C-C chemokine hepatocellular carcinoma motif ligand 5-deficiency promotes hepatocellular carcinoma progression by affecting B cell recruitment.

OBJECTIVE: To evaluate the expression of C-C motif chemokine ligand 5 (CCL5) in hepatocellular carcinoma (HCC) and to explore its role in regulating the immune microenvironment and the related mechanism in tumor immunity. METHODS: The mRNA expression level of CCL5 in HCC and adjacent non-cancerous tissues was measured by quantitative polymerase chain reaction and the protein expression was examined by immunohistochemistry. Serum CCL5 expression was measured by an enzyme-linked immunosorbent assay (ELISA). C57BL/6 wild-type (WT) and Ccl5-knockout (Ccl5-/- ) mice were utilized to conduct the diethylnitrosamine-induced HCC model. The immune cell population was determined by flow cytometry, and peripheral serum immunoglobulin M (IgM) level was quantified by ELISA. RESULTS: CCL5 expression was low in HCC tissue and peripheral blood compared with adjacent non-cancerous tissues or controls, and its expression was correlated with the overall survival, cancer recurrence and distant metastasis. In the HCC mouse model, liver-to-body weight ratio was of the Ccl5-/- group were higher than that of the WT group. Moreover, compared with the WT mice, the number of B cells in the tumor tissue of the Ccl5-/- mice was lower, while there were no significant differences in the other immune cell populations. Furthermore, serum IgM level of the Ccl5-/- mice was significantly lower than that of the WT mice. CONCLUSION: CCL5 expression is decreased in HCC tissues. CCL5 deficiency reduces B cell recruitment and decreases IgM secretion in HCC, potentially leading to tumor progression.

Chemokine receptors coordinately regulate macrophage dynamics and mammary gland development.

Macrophages are key regulators of developmental processes, including those involved in mammary gland development. We have previously demonstrated that the atypical chemokine receptor ACKR2 contributes to the control of ductal epithelial branching in the developing mammary gland by regulating macrophage dynamics. ACKR2 is a chemokine-scavenging receptor that mediates its effects through collaboration with inflammatory chemokine receptors (iCCRs). Here, we reveal reciprocal regulation of branching morphogenesis in the mammary gland, whereby stromal ACKR2 modulates levels of the shared ligand CCL7 to control the movement of a key population of CCR1-expressing macrophages to the ductal epithelium. In addition, oestrogen, which is essential for ductal elongation during puberty, upregulates CCR1 expression on macrophages. The age at which girls develop breasts is decreasing, which raises the risk of diseases including breast cancer. This study presents a previously unknown mechanism controlling the rate of mammary gland development during puberty and highlights potential therapeutic targets.

CCL5 deficiency promotes liver repair by improving inflammation resolution and liver regeneration through M2 macrophage polarization.

Despite the diverse etiologies of drug-induced liver injury (DILI), innate immunity activation is a common feature involved in DILI progression. However, the involvement of innate immunity regulation in inflammation resolution and liver regeneration in DILI remains obscure. Herein, we identified the chemokine CCL5 as a central mediator of innate immunity regulation in the pathogenesis of DILI. First, we showed that serum and hepatic CCL5 levels are elevated in both DILI patients and an APAP-induced liver injury (AILI) mouse model. Interestingly, both nonparenchymal cells and stressed hepatocytes are cell sources of CCL5 induction in response to liver injury. Functional experiments showed that CCL5 deficiency has no effect on the early phase of AILI but promotes liver repair in the late phase mainly by promoting inflammation resolution and liver regeneration, which are associated with an increased number of hepatic M2 macrophages. Mechanistically, CCL5 can directly activate M1 polarization and impede M2 polarization through the CCR1- and CCR5-mediated activation of the MAPK and NF-κB pathways. We then showed that CCL5 inhibition mediated by either a CCL5-neutralizing antibody or the antagonist Met-CCL5 can greatly alleviate liver injury and improve survival in an AILI mouse model. Our data demonstrate CCL5 induction during DILI, identify CCL5 as a novel innate immunity regulator in macrophage polarization, and suggest that CCL5 blockage is a promising therapeutic strategy for the treatment of DILI.

Deletion of C-C Motif Chemokine Ligand 5 Worsens Invariant Natural Killer T-Cell-Mediated Hepatitis via Compensatory Up-regulation of CXCR2-Related Chemokine Activity.

BACKGROUND & AIMS: Chemokine-mediated immune cell recruitment plays pivotal roles in liver inflammation. C-C motif chemokine ligand 5 (CCL5) has been shown to be responsible for the recruitment of monocytes/macrophages and has been implicated in various liver diseases, including nonalcoholic fatty liver disease, fibrosis, and hepatocellular carcinoma. Previous studies have also shown that inhibition of CCL5 appears to be a promising therapeutic approach for several chronic liver diseases. However, whether blocking CCL5 could benefit immune cell-mediated hepatitis remains largely elusive. METHODS: By adopting a specific agonist, alpha-galactosylceramide (α-Galcer), of invariant natural killer T cells (iNKTs), we investigated the function and mechanism of CCL5 in the iNKT induced murine hepatitis model. RESULTS: We found significantly increased CCL5 expression in α-Galcer-induced hepatitis murine model. Such an increase in CCL5 is mainly enriched in non-parenchymal cells such as macrophages and iNKTs but not in hepatocytes. Surprisingly, CCL5 blockage by genetic deletion of Ccl5 does not affect the α-Galcer-induced iNKT activation but greatly worsens α-Galcer-induced liver injury accompanied by an increased hepatic neutrophil infiltration. Mechanistically, we demonstrated that greater neutrophil accumulation in the liver is responsible for the enhanced liver injury in Ccl5-/- mice. Such an increased hepatic neutrophil infiltration is mainly caused by an enhanced CXCL1-CXCR2 signal in Ccl5-/- mice. Therapeutically, either antibody-mediated neutrophil depletion or a CXCR2 antagonist, SB225002, mediated CXCR2 signaling blockage significantly ameliorated α-Galcer-induced liver injury in Ccl5-/- mice. CONCLUSIONS: Our present study demonstrates that (1) α-Galcer-induced murine hepatitis could greatly induce CCL5 production in macrophages and iNKT cells; (2) loss of CCL5 could enhance CXCL1 expression in hepatocytes and activate CXCL1-CXCR2 axis in neutrophils to augment their hepatic infiltration; and (3) neutrophil depletion or blockage of CXCL1-CXCR2 axis greatly improves α-Galcer-induced liver injury in Ccl5-/- mice. This study suggests that clinical utilization of CCL5 blockage may compensatorily induce the activation of other chemokine pathways to enhance neutrophil recruitment and liver injury in hepatitis.

CCL5-deficiency enhances intratumoral infiltration of CD8+ T cells in colorectal cancer.

Colorectal cancer (CRC) is the third most common solid tumor in the world and shows resistance to several immunotherapies, particularly immune checkpoint blockade which has therapeutic effects on many other types of cancer. Cytotoxic CD8+ T cell has been considered as one of the main populations of effector immune cells in antitumor immunity; however, the absence of CD8+ T cells in the central tumor area has become a major obstacle for solid tumor immunotherapy, particularly for CRC. Thus, novel therapeutic strategies that could promote CD8+ T cells to accumulate in the central tumor area are urgently needed. Here, we demonstrated that CCL5-deficiency delayed tumor growth and metastasis via facilitating CD8+ T cells to accumulate into tumor site in CRC mouse models. Furthermore, CCL5-deficiency could upregulate PD-1 and PD-L1 expression and reduce the resistance to anti-PD-1 antibody therapy in CRC mouse model. Mechanically, the results of RNA-sequencing, in vitro coculture system and hypoxia measurements demonstrated that knockdown of CCL5 could result in the metabolic disorders in CD11bhiF4/80low TAMs and suppress the expression of S100a9 to promote the migration of CD8+ T cells in the tumor microenvironment. These findings were verified by the data of clinical samples from CRC patients, suggesting that CCL5 may provide a potential therapeutic target for the combined PD-1-immunotherapy of CRC.

CCL5 deficiency rescues pulmonary vascular dysfunction, and reverses pulmonary hypertension via caveolin-1-dependent BMPR2 activation.

Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder characterized by pulmonary arterial remodeling mainly due to excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with PAH impairs pulmonary arterial endothelial cells (PAECs) function. This can adversely affect PAEC survival and promote PASMCs proliferation. We hypothesized that interventions to normalize the expression of genes that are targets of the BMPR2 signaling could restore PAECs function and prevent or reverse PAH. Here we characterized for the first time, in human PAECs, chemokine (C-C motif) ligand 5 (CCL5/RANTES) deficiency restore BMP-mediated PAECs function. In the cell culture experiments, we found that CCL5 deficiency increased apoptosis and tube formation of PAECs, but suppressed proliferation and migration of PASMCs. Silencing CCL5 expression in PAH PAECs restored bone morphogenetic protein (BMP) signaling responses and promoted phosphorylation of SMADs and transcription of ID genes. Moreover, CCL5 deficiency inhibited angiogenesis by increasing pSMAD-dependent and-independent BMPR2 signaling. This was linked mechanistically to enhanced interaction of BMPR2 with caveolin-1 via CCL5 deficiency-mediated stabilization of endothelial surface caveolin-1. Consistent with these functions, deletion of CCL5 significantly attenuated development of Sugen5416/hypoxia-induced PAH by restoring BMPR2 signaling in mice. Taken together, our findings suggest that CCL5 deficiency could reverse obliterative changes in pulmonary arteries via caveolin-1-dependent amplification of BMPR2 signaling. Our results shed light on better understanding of the disease pathobiology and provide a possible novel target for the treatment of PAH.

Functional role of CCL5/RANTES for HCC progression during chronic liver disease.

BACKGROUND & AIMS: During liver inflammation, triggering fibrogenesis and carcinogenesis immune cells play a pivotal role. In the present study we investigated the role of CCL5 in human and in murine models of chronic liver inflammation leading to hepatocellular carcinoma (HCC) development. METHODS: CCL5 expression and its receptors were studied in well-defined patients with chronic liver disease (CLD) and in two murine inflammation based HCC models. The role of CCL5 in inflammation, fibrosis, tumor initiation and progression was analyzed in different cell populations of NEMOΔhepa/CCL5-/- animals and after bone marrow transplantation (BMT). For therapeutic intervention Evasin-4 was injected for 24h or 8weeks. RESULTS: In CLD patients, CCL5 and its receptor CCR5 are overexpressed - an observation confirmed in the Mdr2-/- and NEMOΔhepa model. CCL5 deletion in NEMOΔhepa mice diminished hepatocyte apoptosis, compensatory proliferation and fibrogenesis due to reduced immune cell infiltration. Especially, CD45+/Ly6G+ granulocytes, CD45+/CD11b+/Gr1.1+/F4/80+ pro-inflammatory monocytes, CD4+ and CD8+ T cells were decreased. One year old NEMOΔhepa/CCL5-/- mice displayed smaller and less malignant tumors, characterized by reduced proliferative capacity and less pronounced angiogenesis. We identified hematopoietic cells as the main source of CCL5, while CCL5 deficiency did not sensitise NEMOΔhepa hepatocytes towards TNFα induced apoptosis. Finally, therapeutic intervention with Evasin-4 over a period of 8weeks ameliorated liver disease progression. CONCLUSION: We identified an important role of CCL5 in human and functionally in mice with disease progression, especially HCC development. A novel approach to inhibit CCL5 in vivo thus appears encouraging for patients with CLD. LAY SUMMARY: Our present study identifies the essential role of the chemoattractive cytokine CCL5 for liver disease progression and especially hepatocellular carcinoma development in men and mice. Finally, the inhibition of CCL5 appears to be encouraging for therapy of human chronic liver disease.

CCL5-Mediated Th2 Immune Polarization Promotes Metastasis in Luminal Breast Cancer.

The tumor-promoting chemokine CCL5 has been implicated in malignant transformation of breast epithelial cells, with studies to date focusing mainly on basal-type breast cancers. In this study, we investigated the consequences of CCL5 deletion in the MMTV-PyMT transgenic mouse model of luminal breast cancer. In this model, primary tumor burden and pulmonary metastases were reduced significantly in CCL5-deficient subjects, an effect found to be associated with a deficit of Th2 (IL4⁺CD4⁺ T) cells. Mechanistic investigations revealed that CCL5 activates CCR3, a highly expressed chemokine receptor on CD4⁺ T cells, and also boosts Gfi1 expression to promote the differentiation of Th2 cells, which enhance the prometastatic activity of tumor-associated myeloid cells. Clinically, polarization toward this immunosuppressive Th2 phenotype was also evident in patients with advanced luminal breast cancer. Thus, our findings showed that CCL5/CCR3 signaling promotes metastasis by inducing Th2 polarization of CD4⁺ T cells, with implications for prognosis and immunotherapy of luminal breast cancer.

Innate immune interleukin-1 receptor-associated kinase 4 exacerbates viral myocarditis by reducing CCR5(+) CD11b(+) monocyte migration and impairing interferon production.

BACKGROUND: Viral myocarditis follows a fatal course in ≈30% of patients. Interleukin-1 receptor-associated kinase 4 (IRAK4), a major nodal signal transducer in innate immunity, can play a pivotal role in host inflammatory response. We sought to determine how IRAK4 modulates inflammation and outcome in a mouse model of viral myocarditis. METHODS AND RESULTS: Myocarditis was induced after intraperitoneal inoculation of coxsackievirus B3 into C57Bl/6 IRAK4-deficient mice and their littermate controls. Mortality and viral proliferation were markedly reduced in IRAK4(-/-) mice compared with their IRAK4(+/+) littermates. Disease resistance of IRAK4(-/-) mice paralleled increased amounts of protective heart-infiltrating CCR5(+) monocytes/macrophages and enhanced interferon-α and interferon-γ production 2 days after infection. Competitive bone marrow chimera demonstrated that intact IRAK4 function inhibited heart-specific migration of bone marrow-derived CCR5(+) cells. Mechanistically, lack of IRAK4 resulted in interferon regulatory factor 5 homodimerization via reduced melanoma differentiation-associated protein 5 degradation and enhanced Stat1 and Stat5 phosphorylation. Consequently, antiviral interferon-α and interferon-γ production, as well as CCR5(+) cell recruitment, increased, whereas the overall proinflammatory response was drastically reduced in the absence of IRAK4. CONCLUSIONS: Innate immunity signal transducer IRAK4 exacerbates viral myocarditis through inhibition of interferon production and reduced mobilization of protective CCR5(+) monocytes/macrophages to the heart. The combination of IRAK4 inhibitors and antiviral adjuvants may become an attractive therapeutic approach against viral myocarditis in the future.

Microbiota-induced activation of epithelial IL-6 signaling links inflammasome-driven inflammation with transmissible cancer.

The microbiota is pivotal in the pathogenesis of inflammatory bowel disease (IBD)-associated inflammation-induced colorectal cancer (CRC), yet mechanisms for these effects remain poorly characterized. Here, we demonstrate that aberrant inflammasome-induced microbiota plays a critical role in CRC development, where mice deficient in the NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammasome feature enhanced inflammation-induced CRC formation. Intriguingly, WT mice cohoused either with inflammasome-deficient mice or with mice lacking IL-18 feature exacerbated inflammation-induced CRC compared with singly housed WT mice. Enhanced tumorigenesis is dependent on microbiota-induced chemokine (C-C motif) ligand 5 (CCL5)-driven inflammation, which in turn promotes epithelial cell proliferation through local activation of the IL-6 pathway, leading to cancer formation. Altogether, our results mechanistically link the altered microbiota with the pathogenesis of inflammation-induced CRC and suggest that in some conditions, microbiota components may transfer CRC susceptibility between individuals.

Impaired bone formation and increased osteoclastogenesis in mice lacking chemokine (C-C motif) ligand 5 (Ccl5).

Chemokines play crucial roles in the recruitment of specific hematopoietic cell types, and some of them have been suggested to be involved in the regulation of bone remodeling. Because we have previously observed that chemokine (C-C motif) ligand 2 (Ccl2) and Ccl5 are direct target genes of noncanonical Wnt signaling in osteoblasts, we analyzed the skeletal phenotypes of Ccl2-deficient and Ccl5-deficient mice. In line with previous studies, Ccl2-deficient mice display a moderate reduction of osteoclastogenesis at the age of 6 months. In contrast, 6-month-old Ccl5-deficient mice display osteopenia associated with decreased bone formation and increased osteoclastogenesis. Moreover, unlike in wild-type and Ccl2-deficient mice, large areas of their trabecular and endocortical bone surfaces are not covered by osteoblasts or bone-lining cells, and this is associated with a severe reduction of endosteal bone formation. Although this phenotype diminishes with age, it is important that we could further identify a reduced number of osteal macrophages in 6-month-old Ccl5-deficient mice, because this cell type has previously been reported to promote endosteal bone formation. Because Ccl5-deficient mice also display increased osteoclastogenesis, we finally addressed the question of whether osteal macrophages could differentiate into osteoclasts and/or secrete inhibitors of osteoclastogenesis. For that purpose we isolated these cells by CD11b affinity purification from calvarial cultures and characterized them ex vivo. Here we found that they are unable to differentiate into osteoblasts or osteoclasts, but that their conditioned medium mediates an antiosteoclastogenic effect, possibly caused by interleukin-18 (IL-18), an inhibitor of osteoclastogenesis expressed by osteal macrophages. Taken together, our data provide in vivo evidence supporting the previously suggested role of Ccl5 in bone remodeling. Moreover, to the best of our knowledge, Ccl5-deficient mice represent the first model with a spontaneous partial deficiency of osteal macrophages, a recently identified cell type, whose impact on bone remodeling is just beginning to be understood.

Absence of C-C motif chemokine ligand 5 in mice leads to decreased local macrophage recruitment and behavioral hypersensitivity in a murine neuropathic pain model.

Accumulated evidence suggests that the C-C motif chemokine ligand 5 (CCL5) modulates migration of inflammatory cells in several pathological conditions. This study tested the hypothesis that lack of CCL5 would modulate the recruitment of inflammatory cells to painful, inflamed sites and could attenuate pain in a murine chronic neuropathic pain model. Nociceptive sensitization, immune cell infiltration, multiple cytokine expression, and opioid peptide expression in damaged nerves were studied in wild-type (CCL5 +/+) and CCL5-deficient (CCL5 -/-) mice after partial sciatic nerve ligation (PSNL). Results indicated that CCL5 -/- mice had less behavioral hypersensitivity after PSNL. Macrophage infiltration and proinflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-1ß, IL-6, and interferon-γ) in damaged nerves following PSNL were significantly decreased in CCL5 -/- mice. Conversely, several antiinflammatory cytokine (IL-4 and IL-10) proteins were significantly increased in CCL5 -/- animals and the expression of enkephalin, ß-endorphin, and dynorphin mRNA was significantly lower than in wild-type control mice. These results represent the first evidence that CCL5 is capable of regulating the pathway that controls hyperalgesia at the level of the peripheral injured site in a murine chronic neuropathic pain model. We demonstrated that lack of CCL5 modulated cell infiltration and the proinflammatory milieu within the injured nerve. Attenuated behavioral hypersensitivity in CCL5 -/- mice observed in the current study could be a result of decreased macrophage infiltration, mobilization, and functional ability at injured sites. Collectively, the present study results suggest that CCL5 receptor antagonists may ultimately provide a novel class of analgesics for therapeutic intervention in chronic neuropathic pain.

CCL5-independent helper T lymphocyte responses to immuno-dominant pneumococcal surface protein A epitopes.

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Several antigens, in addition to the polysaccharide capsule, have been implicated in both the virulence and protective immunity against Streptococcus pneumoniae; one of the best-studied S. pneumoniae antigens is pneumococcal surface protein A (PspA). Recently, it was shown that genetic polymorphisms could diminish CCL5 expression, which results in increased susceptibility to and progression of infectious diseases. We previously showed CCL5 blockade reduced PspA-specific humoral and cellular pneumococcal immunity, during S. pneumoniae strain EF3030-induced carriage, by diminishing IFN-γ and enhancing IL-10 secretion by effector T cells. We also identified immuno-dominant helper T lymphocyte (HTL) epitopes in PspA peptide 19-23 (PspA(199-246)), which caused comparatively more cytokine secretion and proliferation responses by splenic and cervical lymph node (CLN) CD4(+) T cells from mice previously challenged with S. pneumoniae strain EF3030. In this study, we sought to determine if PspA(199-246)-specific CD4(+) T cells responses were resistant to the effect of CCL5 deficiency. In short, T cell responses against these HTL epitopes were resistant to CCL5 inhibition, than compared to cells from control or naïve mice, and unaffected by reduced co-stimulatory molecule expression caused by CCL5 blockade. CCL5 deficiency also corresponded with a higher number of IL-10(+) CD11b(+) CD11c(Lo) and CD11b(+) CD11c(Hi) cells and lower IFN-γ expression by similar cells, than compared to controls. These data confirm CCL5 is an essential factor for optimal pneumococcal adaptive immunity and show CD4(+) T cell responses to PspA(199-246) are largely resistant to CCL5 deficiency.

RANTES deficiency attenuates autoantibody-induced glomerulonephritis.

Experimental autoimmune nephritis in mice and spontaneous lupus nephritis are both associated with elevated expression of several chemokines in the kidneys. Nevertheless, the role that different chemokines play in mediating renal inflammation is far from complete. This study focuses on elucidating the functional role of RANTES, a chemokine that has been noted to be hyper-expressed within the kidneys, both in experimental renal disease as well as in spontaneous lupus nephritis. To elucidate if RANTES was essential for immune-mediated glomerulonephritis, DBA/1 mice that are highly sensitive to nephrotoxic serum nephritis were rendered RANTES-deficient and then tested for disease susceptibility. Nephritis-sensitive DBA/1 mice expressed more RANTES within the diseased kidneys. Compared to wild-type DBA/1 mice, RANTES-deficient DBA/1 mice developed significantly less proteinuria, azotemia, and renal inflammation, with reduced crescent formation and tubulo-interstitial nephritis. These findings indicate that RANTES ablation attenuates immune-mediated nephritis and suggest that this chemokine could be a potential therapeutic target in these diseases.

Mechanisms underlying the cerebral microvascular responses to angiotensin II-induced hypertension.

Angiotensin II (AngII) and AngII type-1 receptors (AT1r) have been implicated in the pathogenesis of hypertension and ischemic stroke. The objectives of this study was to determine if/how chronic AngII administration affects blood-brain barrier (BBB) function and blood cell adhesion in the cerebral microvasculature. AngII-loaded osmotic pumps were implanted in wild type (WT) and mutant mice. Leukocyte and platelet adhesion were monitored in cerebral venules by intravital microscopy and BBB permeability detected by Evans blue leakage. AngII (two week) infusion increased blood pressure in WT mice. This was accompanied by an increased BBB permeability and a high density of adherent leukocytes and platelets. AT1r (on the vessel wall, but not on blood cells) was largely responsible for the microvascular responses to AngII. Immunodeficient (Rag-1(-/-) ) mice exhibited blunted blood cell recruitment responses without a change in BBB permeability. A similar protection pattern was noted in RANTES(-/-) and P-selectin(-/-) mice, with bone marrow chimeras (blood cell deficiency only) yielding responses comparable to the respective knockouts. These findings implicate AT1r in the microvascular dysfunction associated with AngII-induced hypertension and suggest that immune cells and blood cell-associated RANTES and P-selectin contribute to the blood cell recruitment, but not the BBB failure, elicited by AngII.

Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages.

PURPOSE: To evaluate the roles of CCL3 and its specific chemokine receptors, CCR1 and CCR5, in alkali-induced corneal neovascularization (CNV). METHODS: Chemical denudation of corneal and limbal epithelium was performed on wild-type (WT) BALB/c mice and CCL3-, CCR1-, and CCR5-deficienct (knockout [KO]) counterparts. Two weeks after injury CNV was quantified by immunostaining with anti-CD31. Angiogenic factor expression and leukocyte accumulation in the early phase after injury were quantified by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical analysis, respectively. RESULTS: Alkali injury augmented the intraocular mRNA expression of CCL3 and its receptors, CCR1 and CCR5, together with a transient infiltration of F4/80 positive macrophages and Gr-1 positive neutrophils. Compared with WT mice, CCL3-KO and CCR5-KO mice but not CCR1-KO mice exhibited reduced CNV two weeks after injury both macroscopically and microscopically as evidenced by CD31 positive areas. Concomitantly, the infiltration of F4/80 positive macrophages but not Gr-1 positive neutrophils was significantly attenuated in CCL3-KO mice compared with WT mice. Intracorneal infiltration of CCR5 expressing cells was significantly impaired in CCL3-KO mice compared with WT mice. Alkali injury induced a massive increase in the intraocular mRNA expression of a potent angiogenic factor, vascular endothelial growth factor (VEGF), in WT mice whereas these increments were severely retarded in CCL3-KO mice. Moreover, CCL3 enhanced VEGF expression by murine peritoneal macrophages at both the mRNA and the protein level. Furthermore, topical CCL3 application restored CNV, which was macroscopically and microscopically reduced in CCL3-KO mice after two weeks to levels similar to those found in WT mice. CONCLUSIONS: In alkali-induced CNV, CCL3 induced macrophages to infiltrate and produce VEGF by binding to CCR5 but not to CCR1 and eventually promoted angiogenesis.

Donor T-cell production of RANTES significantly contributes to the development of idiopathic pneumonia syndrome after allogeneic stem cell transplantation.

Idiopathic pneumonia syndrome (IPS) is a major cause of mortality following allogeneic stem cell transplantation (allo-SCT). Clinical and experimental data support a role for conditioning-induced inflammation and alloreactive T-cell responses in IPS pathophysiology, but the mechanisms by which donor leukocytes are ultimately recruited to the lung are not fully understood. RANTES is a chemokine ligand that is up-regulated during inflammation and promotes the recruitment of T cells and macrophages to sites of tissue damage. Using a lethally irradiated murine SCT model (B6 --> B6D2F1), we evaluated the role of donor leukocyte-derived RANTES in the development of IPS. Pulmonary mRNA and protein levels of RANTES were significantly elevated in allo-SCT recipients compared to syngeneic controls and were associated with enhanced mRNA expression of CCR5 and CCR1 and with inflammatory cell infiltration into the lung. Allo-SCT with RANTES-/- donor cells significantly decreased IPS and improved survival. Combinations of allogeneic wild-type or RANTES-/- bone marrow with wild-type or RANTES-/- T cells demonstrated that the expression of RANTES by donor T cells was critical to the development of lung injury after SCT. These data reveal that donor T cells can help regulate leukocyte recruitment to the lung after allo-SCT and provide a possible mechanism through which inflammation engendered by SCT conditioning regimens is linked to allo-specific T-cell responses during the development of IPS.

CCR1 and CC chemokine ligand 5 interactions exacerbate innate immune responses during sepsis.

CCR1 has previously been shown to play important roles in leukocyte trafficking, pathogen clearance, and the type 1/type 2 cytokine balance, although very little is known about its role in the host response during sepsis. In a cecal ligation and puncture model of septic peritonitis, CCR1-deficient (CCR1(-/-)) mice were significantly protected from the lethal effects of sepsis when compared with wild-type (WT) controls. The peritoneal and systemic cytokine profile in CCR1(-/-) mice was characterized by a robust, but short-lived and regulated antibacterial response. CCR1 expression was not required for leukocyte recruitment, suggesting critical differences extant in the activation of WT and CCR1(-/-) resident or recruited peritoneal cells during sepsis. Peritoneal macrophages isolated from naive CCR1(-/-) mice clearly demonstrated enhanced cytokine/chemokine generation and antibacterial responses compared with similarly treated WT macrophages. CCR1 and CCL5 interactions markedly altered the inflammatory response in vivo and in vitro. Administration of CCL5 increased sepsis-induced lethality in WT mice, whereas neutralization of CCL5 improved survival. CCL5 acted in a CCR1-dependent manner to augment production of IFN-gamma and MIP-2 to damaging levels. These data illustrate that the interaction between CCR1 and CCL5 modulates the innate immune response during sepsis, and both represent potential targets for therapeutic intervention.

Impaired T cell function in RANTES-deficient mice.

The chemokine RANTES is a chemoattractant for monocytes and T cells and is postulated to participate in many aspects of the immune response. To evaluate the biological roles of RANTES in vivo, we generated RANTES-deficient (-/-) mice and characterized their T cell function. In cutaneous delayed-type hypersensitivity assays, a 50% reduction in ear and footpad swelling was seen in -/- mice compared to +/+ mice. In vitro, polyclonal and antigen-specific T cell proliferation was decreased. Quantitative analysis using the fluorescent dye carboxy-fluorescein succinimidyl ester revealed that this proliferative defect was due both to fewer antigen-reactive T cells and to a reduction in the capacity of these cells to proliferate. In addition, IFN-gamma and IL-2 production by the -/- T cells was dramatically decreased. Together, these data suggest that RANTES is required for normal T cell functions as well as for recruiting monocytes and T cells to sites of inflammation.