Heterocomplexes between the Atypical Chemokine MIF and the CXC-Motif Chemokine CXCL4L1 Regulate Inflammation and Thrombus Formation
Markus BrandhoferAdrian HoffmannXavier BlanchetElena SiminkovitchAnne‐Katrin RohlfingOmar El BounkariJeremy NesteleAlexander BildChristos K. KontosKathleen HilleVanessa RohdeAdrian FröhlichJona GolemiÖzgün GökçeChristine KrammerPatrick ScheiermannNikolaos TsilimparisW. KempfLars MäegdefesselRemco T. A. MegensHans IppelRory R. KoenenKevin H. MayoMeinrad GawazAphrodite KapurniotuChristian WeberPhilipp von HundelshausenJürgen Bernhagen
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Abstract To fulfil its orchestrating function in immune cell trafficking, a network of chemokines and receptors capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within the network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to the chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine-chemokine interactions extend to ACKs and that MIF may form heterocomplexes with classical chemokines. We tested this hypothesis by an unbiased chemokine protein array. The platelet chemokine CXCL4L1, but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12, was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation abrogated MIF-elicited T-cell chemotaxis as assessed in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ . Of note, CXCL4L1 blocked the binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. As MIF and CXCL4L1 are platelet products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and clinical human thrombus sections. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction, adding to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures to modulate inflammation and thrombosis.Keywords:
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ABSTRACT Chemokines are involved in recruitment and activation of hematopoietic cells at sites of infection and inflammation. The M3 gene of γHV68, a gamma-2 herpesvirus that infects and establishes a lifelong latent infection and chronic vasculitis in mice, encodes an abundant secreted protein during productive infection. The M3 gene is located in a region of the genome that is transcribed during latency. We report here that the M3 protein is a high-affinity broad-spectrum chemokine scavenger. The M3 protein bound the CC chemokines human regulated upon activation of normal T-cell expressed and secreted (RANTES), murine macrophage inflammatory protein 1α (MIP-1α), and murine monocyte chemoattractant protein 1 (MCP-1), as well as the human CXC chemokine interleukin-8, the murine C chemokine lymphotactin, and the murine CX 3 C chemokine fractalkine with high affinity ( K d = 1.6 to 18.7 nM). M3 protein chemokine binding was selective, since the protein did not bind seven other CXC chemokines ( K d > 1 μM). Furthermore, the M3 protein abolished calcium signaling in response to murine MIP-1α and murine MCP-1 and not to murine KC or human stromal cell-derived factor 1 (SDF-1), consistent with the binding data. The M3 protein was also capable of blocking the function of human CC and CXC chemokines, indicating the potential for therapeutic applications. Since the M3 protein lacks homology to known chemokines, chemokine receptors, or chemokine binding proteins, these studies suggest a novel herpesvirus mechanism of immune evasion.
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Abstract Tissue-resident macrophages in the lung comprising alveolar and interstitial macrophages (IMs) display a high degree of heterogeneity. In general, macrophage heterogeneity is thought to arise from various forms of activation that are heavily confounded by the recruitment of monocytes to the tissue-resident macrophage pool. To better understand the functional heterogeneity of IMs in the lung, we profiled the transcription of resident CD206 hi and CD206 lo IMs under steady-state and inflammatory conditions, excluding recruited macrophages. Rather than observing conventional in vitro M1 and M2 activation states, we identified seven chemokine-expressing IM subsets: IMck1 ( Ccl2, Ccl7, Ccl12, and some Cxcl14 ), IMck2-4 ( Ccl3, Ccl4, Ccl5, Cxcl1, Cxcl2, and Cxcl3 ), IMck5 ( Ccl8 ), IMck6 ( Ccl6 and Ccl9 ), IMck7 ( Cxcl9 and Cxcl10 ), IMck8 ( Cxcl13 ), and IMck9 ( Ccl24 ), which were found in steady-state or induced by acute inflammation. Beyond the mouse lung, similar coordinated chemokine signatures were observed in macrophages and monocytes from other tissues and across species. Although all IMs expressed Pf4 (CXCL4), mainly CD206 hi IMs were selectively depleted in Pf4 Cre R26 EYFP-DTR mice. Loss of CD206 hi IMs resulted in significantly reduced inflammatory cell influx in allergen- and infection-driven models, as well as significantly diminished tertiary lymphoid formation and subsequent accumulation of GL7 + germinal center B cells. Overall, our study highlights a division of labor among interstitial macrophages, reflected by the coordinated production of chemokines to control inflammatory cell influx and organize tertiary lymphoid tissue architecture. One Sentence Summary The study highlights a division of labor among interstitial macrophages, reflected by the coordinated production of chemokines to control inflammatory cell influx and organize tertiary lymphoid tissue architecture.
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In vivo leukocyte recruitment is not fully understood and may result from interactions of chemokines with glycosaminoglycans/GAGs. We previously showed that chlorite-oxidized oxyamylose/COAM binds the neutrophil chemokine GCP-2/CXCL6. Here, mouse chemokine binding by COAM was studied systematically and binding affinities of chemokines to COAM versus GAGs were compared. COAM and heparan sulphate bound the mouse CXC chemokines KC/CXCL1, MIP-2/CXCL2, IP-10/CXCL10 and I-TAC/CXCL11 and the CC chemokine RANTES/CCL5 with affinities in the nanomolar range, whereas no binding interactions were observed for mouse MCP-1/CCL2, MIP-1α/CCL3 and MIP-1β/CCL4. The affinities of COAM-interacting chemokines were similar to or higher than those observed for heparan sulphate. Although COAM did not display chemotactic activity by itself, its co-administration with mouse GCP-2/CXCL6 and MIP-2/CXCL2 or its binding of endogenous chemokines resulted in fast and cooperative peritoneal neutrophil recruitment and in extravasation into the cremaster muscle in vivo. These local GAG mimetic features by COAM within tissues superseded systemic effects and were sufficient and applicable to reduce LPS-induced liver-specific neutrophil recruitment and activation. COAM mimics glycosaminoglycans and is a nontoxic probe for the study of leukocyte recruitment and inflammation in vivo.
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Abstract HIV-1 Tat has been proposed as a key agent in many AIDS-related disorders, including HIV-1-associated neurological diseases. We have recently shown that Tat expression induces a significant increase in T lymphocytes in the brains of Tat transgenic mice. The CNS infiltration of T lymphocytes has been noted in AIDS patients. In the present study using this unique genetic system we attempted to understand the underlying mechanisms of Tat expression-induced infiltration of T lymphocytes by examining chemokine expression. RNase protection assay revealed that in addition to CCL2 (monocyte chemoattractant protein-1), CCL3 (macrophage inflammatory protein-1α (MIP-1α)), CCL4 (MIP-1β), CCL5 (RANTES), CXCL2 (MIP-2), and CXCL10 (inducing protein-10), XCL1 (lymphotactin/single C motif-1α/activation-induced, T cell-derived and chemokine-related cytokine) was identified to be up-regulated by Tat expression. XCL1 is a C chemokine and plays a specific and important role in tissue-specific recruitment of T lymphocytes. Thus, we further determined the relationship between Tat and XCL1 expression. Tat-induced XCL1 expression was further confirmed by XCL1-specific RT-PCR and ELISA. Combined in situ hybridization and immunohistochemical staining identified astrocytes, monocytes, and macrophages/microglia as XCL1-producing cells in vivo. Using human astrocytes, U87.MG cells, as an in vitro model, activation of XCL1 expression was positively correlated with Tat expression. Moreover, the XCL1 promoter-driven reporter gene assay showed that Tat-induced XCL1 expression occurred at the transcriptional level. Taken together, these results demonstrate that Tat directly trans-activated XCL1 expression and suggest potential roles of Tat-induced XCL1 expression in the CNS infiltration of T lymphocytes during HIV-1 infection and subsequent HIV-1-induced neurological diseases.
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During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, −2, and −5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine’s ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.
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Neuropeptides play an important role in the active communication between the nervous and immune systems. Substance P (SP) is a prominent neuropeptide involved in neurogenic inflammation and has been reported to exert various proinflammatory actions on inflammatory leukocytes including neutrophils. The present study further investigated the modulatory effect of SP (1 μM) on chemokine production and chemokine receptor expression in primary mouse neutrophils. Our results showed that SP primed neutrophils for chemotactic responses not only to the CXC chemokine macrophage inflammatory protein (MIP)-2/CXCL2 but also to the CC chemokine MIP-1α/CCL3. The activating effect of SP on neutrophils was further evidenced by upregulation of the CD11b integrin, the activation marker of neutrophils. SP induced both the mRNA and protein expression of the chemokines MIP-1α/CCL3 and MIP-2/CXCL2 in neutrophils and upregulated the chemokine receptors CC chemokine receptor (CCR)-1 and CXC chemokine receptor (CXCR)-2. This stimulatory effect on chemokine and chemokine receptor expression in neutrophils was further found to be neurokinin-1 receptor (NK-1R) specific. Pretreatment with selective NK-1R antagonists inhibited SP-triggered activation of neutrophils and chemokine and chemokine receptor upregulation. Moreover, SP-induced chemokine upregulation was NF-κB dependent. SP time dependently induced NF-κB p65 binding activity, IκBα degradation, and NF-κB p65 nuclear translocation in neutrophils. Inhibition of NF-κB activation with its inhibitor Bay11-7082 (10 μM) abolished SP-induced NF-κB binding activity and upregulation of MIP-1α/CCL3 and MIP-2/CXCL2 in neutrophils. Together, these results suggest that SP exerts a direct stimulatory effect on the expression of chemokines and chemokine receptors in mouse neutrophils. The effect is NK-1R mediated, involving NF-κB activation.
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