Secretion of oncostatin M by infiltrating neutrophils: regulation of IL-6 and chemokine expression in human mesothelial cells

Recently, we identified that regulation of leukocyte recruitment by IL-6 requires shedding of the IL-6R from infiltrating neutrophils. In this study, experiments have examined whether other IL-6-related cytokines possess similar properties. Levels of oncostatin M (OSM) and leukemia inhibitory factor were analyzed in patients with overt bacterial peritonitis during the first 5 days of infection. Although no change in leukemia inhibitory factor was observed throughout the duration of infection, OSM was significantly elevated on day 1 and rapidly returned to baseline by days 2-3. The source of OSM was identified as the infiltrating neutrophils, and OSM levels correlated both with leukocyte numbers and i.p. soluble IL-6R (sIL-6R) levels. FACS analysis revealed that OSM receptor beta expression was restricted to human peritoneal mesothelial cells. Stimulation of human peritoneal mesothelial cells with OSM induced phosphorylation of gp130 and OSM receptor beta, which was accompanied by activation of STAT3 and secretion of CC chemokine ligand 2/monocyte chemoattractant protein-1 and IL-6. Although OSM itself did not modulate CXC chemokine ligand 8/IL-8 release, it effectively suppressed IL-1beta-mediated expression of this neutrophil-activating CXC chemokine. Moreover, OSM synergistically blocked IL-1beta-induced CXC chemokine ligand 8 secretion in combination with the IL-6/sIL-6R complex. Thus suggesting that OSM and sIL-6R release from infiltrating neutrophils may contribute to the temporal switch between neutrophil influx and mononuclear cell recruitment seen during acute inflammation.     

Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo

The Duffy Ag expressed on RBCs, capillaries, and postcapillary venular endothelial cells binds selective CXC and CC chemokines with high affinity. Cells transfected with the Duffy Ag internalize but do not degrade chemokine ligand. It has been proposed that Duffy Ag transports chemokines across the endothelium. We hypothesized that Duffy Ag participates in the movement of chemokines across the endothelium and, by doing so, modifies neutrophil transmigration. We found that the Duffy Ag transfected into human endothelial cells facilitates movement of the radiolabeled CXC chemokine, growth related oncogene-alpha/CXC chemokine ligand 1 (GRO-alpha/CXCL1), across an endothelial monolayer. In addition, neutrophil migration toward GRO-alpha/CXCL1 and IL-8 (IL-8/CXCL8) was enhanced across an endothelial monolayer expressing the Duffy Ag. Furthermore, GRO-alpha/CXCL1 stimulation of endothelial cells expressing the Duffy Ag did not affect gene expression by oligonucleotide microarray analysis. These in vitro observations are supported by the finding that IL-8/CXCL8-driven neutrophil recruitment into the lungs was markedly attenuated in transgenic mice lacking the Duffy Ag. We conclude that Duffy Ag has a role in enhancing leukocyte recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium.     

Viral IL-6 blocks neutrophil infiltration during acute inflammation

Pathologies arising as a consequence of human herpesvirus-8 (HHV8) infections are closely associated with the autocrine activity of a HHV8 encoded IL-6 (vIL-6), which promotes proliferation of infected cells and their resistance to apoptosis. In this present report, studies show that vIL-6 may also be important in influencing the host's immunological response to secondary infections. Using peritoneal inflammation as a model of acute bacterial infection, vIL-6 was found to specifically block neutrophil recruitment in vivo through regulation of inflammatory chemokine expression. This response was substantiated in vitro where activation of STAT3 in human peritoneal mesothelial cells by vIL-6 was associated with enhanced CCL2 release. Although vIL-6 did not effect CXCL8 production, IL-1beta-induced secretion of this neutrophil-activating chemokine was significantly suppressed by vIL-6. These data suggest that vIL-6 has the capacity to suppress innate immune responses and thereby influence the outcome of opportunistic infections in HHV8-associated disease.     

Eotaxin/CCL11 suppresses IL-8/CXCL8 secretion from human dermal microvascular endothelial cells

The CC chemokine eotaxin/CCL11 is known to bind to the receptor CCR3 on eosinophils and Th2-type lymphocytes. In this study, we demonstrate that CCR3 is expressed on a subpopulation of primary human dermal microvascular endothelial cells and is up-regulated by TNF-alpha. We found that incubation of human dermal microvascular endothelial cells with recombinant eotaxin/CCL11 suppresses TNF-alpha-induced production of the neutrophil-specific chemokine IL-8/CXCL8. The eotaxin/CCL11-suppressive effect on endothelial cells was not seen on IL-1beta-induced IL-8/CXCL8 release. Eotaxin/CCL11 showed no effect on TNF-alpha-induced up-regulation of growth-related oncogene-alpha or IFN-gamma-inducible protein-10, two other CXC chemokines tested, and did not affect production of the CC chemokines monocyte chemoattractant protein-1/CCL2 and RANTES/CCL5, or the adhesion molecules ICAM-1 and E-selectin. These results suggest that eotaxin/CXCL11 is not effecting a general suppression of TNF-alphaR levels or signal transduction. Suppression of IL-8/CXCL8 was abrogated in the presence of anti-CCR3 mAb, pertussis toxin, and wortmannin, indicating it was mediated by the CCR3 receptor, G(i) proteins, and phosphatidylinositol 3-kinase signaling. Eotaxin/CCL11 decreased steady state levels of IL-8/CXCL8 mRNA in TNF-alpha-stimulated cells, an effect mediated in part by an acceleration of IL-8 mRNA decay. Eotaxin/CCL11 may down-regulate production of the neutrophil chemoattractant IL-8/CXCL8 by endothelial cells in vivo, acting as a negative regulator of neutrophil recruitment. This may play an important biological role in the prevention of overzealous inflammatory responses, aiding in the resolution of acute inflammation or transition from neutrophilic to mononuclear/eosinophilic inflammation.     

Fas ligation and tumor necrosis factor α activation of murine astrocytes promote heat shock factor-1 activation and heat shock protein expression leading to chemokine induction and cell survival

Death-inducing ligands tumor necrosis factor alpha (TNFα) and Fas ligand (FasL) do not kill cultured astrocytes; instead they induce a variety of chemokines including macrophage-inflammatory protein-1α/CC chemokine ligand 3 (CCL3), monocyte chemoattractant protein-1 (CC CCL-2), macrophage-inflammatory protein-2/CXC chemokine ligand 2 (CXCL2, a murine homologue of interleukin 8), and interferon-induced protein of 10 kDa (CXCL10). Induction is enhanced by protein synthesis inhibition suggesting the existence of endogenous inhibitors. ERK, NF-κB, heat shock factor-1 (HSF-1) and heat shock proteins were examined for their possible roles in signal transduction. Inhibition of ERK activation by PD98059 partially inhibited expression of all but FasL-induced CXCL10. Although inhibition of NF-κB DNA binding inhibited chemokine induction, PD98059 did not inhibit TNFα-induced NF-κB DNA binding suggesting that ERK serves an NF-κB-independent pathway. Heat shock itself induced astrocytic chemokine expression; both TNFα and FasL induced HSF-1 DNA binding and Hsp72 production; and Hsp72-induced chemokine expression. Inhibition of either HSF-1 binding with quercetin or heat shock protein synthesis with KNK437 compromised chemokine induction without compromising cell survival. These data suggest that the induction of heat shock proteins via HSF-1 contribute to the TNFα- and FasL-induced expression of chemokines in astrocytes.     

Differential regulation of chemokine production in human peritoneal mesothelial cells: IFN-gamma controls neutrophil migration across the mesothelium in vitro and in vivo.

Leukocyte recruitment into the infected peritoneal cavity consists of an early, predominant polymorphonuclear leukocyte (PMN) influx and subsequent, prolonged mononuclear cell migration phase. Although chemokine secretion by resident peritoneal cells plays a primary role in mediating this migration, the mechanisms involved in controlling the switch in phenotype of cell infiltrate remain unclear. The present study investigates a potential role for the Th1-type cytokine IFN-gamma in the process of leukocyte recruitment into the peritoneal cavity. Stimulation of cultured human peritoneal mesothelial cells with IFN-gamma (1-100 U/ml) alone or in combination with IL-1beta (100 pg/ml) or TNF-alpha (1000 pg/ml) resulted in significant up-regulation of monocyte chemoattractant protein-1 and RANTES protein secretion. In contrast, IFN-gamma inhibited basal and IL-1beta-, and TNF-alpha-induced production of IL-8. The modulating effects of IFN-gamma on chemokine production occurred at the level of gene expression, and the degree of regulation observed was dependent on the doses of IL-1beta and TNF-alpha used. Analysis of the functional effects of IFN-gamma on IL-1beta-induced transmesothelial PMN migration with an in vitro human transmigration system and an in vivo murine model of peritoneal inflammation demonstrated that IFN-gamma was able to down-regulate PMN migration induced by optimal doses of IL-1beta. These effects were mediated in vivo via down-regulation of CXC chemokine synthesis. These findings suggest that IFN-gamma may play a role in controlling the phenotype of infiltrating leukocyte during the course of an inflammatory response, in part via regulation of resident cell chemokine synthesis.     

Coexpression and interaction of CXCL10 and CD26 in mesenchymal cells by synergising inflammatory cytokines: CXCL8 and CXCL10 are discriminative markers for autoimmune arthropathies

Leukocyte infiltration during acute and chronic inflammation is regulated by exogenous and endogenous factors, including cytokines, chemokines and proteases. Stimulation of fibroblasts and human microvascular endothelial cells with the inflammatory cytokines interleukin-1beta (IL-1beta) or tumour necrosis factor alpha (TNF-alpha) combined with either interferon-alpha (IFN-alpha), IFN-beta or IFN-gamma resulted in a synergistic induction of the CXC chemokine CXCL10, but not of the neutrophil chemoattractant CXCL8. In contrast, simultaneous stimulation with different IFN types did not result in a synergistic CXCL10 protein induction. Purification of natural CXCL10 from the conditioned medium of fibroblasts led to the isolation of CD26/dipeptidyl peptidase IV-processed CXCL10 missing two NH2-terminal residues. In contrast to intact CXCL10, NH2-terminally truncated CXCL10(3-77) did not induce extracellular signal-regulated kinase 1/2 or Akt/protein kinase B phosphorylation in CXC chemokine receptor 3-transfected cells. Together with the expression of CXCL10, the expression of membrane-bound CD26/dipeptidyl peptidase IV was also upregulated in fibroblasts by IFN-gamma, by IFN-gamma plus IL-1beta or by IFN-gamma plus TNF-alpha. This provides a negative feedback for CXCL10-dependent chemotaxis of activated T cells and natural killer cells. Since TNF-alpha and IL-1beta are implicated in arthritis, synovial concentrations of CXCL8 and CXCL10 were compared in patients suffering from crystal arthritis, ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis. All three groups of autoimmune arthritis patients (ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis) had significantly increased synovial CXCL10 levels compared with crystal arthritis patients. In contrast, compared with crystal arthritis, only rheumatoid arthritis patients, and not ankylosing spondylitis or psoriatic arthritis patients, had significantly higher synovial CXCL8 concentrations. Synovial concentrations of the neutrophil chemoattractant CXCL8 may therefore be useful to discriminate between autoimmune arthritis types.     

Gelatinase B/MMP-9 and neutrophil collagenase/MMP-8 process the chemokines human GCP-2/CXCL6, ENA-78/CXCL5 and mouse GCP-2/LIX and modulate their physiological activities.

On chemokine stimulation, leucocytes produce and secrete proteolytic enzymes for innate immune defence mechanisms. Some of these proteases modify the biological activity of the chemokines. For instance, neutrophils secrete gelatinase B (matrix metalloproteinase-9, MMP-9) and neutrophil collagenase (MMP-8) after stimulation with interleukin-8/CXCL8 (IL-8). Gelatinase B cleaves and potentiates IL-8, generating a positive feedback. Here, we extend these findings and compare the processing of the CXC chemokines human and mouse granulocyte chemotactic protein-2/CXCL6 (GCP-2) and the closely related human epithelial-cell derived neutrophil activating peptide-78/CXCL5 (ENA-78) with that of human IL-8. Human GCP-2 and ENA-78 are cleaved by gelatinase B at similar rates to IL-8. In addition, GCP-2 is cleaved by neutrophil collagenase, but at a lower rate. The cleavage of GCP-2 is exclusively N-terminal and does not result in any change in biological activity. In contrast, ENA-78 is cleaved by gelatinase B at eight positions at various rates, finally generating inactive fragments. Physiologically, sequential cleavage of ENA-78 may result in early potentiation and later in inactivation of the chemokine. Remarkably, in the mouse, which lacks IL-8 which is replaced by GCP-2/LIX as the most potent neutrophil activating chemokine, N-terminal clipping and twofold potentiation by gelatinase B was also observed. In addition to the similarities in the potentiation of IL-8 in humans and GCP-2 in mice, the conversion of mouse GCP-2/LIX by mouse gelatinase B is the fastest for any combination of chemokines and MMPs so far reported. This rapid conversion was also performed by crude neutrophil granule secretion under physiological conditions, extending the relevance of this proteolytic cleavage to the in vivo situation.     

ELR+ CXC chemokines in human milk

CXC chemokines bearing the glutamic acid-leucine-arginine (ELR) motif are crucial mediators in neutrophil-dependent acute inflammation. Interestingly, however, Interleukin (IL)-8/CXC ligand (CXCL) 8 is expressed in human milk in biologically significant concentrations, and may play a local maturational role in the developing human intestine. In this chemokine subfamily, there are six other known peptides beside IL-8/CXCL8, all sharing similar effects on neutrophil chemotaxis and angiogenesis. In this study, we measured the concentrations of these chemokines in human milk, sought their presence in human mammary tissue by immunohistochemistry, and confirmed chemokine expression in cultured human mammary epithelial cells (HMECs). Each of the seven ELR(+) CXC chemokines was measurable in milk, and except for NAP-2/CXCL7, these concentrations were higher than serum. The concentrations were higher in colostrum (except for GRO-beta/CXCL2 and NAP-2/CXCL7), and correlated negatively with time elapsed postpartum. IL-8/CXCL8, GRO-gamma/CXCL3, and ENA-78/CXCL5 concentrations were higher in preterm milk. There was intense immunoreactivity in mammary epithelial cells for all ELR(+) CXC chemokines, and the intensity of staining was higher in breast tissue with lactational changes. The supernatants from confluent HMEC cultures also contained measurable concentrations of all the seven ELR(+) CXC chemokines. These results confirm that all ELR(+) CXC chemokines are actively secreted by the mammary epithelial cells into human milk. Further studies are needed to determine if these chemokines share with IL-8/CXCL8 the protective effects on intestinal epithelial cells.     

NH2- and COOH-terminal truncations of murine granulocyte chemotactic protein-2 augment the in vitro and in vivo neutrophil chemotactic potency

Chemokines are important mediators of leukocyte migration during the inflammatory response. Post-translational modifications affect the biological potency of chemokines. In addition to previously identified NH2-terminally truncated forms, COOH-terminally truncated forms of the CXC chemokine murine granulocyte chemotactic protein-2 (GCP-2) were purified from conditioned medium of stimulated fibroblasts. The truncations generated 28 natural murine GCP-2 isoforms containing 69-92 residues, including most intermediate forms. Both NH2- and COOH-terminal truncations of GCP-2 resulted in enhanced chemotactic potency for human and murine neutrophils in vitro. The truncated isoform GCP-2(9-78) was 30-fold more potent than intact GCP-2(1-92)/LPS-induced CXC chemokine (LIX) at inducing an intracellular calcium increase in human neutrophils. After intradermal injection in mice, GCP-2(9-78) was also more effective than GCP-2(1-92)/LIX at inducing neutrophil infiltration. Similar to human IL-8 and GCP-2, murine GCP-2(9-78) and macrophage inflammatory protein-2 (MIP-2) induced calcium increases in both CXCR1 and CXCR2 transfectants. Murine GCP-2(9-78) could desensitize the calcium response induced by MIP-2 in human neutrophils and vice versa. Furthermore, MIP-2 and truncated GCP-2(9-78), but not intact GCP-2(1-92)/LIX, partially desensitized the calcium response to human IL-8 in human neutrophils. Taken together, these findings point to an important role of post-translationally modified GCP-2 to replace IL-8 in the mouse.     

Differential expression and polarized secretion of CXC and CC chemokines by human intestinal epithelial cancer cell lines in response to Clostridium difficile toxin A

Intestinal epithelial cells are the initial sites of host response to Clostridium difficile infection and can play a role in signaling the influx of inflammatory cells. To further explore this role, the regulated expression and polarized secretion of CXC and CC chemokines by human intestinal epithelial cells were investigated. An expression of the CXC chemokines, including IL-8 and growth-related oncogene (GRO)-alpha, and the CC chemokine monocyte chemoattractant protein (MCP)-1 from HT-29 cells increased in the 1-6 hr following C. difficile toxin A stimulation, assessed by quantitative RT-PCR. In contrast, the expression of neutrophil activating protein-78 (ENA-78) was delayed for 18 hr. The up-regulated mRNA expression of chemokines was paralleled by the increase of protein levels. However, the expression of macrophage inflammatory protein (MIP)-1alpha, RANTES (regulated on activation normal T cells expressed and secreted), and interferon-gamma-inducible protein-10 (IP-10) was not changed in HT-29 or Caco-2 cells stimulated with toxin A. Upon stimulation of the polarized Caco-2 epithelial cells in a transwell chamber with toxin A, CXC and CC chemokines were released predominantly into the basolateral compartment. Moreover, the addition of IFN-gamma and TNF-alpha to toxin A stimulated Caco-2 cells increased the basolateral release of CC chemokine MCP-1. In contrast, IFN-gamma and TNF-alpha had no effect on the expression of the CXC chemokines IL-8 and GRO-alpha. These results suggest that a CXC and CC chemokine expression from epithelial cells infected with C. difficile may be an important factor in the mucosal inflammatory response.     

Absence of CXCL10 Aggravates Herpes Stromal Keratitis with Reduced Primary Neutrophil Influx in Mice

Herpes simplex virus 1 (HSV-1) replication initiates inflammation and angiogenesis responses in the cornea to result in herpetic stromal keratitis (HSK), which is a leading cause of infection-induced vision impairment. Chemokines are secreted to modulate HSK by recruiting leukocytes, which affect virus growth, and by influencing angiogenesis. The present study used a murine infection model to investigate the significance of the chemokine CXC chemokine ligand 10 (CXCL10; gamma interferon-inducible protein 10 [IP-10]) in HSK. Here, we show that HSV-1 infection of the cornea induced CXCL10 protein expression in epithelial cells. The corneas of mice with a targeted disruption of the gene encoding CXCL10 displayed decreases in levels of neutrophil-attracting cytokine (interleukin-6), primary neutrophil influx, and viral clearance 2 or 3 days postinfection. Subsequently, absence of CXCL10 aggravated HSK with elevated levels of interleukin-6, chemokines for CD4⁺ T cells and/or neutrophils (macrophage inflammatory protein-1α and macrophage inflammatory protein-2), angiogenic factor (vascular endothelial growth factor A), and secondary neutrophil influx, as well as infiltration of CD4⁺ T cells to exacerbate opacity and angiogenesis in the cornea at 14 and up to 28 days postinfection. Our results collectively show that endogenous CXCL10 contributes to recruit the primary neutrophil influx and to affect the expression of cytokines, chemokines, and angiogenic factors as well as to reduce the viral titer and HSK severity.     

Oncostatin M receptor-beta signaling limits monocytic cell recruitment in acute inflammation

Although the IL-6-related cytokine oncostatin M (OSM) affects processes associated with disease progression, the specific function of OSM in the face of an inflammatory challenge remains unclear. In this report, a peritoneal model of acute inflammation was used to define the influence of OSM on chemokine-mediated leukocyte recruitment. When compared with wild-type and IL-6-deficient mice, peritoneal inflammation in oncostatin M receptor-beta-deficient (OSMR-KO) mice resulted in enhanced monocytic cell trafficking. In contrast to IL-6-deficient mice, OSMR-KO mice displayed no difference in neutrophil and lymphocyte migration. Subsequent in vitro studies using human peritoneal mesothelial cells and an in vivo appraisal of inflammatory chemokine expression after peritoneal inflammation identified OSM as a prominent regulator of CCL5 expression. Specifically, OSM inhibited IL-1beta-mediated NF-kappaB activity and CCL5 expression in human mesothelial cells. This was substantiated in vivo where peritoneal inflammation in OSMR-KO mice resulted in a temporal increase in both CCL5 secretion and NF-kappaB activation. These findings suggest that IL-6 and OSM individually affect the profile of leukocyte trafficking, and they point to a hitherto unidentified interplay between OSM signaling and the inflammatory activation of NF-kappaB.     

Early enhanced local neutrophil recruitment in peritonitis-induced sepsis improves bacterial clearance and survival

Neutrophils are critical for the rapid eradication of bacterial pathogens, but they also contribute to the development of multiple organ failure in sepsis. We hypothesized that increasing early recruitment of neutrophils to the focus of infection will increase bacterial clearance and improve survival. Sepsis was induced in mice, using cecal ligation and puncture (CLP); blood samples were collected at 6 and 24 h; and survival was followed for 28 d. In separate experiments, peritoneal bacteria and inflammatory cells were measured. Septic mice predicted to die based on IL-6 levels (Die-P) had higher concentrations of CXCL1 and CXCL2 in the peritoneum and plasma compared with those predicted to live (Live-P). At 6 h, Live-P and Die-P had equivalent numbers of peritoneal neutrophils and bacteria. In Die-P mice the number of peritoneal bacteria increased between 6 and 24 h post-CLP, whereas in Live-P it decreased. The i.p. injection of CXCL1 and CXCL2 in naive mice resulted in local neutrophil recruitment. When given immediately after CLP, CXC chemokines increased peritoneal neutrophil recruitment at 6 h after CLP. This early increase in neutrophils induced by exogenous chemokines resulted in significantly fewer peritoneal bacteria by 24 h [CFU (log) = 6.04 versus 4.99 for vehicle versus chemokine treatment; p < 0.05]. Chemokine treatment significantly improved survival at both 5 d (40 versus 72%) and 28 d (27 versus 52%; p < 0.02 vehicle versus chemokines). These data demonstrate that early, local treatment with CXC chemokines enhances neutrophil recruitment and clearance of bacteria as well as improves survival in the CLP model of sepsis.     

The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells

Toll-like receptors (TLRs) are pattern recognition receptors that serve an important function in detecting pathogens and initiating inflammatory responses. Upon encounter with foreign Ag, dendritic cells (DCs) go through a maturation process characterized by an increase in surface expression of MHC class II and costimulatory molecules, which leads to initiation of an effective immune response in naive T cells. The innate immune response to bacterial flagellin is mediated by TLR5, which is expressed on human DCs. Therefore, we sought to investigate whether flagellin could induce DC maturation. Immature DCs were cultured in the absence or presence of flagellin and monitored for expression of cell surface maturation markers. Stimulation with flagellin induced increased surface expression of CD83, CD80, CD86, MHC class II, and the lymph node-homing chemokine receptor CCR7. Flagellin stimulated the expression of chemokines active on neutrophils (IL-8/CXC chemokine ligand (CXCL)8, GRO-alpha/CXCL1, GRO-beta/CXCL2, GRO-gamma/CXCL3), monocytes (monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2), and immature DCs (macrophage-inflammatory protein-1 alpha/CCL3, macrophage-inflammatory protein-1 beta/CCL4), but not chemokines active on effector T cells (IFN-inducible protein-10 kDa/CXCL10, monokine induced by IFN-gamma/CXCL9, IFN-inducible T cell alpha chemoattractant/CXCL11). However, stimulating DCs with both flagellin and IFN-inducible protein-10 kDa, monokine induced by IFN-gamma, and IFN-inducible T cell alpha chemoattractant expression, whereas stimulation with IFN-beta or flagellin alone failed to induce these chemokines. In functional assays, flagellin-matured DCs displayed enhanced T cell stimulatory activity with a concomitant decrease in endocytic activity. Finally, DCs isolated from mouse spleens or bone marrows were shown to not express TLR5 and were not responsive to flagellin stimulation. These results demonstrate that flagellin can directly stimulate human but not murine DC maturation, providing an additional mechanism by which motile bacteria can initiate an acquired immune response.     

Cutting edge: activity of human adult microglia in response to CC chemokine ligand 21

The approximately 50 known chemokines are classified in distinct subfamilies: CXC, CC, CX3C, and C. Although the signaling of chemokines often is promiscuous, signaling events between members of these distinct chemokine classes are hardly observed. The only known exception so far is the murine CC chemokine ligand (CCL)21 (secondary lymphoid tissue chemokine, Exodus-2, 6Ckine), which binds and activates the murine CXC chemokine receptor CXCR3. However, this exception has not been found in humans. In this study, we provide evidence that human CCL21 is a functional ligand for endogenously expressed CXCR3 in human adult microglia. In absence of CCR7 expression, CCL21 induced chemotaxis of human microglia with efficiency similar to the CXCR3 ligands CXC chemokine ligand 9 (monokine induced by IFN-gamma) and CXC chemokine ligand 10 (IFN-gamma-inducible protein-10). Because human CCL21 did not show any effects in CXCR3-transfected HEK293 cells, it is indicated that CXCR3 signaling depends on the cellular background in which the CXCR3 is expressed.     

Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles

Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.