR2 specifically transports C5a into the blood vessel lumen to induce neutrophil arrest, whereas ACKR1 specifically transports CXCR2 ligands into the lumen of blood vessel in vivo to induce neutrophil adhesion and extravasation.

Fig. 6 Sequential in vivo superfusion assays.

WT-LysM-GFP BM was transferred into lethally irradiated WT, Ackr1-KO, or C5ar2-KO mice as in Fig. 4. After reconstitution, the indicated chemoattractants (0.1 pmol) dissolved in sterile PBS (100 μl) were superfused onto surgically exposed joints. (A and B) C5a application followed 35 min later by the application of other chemoattractants (CAs), including CXCL1, LTB4, or CXCL2 onto joints of C5ar2-KO BMC. (C and D) CXCL1 application followed 35 min later by the application of other CAs, inclduing C5a, LTB4, CXCL2, or PBS onto joints of WT and Ackr1-KO BMC. Data indicate means ± SEM. n = 3 mice per group and P value calculated using ordinary one-way ANOVA with a post hoc Tukey’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

C5aR2 transports C5a into the lumen of blood vessels in vivo

Our previous work demonstrated that C5a was expressed on VWF+ EC in the arthritic joints (24). To determine whether C5aR2 was required for the deposition of C5a on the joint endothelium in arthritic mice, we analyzed the localization of C5a in the joints of WT and C5ar2-KO mice on day 7 after AST. C5a was detected on VWF+ blood vessels in the joints of WT but not C5ar2-KO mice on day 7 after AST (fig. S5). Last, we visualized the requirement for C5aR2 for the transport of C5a from the extravascular space into the lumen of blood vessels overlying the joint. To do so, we followed the fate of Alexa Fluor (AF) 594–labeled C5a (AF594-C5a) applied to the surgically exposed joint of WT, C5ar2-KO, or Ackr1-KO mice using our in vivo superfusion assay and MP-IVM (Fig. 7A). AF594-C5a was active in the in vivo superfusion assay, demonstrating that it retained its biological activity (fig. S6, A and B). Before superfusing AF594-C5a, fluorescently labeled CD31 and C5aR2 monoclonal antibodies (mAbs) were intravenously injected into WT, C5ar2-KO, or Ackr1-KO mice to label ECs and C5aR2, respectively. After 30 minutes, AF594-C5a was superfused onto the surface of surgically exposed ankle joints of live anesthetized mice, which were then imaged for 30 min (Fig. 7A). In the joints of WT and Ackr1-KO mice, AF594-C5a was visualized on the luminal surface of the joint endothelium and colocalized with C5aR2 and CD31 staining (Fig. 7, B and C), demonstrating that C5a was transported into the blood vessel lumen. Unconjugated AF594 dye superfused onto the exposed ankle joint of WT mice was not transported into the lumen of joint blood vessels, demonstrating that the transport of the label was the result of C5a transport and not leakage of dye into the blood vessels (fig. S6C). However, in C5ar2-KO mice, C5a was only found within ST and was not detected within the blood vessels of the joint (Fig. 7, B and C), demonstrating that C5aR2 was required for C5a transport into the blood vessel lumen.

Fig. 7 Imaging C5ar2-dependent transport of C5a into the blood vessel lumen in vivo.

(A) Schematic of experimental design. WT, C5ar2-KO, and Ackr1-KO mice were intravenously injected with AF647-conjugated CD31 mAb (20 μg per mouse) and AF488-conjugated C5aR2 mAb (15 μg per mouse) 30 min before AF594-conjugated C5a was applied onto surgically exposed joints. Joints were then imaged by MP-IVM for 30 min in live mice. (B) Representative frames from in vivo imaging videos. Scale bars, 10 μm. J, joint tissue; B, blood vessel. (C) Mean fluorescence intensity of C5a deposition on joint endothelium. Data indicate means ± SEM. n = 4 mice per group and P value calculated using ordinary one-way ANOVA with a post hoc Tukey’s test for multiple comparisons. ****P < 0.0001.

We also visualized the requirement for ACKR1 for the transport of CXCL1 into the blood vessel lumen in vivo (fig. S7A). WT, C5ar2-KO, and Ackr1-KO mice were intravenously treated with fluorescently labeled antibodies (Abs) to CD31 and CXCL1. After 30 minutes, CXCL1 was superfused onto surgically exposed ankle joints, which were then imaged for 30 min (fig. S7B). CXCL1 immunoreactivity was detected within the joint endothelium of WT and C5ar2-KO mice but not in Ackr1-KO mice, suggesting that CXCL1 was transported by ACKR1 into the blood vessel lumen (fig. S7B). These data demonstrate that C5a is transported by C5aR2, whereas CXCL1 is transported by ACKR1 into the lumen of overlying blood vessels in vivo.

DISCUSSION

The recruitment of blood-borne leukocytes into tissue is an essential element of inflammation. Chemoattractants instruct circulating leukocytes to enter specific tissue beds by inducing their firm arrest on the endothelium, followed by TEM. For the most part, chemoattractants that act as such tissue gate keepers are produced by activated ECs where they guide localized leukocyte recruitment. However, how circulating leukocytes receive such signals when the endothelium has not been activated to produce chemoattractants is not entirely clear. We now demonstrate that the atypical C5a receptor, C5aR2, is required for specifically transporting tissue-derived C5a into the lumen of overlying blood vessels to initiate the adhesion of the very first neutrophils in the K/BxN AST model of arthritis. C5aR2 was expressed on joint ECs and was required to transport superfused C5a into the blood vessel lumen to induce neutrophil arrest. We therefore conclude that C5aR2 transports locally generated C5a into the blood vessel lumen where it binds cell surface proteoglycans decorating the joint endothelium and is presented to circulating neutrophils to induce their initial arrest, igniting joint inflammation (Fig. 8).

Fig. 8 Sequential roles of atypical chemoattractant receptors C5aR2 and ACKR1 required for neutrophil arrest and subsequent TEM.

The atypical chemoattractant receptors C5aR2 and ACKR1 are required for neutrophil entry into the joint and the development of inflammatory arthritis. In the KxB/N model of IC-induced inflammatory arthritis, the atypical C5a receptor, C5aR2, expressed on ECs transports C5a generated within the joint into the vessel lumen ①. C5a is then captured on the surface of ECs by heparan sulfate proteoglycans (HSPGs) where it is presented to circulating neutrophils and initiates C5aR1-mediated arrest ②. The atypical chemokine receptor, ACKR1, is also expressed on ECs where it transports CXCR2 ligands produced within the joint into the lumen of the overlying blood vessel ③ to initiate CXCR2-mediated neutrophil TEM and entry into the joint ④. Thus, atypical chemoattractant receptors collaborate with classical signaling chemoattractant receptors to control the recruitment of neutrophils into tissue sites of inflammation.

C5aR2 deficiency did not completely inhibit the development of IC-induced arthritis, although tissue-derived C5a could not be transported into the blood vessel lumen in the joints. It is likely that other chemoattractants were able to be transported into the blood vessel lumen to initiate neutrophil recruitment in C5ar2-KO mice in the arthritis model. For example, ICs have been shown to directly stimulate macrophages to produce CXCL1 in vitro (41), suggesting that macrophages in the joint might be able to generate CXCL1 under this condition. Consistent with this, we demonstrated that CXCL1 superfused on the joints of C5ar2-KO host BMC mice induced neutrophil adhesion and extravasation. In addition, once neutrophils enter into the joint, they are activated to release interleukin-1β, LTB4, and CXCL2, which then amplifies neutrophil recruitment and inflammation (42, 43). Thus, once neutrophils enter into the joint, C5aR1 and C5aR2 appear to become less critical as ACKR1 transports CXCL1 and CXCL2 released by immune cells within the joint and as activated joint ECs produce CXCL1 (42).

Although C5a was a potent inducer of neutrophil arrest, it was not able to induce TEM. This is in contrast to other neutrophil chemoattractants, such as LTB4, CXCL1, and CXCL2, which were able to induce neutrophil TEM. We do not understand why C5aR1 signaling is able to induce neutrophil arrest but not TEM, whereas the LTB4 receptor BLT1 and CXCR2 are able to induce both arrest and TEM. The difference in the signaling pathways induced by C5aR1 versus CXCR2 and BLT1 in neutrophils responsible for this different biology will be interesting to explore in the future.

There was no intrinsic global defect in the ability of C5ar2-KO and Ackr1-KO mice to support neutrophil arrest and TEM. The defects in neutrophil arrest in C5ar2-KO mice and neutrophil arrest and TEM in Ackr1-KO mice were specific for C5a and CXCR2 ligands, respectively. C5ar2-KO and Ackr1-KO mice are able to support neutrophil arrest and TEM; they are just unable to specifically transport tissue-derived C5a and CXCR2 chemokine ligands, respectively, to induce these processes. Our data therefore suggest that inhibition of both C5aR2 and ACKR1 may have an additive or synergistic effect on suppressing neutrophil recruitment.

C5aR2 has been implicated in neutrophil recruitment into tissue in some models of inflammation, although the mechanism has not been appreciated. In models of acute lung injury, including those induced by intratracheal administration of IC or C5a, deficiency in C5aR2 decreased neutrophil recruitment into the lung (36). Our data now provide a molecular mechanism for the role of C5aR2 in mediating neutrophil recruitment into tissue. However, other mechanisms are possible such as loss of a C5a sink, resulting in increased concentrations of C5a in the blood and C5aR1 desensitization. Furthermore, C5aR2 is likely not required for all IC- or C5a-induced neutrophil recruitment. For instance, C5a-induced recruitment of neutrophils into the peritoneal cavity was not completely dependent on C5aR2 (35). We suspect that C5aR2 may play a more important role in tissues that have a paucity of resident immune cells, such as the joint, where C5aR2 plays an important role in transporting C5a into the blood vessel lumen to “directly” initiate neutrophil arrest. However, in other tissues with larger resident immune cell populations, C5a can also “indirectly” contribute to neutrophil recruitment by activating tissue-resident cells, such as macrophages and mast cells, that then release cytokines, chemokines, and lipid mediators that induce neutrophil recruitment.

We demonstrated that C5aR2 expressed on nonhematopoietic cells is required for transporting tissue-derived C5a into vessel lumens. We suspect that C5aR2 expressed on ECs transcytoses C5a across the endothelium, analogous to what has been described for ACKR1 and chemokines (4). In vitro experiments using human C5aR2–transfected human embryonic kidney cells demonstrated that the carboxyl tail of C5aR2 is phosphorylated after C5a activation, associates with β-arrestin 2, and is internalized in vesicles that associates with the Rab proteins (Ras-like small G proteins) 5, 7, and 11, which are known to be involved with intracellular vesicle trafficking (44). Determining the mechanism of C5aR2-mediated C5a transcytosis will be interesting to explore in the future.

In summary, we have identified a previously unknown role for C5aR2 in transporting tissue-derived C5a into the lumen of blood vessels to induce the initial arrest and adhesion of neutrophils, igniting a type III hypersensitivity reaction in the joint (Fig. 8). We have also found that ACKR1 contributes to the pathogenesis of IC-induced arthritis by transporting CXCR2 chemokine ligands into the blood vessel lumen to induce neutrophil extravasation. Thus, we have provided insights into how atypical chemoattractant receptors collaborate with classical chemoattractant receptors to control the recruitment of neutrophils into tissue.

MATERIALS AND METHODS

Study design

The aim of this study was to determine how tissue-derived chemoattractants initiate the recruitment of neutrophils into the joint in an IC-induced model of arthritis. Age (8 to 10 weeks old)– and sex (only male)–matched WT, C5ar2-KO, and Ackr1-KO mice bred and housed in the same facility at the Massachusetts General Hospital were used for this study. The sample size was determined to achieve a power of 90%, accepting a type I error rate of 0.05. Analysis of arthritis, histological scores, and movies, as well as flow cytometry, was performed by an investigator blinded to the experimental groups. Randomization and blinding were not used for in vivo joint imaging.

Mice

K/BxN mice were obtained by crossing KRN with NOD/LtJ mice (the Jackson Laboratory). Ackr1-KO (14), C5ar2-KO (45), and WT-LysM-GFP (46) mice were maintained in our laboratory. WT C57BL/6 mice were purchased from the Charles River Laboratories. Genotypes were confirmed by Transnetyx Inc. All experiments were performed according to protocols approved by the Massachusetts General Hospital Subcommittee on Research Animal Care.

Induction of K/BxN serum transfer model of arthritis

Pooled serum from 8-week-old arthritic K/BxN mice was collected as described (47) and was intraperitoneally transferred into recipient mice (100 μl) on days 0 and 2.

Clinical arthritis score and measurement of ankle thickness

Clinical arthritis score for each limb was recorded every other day by one investigator who was blinded to the mouse genotypes on a scale of 0 to 4 as previously described (47). The clinical arthritis score is defined as the sum of the scores for all four paws of each mouse (maximum possible score, 16). Ankle thickness was measured using digital slide calipers.

Histological analysis

Mouse ankle joints, including the tibiotalar joint to the midfoot, were harvested on day 7 after AST and fixed with 10% formalin overnight. Decalcification, paraffin embedding of sample, and hematoxylin and eosin (H&E) staining were performed by the Pathology Core Laboratory at the Brigham and Women’s Hospital. Histology was evaluated blindly and scored as previously described (47).

Isolation of blood, ST and SF neutrophils, and flow cytometry

The ankle joint, synovium, and SF cells were prepared as described (47). We also isolated blood from the inferior vena cava. Blood or SF cells were incubated with anti-CD16/32 mAb (BioLegend, #101302)as an Fc-blocker for 5 min at room temperature and stained with allophycocyanin-conjugated anti-Ly6G mAb (BioLegend, #127614) for 20 min at 4°C. CountBright Absolute Counting Beads (Thermo Fisher Scientific) were used for counting cells. Flow cytometry was performed with FACS LSRFortessa (BD Biosciences) and analyzed with FlowJo software. Neutrophils were identified as Ly6G+ cells in the granulocyte gate of forward and side scatterplots (fig. S8).

Real-time qPCR

ST from the ankle joint was collected as described above and homogenized in TRIzol (Sigma-Aldrich), and RNA was isolated according to the manufacturer’s instruction. Purified RNA was then converted to complementary DNA by reverse transcription (TaqMan Reverse Transcription Reagents, Thermo Fisher Scientific). qPCR reactions in the presence of SYBR Green (FastStart Essential DNA Green Master Mix, Roche) were performed on the LightCycler 96 Instrument (Roche) and normalized to B2m using the following the primers: C5ar2, 5′-ACCACCAGCGAGTATTATGACT-3′ (forward) and 5′-GCAGGACTATCAGGTAGACATCA-3′ (reverse).

MP-IVM and image analysis

MP-IVM was performed as described (23). Imaged venules were identified by the direction of blood flow. Multiphoton excitation was obtained through DeepSee and MaiTai Ti:sapphire lasers (Newport/Spectra-Physics) tuned to 820 and 920 nm to excite all fluorescent probes used. Stacks of 11 square optical sections with 4-μm z spacing were acquired on an Ultima IV multiphoton microscope (Prairie Technologies) every 17 s with optical zoom of 2× to provide image volumes 40 μm in depth and 307 μm in width. Emitted fluorescence was detected through 460/50, 525/50, 595/50, and 660/40 band-pass filters and non-descanned detectors to generate four-color images. Sequences of image stacks were transformed into volume-rendered, time-lapse movies with Imaris software (Bitplane).

For the imaging of endogenous neutrophils within joints of LysM-GFP mice, neutrophils were distinguished from monocytes on the basis of their increased intensity of GFP fluorescence (48), their smaller size, and their increased mobility (24). More than 80% of GFP+ cells expressed Ly6G in the arthritic joint (fig. S8B). Neutrophils were scored on the basis of the number that adhered and extravasated during the observation period. Adhesion included both arrest and crawling. Arrest was defined as a cell that maintained a round shape and that remained in the same position for at least 30 s. A crawling cell was defined as a cell that took on an amoeboid shape cell that crawled along the inside of blood vessel. Extravasation was defined as a neutrophil that had entered into the tissue (23).

For the in vivo superfusion assays, chemoattractants [0.1 pmol; C5a (R&D Systems), CXCL1 and CXCL2 (PeproTech), and LTB4 (Tocris Bioscience)] dissolved in sterile phosphate-buffered saline (PBS) (100 μl) were placed on the joints of WT, C5ar2-KO, and Ackr1-KO BMC mice and imaged for 70 min. The number of adherent neutrophils and extravasated neutrophils was quantitated.

For sequential in vivo superfusion assays, (i) C5a (0.1 pmol) dissolved in sterile PBS (100 μl) was placed on the joints of C5ar2-KO BMC mice and imaged for 35 min. After gently washing three times with sterile PBS, CXCL1, CXCL2, or LTB4 (0.1 pmol) dissolved in sterile PBS (100 μl) was superfused on the joints of C5ar2-KO BMC mice and imaged for 35 min. (ii) CXCL1 (0.1 pmol) dissolved in sterile PBS (100 μl) was placed on the joints of Ackr1-KO BMC mice and imaged for 35 min. After gently washing three times with sterile PBS, C5a, CXCL2, or LTB4 (0.1 pmol) dissolved with sterile PBS (100 μl) was superfused on the joints of Ackr1-KO BMC mice and imaged for 35 min. The number of adherent neutrophils and extravasated neutrophils was quantitated.

For AF594-conjugated C5a transporting assay in vivo, C5a was labeled with AF594 using the AF594 Microscale Protein Labeling Kit (Thermo Fisher Scientific), according to the manufacturer’s instruction. The conjugates were spectrophotometrically analyzed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). WT, C5ar2-KO, or Ackr1-KO mice were intravenously injected with AF647-conjugated anti-mouse CD31 mAb (BioLegend, #102516) (20 μg per mouse) and AF488-conjugated anti-mouse C5aR2 mAb (R&D Systems, #IC4729G) (20 μg per mouse) 30 min before in vivo joint imaging. AF594-conjugated C5a (0.1 pmol) dissolved in sterile PBS (100 μl) was superfused on the surface of surgically exposed joints of WT, C5ar2-KO, or Ackr1-KO mice and then imaged for 30 min after the application of C5a. We measured the mean fluorescence intensity of C5a deposition on joint endothelium by ImageJ (49). All data were analyzed by Imaris software.

Generation of BMC mice

BMC mice were generated according to established protocols in the laboratory (24, 43) and used for experiments 4 weeks after reconstitution. BMC mice were used if ≥95% blood neutrophils were of donor origin.

Analysis of C5aR2 expression on joint endothelium

Flow cytometry

ST from WT or C5ar2-KO mice was digested as described (47). A cell suspension was obtained by mashing synovium through a 70-μm strainer. The cells were incubated with anti-mouse CD16/32 mAb (BioLegend) as an Fc-blocker for 5 min at room temperature and then incubated with AF647-conjgated anti-mouse CD31 mAb (BioLegend), AF488-conjugated anti-mouse C5aR2 mAb (R&D), and Brilliant Violet 421–conjugated anti-mouse CD45 mAb (BioLegend, #103134) for 20 min at 4°C. The cells were incubated with Fixable Viability Dye eFluor 780 (eBioscience) to exclude dead cells, according to the instruction of the manufacturer. Flow cytometry analysis was performed with a BD LSRFortessa.

Immunofluorescence staining

Paraffin-embedded tissue sections (thickness, 4 μm) from WT or C5ar2-KO mice with AST on day 7 were prepared as described (47). Sections were then blocked with protein block (DakoCytomation) for 15 min and stained with AF488-conjugated rat anti-C5aR2 mAb (R&D Systems) or AF647-conjugated rabbit anti-VWF polyclonal Ab (Bioss Antibodies, #bs-4754R-A647) for 2 hours at 25°C. The slides were examined using a fluorescence microscope (Carl Zeiss).

Statistics

Data are expressed as the means ± SEM. All statistical analyses were performed in Prism 8 (GraphPad Software). P value in multiple groups was calculated using ordinary one-way analysis of variance (ANOVA) with a post hoc Tukey’s test for multiple comparisons. Means between two groups were compared with unpaired two-tailed Student’s t test.

SUPPLEMENTARY MATERIALS

immunology.sciencemag.org/cgi/content/full/4/35/eaav5951/DC1

Fig. S1. C5aR2 contributes to the pathogenesis of IC-induced arthritis.

Fig. S2. ACKR1 contributes to the pathogenesis of IC-induced arthritis.

Fig. S3. C5aR2 expression on joint endothelium.

Fig. S4. In vivo superfusion assays.

Fig. S5. Immunofluorescence analysis of C5a expression on joint tissue harvested from WT and C5ar2-KO mice on day 7 after AST.

Fig. S6. In vivo superfusion assay using AF594-C5a.

Fig. S7. Analysis of CXCL1 transport into the lumen of joint blood vessels in WT, C5ar2-KO, and Ackr1-KO mice.

Fig. S8. Representative gating strategy of Ly6G+ neutrophil.

Movie S1. In vivo MP-IVM joint imaging of live WT, C5ar2-KO, and Ackr1-KO BMC mice on day 3 after AST.

Movie S2. In vivo MP-IVM joint imaging of live WT, C5ar2-KO, and Ackr1-KO BMC mice on day 7 after AST.

Movie S3. In vivo superfusion assay (application of C5a).

Movie S4. In vivo superfusion assay (application of CXCL1).

Movie S5. In vivo superfusion assay (application of CXCL2).

Movie S6. Sequential in vivo superfusion assay (application of C5a followed by application of CXCL1, CXCL2, or LTB4).

Movie S7. Sequential in vivo superfusion assay (application of CXCL1 followed by application of CXCL1, CXCL2, or LTB4).

REFERENCES AND NOTES

Acknowledgments: We thank D. Mathis and C. Benoist (Harvard Medical School) for providing KRN mice, C. Gerard (Boston Children’s Hospital) for providing C5ar2-deficient mice, S. Peiper (Thomas Jefferson University School of Medicine) for providing Ackr1-deficent mice, and T. Graf (Albert Einstein College of Medicine). Funding: This work was supported by the National Institutes of Health (R01AI050892 to A.D.L. and R01AI097053 to T.R.M.), Rheumatology Research Foundation (to A.D.L.), Mallinckrodt Pharmaceuticals Research Fellowship Award in Rheumatology Research, Pfizer ASPIRE Rheumatology Award, JSPS KAKENHI Grant Number JP19K08895 and Japan Rheumatism Foundation Research Grant (Y.M.). Author contributions: Y.M. designed and performed most experiments, statistically analyzed and interpreted the data, and contributed to writing the manuscript. C.M. provided technical assistance with the K/BxN serum transfer model of arthritis. V.M. and T.R.M. provided technical assistance for the MP-IVM experiments. A.D.L. provided overall project supervision, contributed to the design of the experiments, and wrote the manuscript. Competing interests: The authors declare that they have no competing financial interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. All reagents used in this study were commercially available or derived from commercial reagents. All mouse strains used were available from the Jackson Laboratory unless otherwise indicated. KRN mice were available from D. Mathis and C. Benoist (Harvard Medical School, Boston, MA). C5ar2-deficient mice were available from C. Gerard and B. Lu (Boston Children’s Hospital, Boston, MA). Ackr1-deficent mice were available from S. Peiper (Thomas Jefferson University School of Medicine, Philadelphia, PA) under a material transfer agreement from the National Institutes of Health. LysM-GFP mice were available from T. Graf (Centre for Genomic Regulation, Barcelona, Spain) under a material transfer agreement from the Albert Einstein College of Medicine.
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