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.

CCL3/Macrophage Inflammatory Protein-1α Is Dually Involved in Parasite Persistence and Induction of a TNF- and IFNγ-Enriched Inflammatory Milieu in Trypanosoma cruzi-Induced Chronic Cardiomyopathy.

CCL3, a member of the CC-chemokine family, has been associated with macrophage recruitment to heart tissue and parasite control in the acute infection of mouse with Trypanosoma cruzi, the causative agent of Chagas disease. Here, we approached the participation of CCL3 in chronic chagasic cardiomyopathy (CCC), the main clinical form of Chagas disease. We induced CCC in C57BL/6 (ccl3+/+) and CCL3-deficient (ccl3-/-) mice by infection with the Colombian Type I strain. In ccl3+/+ mice, high levels of CCL3 mRNA and protein were detected in the heart tissue during the acute and chronic infection. Survival was not affected by CCL3 deficiency. In comparison with ccl3+/+, chronically infected ccl3-/- mice presented reduced cardiac parasitism and inflammation due to CD8+ cells and macrophages. Leukocytosis was decreased in infected ccl3-/- mice, paralleling the accumulation of CD8+ T cells devoid of activated CCR5+ LFA-1+ cells in the spleen. Further, T. cruzi-infected ccl3-/-mice presented reduced frequency of interferon-gamma (IFNγ)+ cells and numbers of parasite-specific IFNγ-producing cells, while the T. cruzi antigen-specific cytotoxic activity was increased. Stimulation of CCL3-deficient macrophages with IFNγ improved parasite control, in a milieu with reduced nitric oxide (NOx) and tumor necrosis factor (TNF), but similar interleukin-10 (IL-10), concentrations. In comparison with chronically T. cruzi-infected ccl3+/+ counterparts, ccl3-/- mice did not show enlarged heart, loss of left ventricular ejection fraction, QTc prolongation and elevated CK-MB activity. Compared with ccl3+/+, infected ccl3-/- mice showed reduced concentrations of TNF, while IL-10 levels were not affected, in the heart milieu. In spleen of ccl3+/+ NI controls, most of the CD8+ T-cells expressing the CCL3 receptors CCR1 or CCR5 were IL-10+, while in infected mice these cells were mainly TNF+. Lastly, selective blockage of CCR1/CCR5 (Met-RANTES therapy) in chronically infected ccl3+/+ mice reversed pivotal electrical abnormalities (bradycardia, prolonged PR, and QTc interval), in correlation with reduced TNF and, mainly, CCL3 levels in the heart tissue. Therefore, in the chronic T. cruzi infection CCL3 takes part in parasite persistence and contributes to form a CD8+ T-cell and macrophage-enriched cardiac inflammation. Further, increased levels of CCL3 create a scenario with abundant IFNγ and TNF, associated with cardiomyocyte injury, heart dysfunction and QTc prolongation, biomarkers of severity of Chagas' heart disease.

Differential regulation of breast cancer bone metastasis by PARP1 and PARP2.

PARP1 and PARP2 dual inhibitors, such as olaparib, have been recently FDA approved for the treatment of advanced breast and ovarian cancers. However, their effects on bone mass and bone metastasis are unknown. Here we show that olaparib increases breast cancer bone metastasis through PARP2, but not PARP1, specifically in the myeloid lineage, but not in the cancer cells. Olaparib treatment or PARP1/2 deletion promotes osteoclast differentiation and bone loss. Intriguingly, myeloid deletion of PARP2, but not PARP1, increases the population of immature myeloid cells in bone marrow, and impairs the expression of chemokines such as CCL3 through enhancing the transcriptional repression by ß-catenin. Compromised CCL3 production in turn creates an immune-suppressive milieu by altering T cell subpopulations. Our findings warrant careful examination of current PARP inhibitors on bone metastasis and bone loss, and suggest cotreatment with CCL3, ß-catenin inhibitors, anti-RANKL or bisphosphonates as potential combination therapy for PARP inhibitors.

The Chemokine CCL3 Regulates Myeloid Differentiation and Hematopoietic Stem Cell Numbers.

The chemokine CCL3 is frequently overexpressed in malignancies and overexpression leads to microenvironmental dysfunction. In murine models of chronic myelogenous leukemia (CML), CCL3 is critical for the maintenance of a leukemia stem cell population, and leukemia progression. With CCL3 implicated as a potentially viable therapeutic target, it is important to carefully characterize its role in normal hematopoietic homeostasis. CCL3-/- mice were used to evaluate the role of CCL3 in regulating hematopoietic stem and progenitor cell (HSPC) populations. CCL3-/- mice had loss of mature myeloid populations, while myeloid progenitors and HSPCs were increased, and microenvironmental populations were unchanged. These data show that CCL3 promotes myeloid lineage differentiation and the size of the HSPC pool independent of the supportive bone marrow microenvironment. Our results demonstrate a previously unrecognized role of CCL3 in the maintenance of homeostatic hematopoiesis that should be evaluated when targeting CCL3 signaling for the treatment of hematologic malignancy.

Otitis Media and Nasopharyngeal Colonization in ccl3-/- Mice.

We previously found CC chemokine ligand 3 (CCL3) to be a potent effector of inflammation during otitis media (OM): exogenous CCL3 rescues the OM phenotype of tumor necrosis factor-deficient mice and the function of macrophages deficient in several innate immune molecules. To further delineate the role of CCL3 in OM, we evaluated middle ear (ME) responses of ccl3-/-mice to nontypeable Haemophilus influenzae (NTHi). CCL chemokine gene expression was evaluated in wild-type (WT) mice during the complete course of acute OM. OM was induced in ccl3-/- and WT mice, and infection and inflammation were monitored for 21 days. Phagocytosis and killing of NTHi by macrophages were evaluated by an in vitro assay. The nasopharyngeal bacterial load was assessed in naive animals of both strains. Many CCL genes showed increased expression levels during acute OM, with CCL3 being the most upregulated, at levels 600-fold higher than the baseline. ccl3-/- deletion compromised ME bacterial clearance and prolonged mucosal hyperplasia. ME recruitment of leukocytes was delayed but persisted far longer than in WT mice. These events were linked to a decrease in the macrophage capacity for NTHi phagocytosis and increased nasopharyngeal bacterial loads in ccl3-/- mice. The generalized impairment in inflammatory cell recruitment was associated with compensatory changes in the expression profiles of CCL2, CCL7, and CCL12. CCL3 plays a significant role in the clearance of infection and resolution of inflammation and contributes to mucosal host defense of the nasopharyngeal niche, a reservoir for ME and upper respiratory infections. Therapies based on CCL3 could prove useful in treating or preventing persistent disease.

Absence of CCL2 and CCL3 Ameliorates Central Nervous System Grey Matter But Not White Matter Demyelination in the Presence of an Intact Blood-Brain Barrier.

A broad spectrum of diseases is characterized by myelin abnormalities, oligodendrocyte pathology, and concomitant glia activation, among multiple sclerosis (MS). Our knowledge regarding the factors triggering gliosis and demyelination is scanty. Chemokines are pivotal for microglia and astrocyte activation and orchestrate critical steps during the formation of central nervous system (CNS) demyelinating lesions. Redundant functions of chemokines complicate, however, the study of their functional relevance. We used the cuprizone model to study redundant functions of two chemokines, CCL2/MCP1 and CCL3/MIP1α, which are critically involved in the pathological process of cuprizone-induced demyelination. First, we generated a mutant mouse strain lacking functional genes of both chemokines and demonstrated that double-mutant animals are viable, fertile, and do not present with gross abnormalities. Astrocytes and peritoneal macrophages, cultured form tissues of these animals did neither express CCL2 nor CCL3. Exposure to cuprizone resulted in increased CCL2 and CCL3 brain levels in wild-type but not mutant animals. Cuprizone-induced demyelination, oligodendrocyte loss, and astrogliosis were significantly ameliorated in the cortex but not corpus callosum of chemokine-deficient animals. In summary, we provide a novel powerful model to study the redundant function of two important chemokines. Our study reveals that chemokine function in the CNS redounds to region-specific pathophysiological events.

CCL3 production by microglial cells modulates disease severity in murine models of retinal degeneration.

Many degenerative retinal diseases illustrate retinal inflammatory changes that include infiltration of microglia and macrophages into the subretinal space. In this study, we examined the role of chemokines in the Abca4(-/-)Rdh8(-/-) mouse model of Stargardt disease and the Mertk(-/-) mouse model of retinitis pigmentosa. PCR array analysis of 84 chemokines and related molecules revealed 84.6-fold elevated expression of Ccl3 (MIP-1a) 24 h after light exposure in Abca4(-/-)Rdh8(-/-) mice. Only MIP-1 chemokines, including Ccl3 and Ccl4, displayed peak expression 24 h after light exposure, and peaked earlier than the other chemokines. Secretion of Ccl3 was documented only in microglia, whereas both microglia and retinal pigment epithelium cells produced Ccl2. Exposure of Cx3Cr1(gfp/Δ)Abca4(-/-)Rdh8(-/-) mice to intense light resulted in the appearance of Cx3Cr1GFP(+) monocytes in the subretinal space. To address the in vivo role of CCL3 in retinal degeneration, Ccl3(-/-)Abca4(-/-)Rdh8(-/-) mice and Ccl3(-/-)Mertk(-/-) mice were generated. Following intense light exposure, Ccl3(-/-)Abca4(-/-)Rdh8(-/-) mice displayed persistent retinal inflammation with appearance of Iba-1(+) cells in the subretinal space, severe photoreceptor cell death, and increased Ccl4 expression compared with Abca4(-/-)Rdh8(-/-) mice. In contrast, Ccl3(-/-)Abca4(-/-)Rdh8(-/-) mice exhibited a milder retinal inflammation and degeneration than Abca4(-/-)Rdh8(-/-) mice did in age-related chronic retinal degeneration under room light conditions. The deficiency of Ccl3 also attenuated the severity of retinal degeneration in Mertk(-/-) mice. Taken together, our results indicate that Ccl3 has an essential role in regulating the severity of retinal inflammation and degeneration in these mouse models.

CCR1 inhibition ameliorates the progression of lupus nephritis in NZB/W mice.

Systemic lupus erythematosus is a chronic inflammatory autoimmune disease, the development of which is characterized by a progressive loss of renal function. Such dysfunction is associated with leukocyte infiltration in the glomerular and tubulointerstitial compartments in both human and experimental lupus nephritis. In this study, we investigated the role of the Ccr1 chemokine receptor in this infiltration process during the progression of nephritis in the lupus-prone New Zealand Black/New Zealand White (NZB/W) mouse model. We found that peripheral T cells, mononuclear phagocytes, and neutrophils, but not B cells, from nephritic NZB/W mice were more responsive to Ccr1 ligands than the leukocytes from younger prenephritic NZB/W mice. Short-term treatment of nephritic NZB/W mice with the orally available Ccr1 antagonist BL5923 decreased renal infiltration by T cells and macrophages. Longer Ccr1 blockade decreased kidney accumulation of effector/memory CD4(+) T cells, Ly6C(+) monocytes, and both M1 and M2 macrophages; reduced tubulointerstitial and glomerular injuries; delayed fatal proteinuria; and prolonged animal lifespan. In contrast, renal humoral immunity was unaffected in BL5923-treated mice, which reflected the unchanged numbers of infiltrated B cells in the kidneys. Altogether, these findings define a pivotal role for Ccr1 in the recruitment of T and mononuclear phagocyte cells to inflamed kidneys of NZB/W mice, which in turn contribute to the progression of renal injury.

Influence of the CCR-5/MIP-1 α axis in the pathogenesis of Rocio virus encephalitis in a mouse model.

Rocio virus (ROCV) caused an outbreak of human encephalitis during the 1970s in Brazil and its immunopathogenesis remains poorly understood. CC-chemokine receptor 5 (CCR5) is a chemokine receptor that binds to macrophage inflammatory protein (MIP-1 α). Both molecules are associated with inflammatory cells migration during infections. In this study, we demonstrated the importance of the CCR5 and MIP-1 α, in the outcome of viral encephalitis of ROCV-infected mice. CCR5 and MIP-1 α knockout mice survived longer than wild-type (WT) ROCV-infected animals. In addition, knockout mice had reduced inflammation in the brain. Assessment of brain viral load showed mice virus detection five days post-infection in wild-type and CCR5-/- mice, while MIP-1 α-/- mice had lower viral loads seven days post-infection. Knockout mice required a higher lethal dose than wild-type mice as well. The CCR5/MIP-1 α axis may contribute to migration of infected cells to the brain and consequently affect the pathogenesis during ROCV infection.

MIP-1α/CCL3-mediated maintenance of leukemia-initiating cells in the initiation process of chronic myeloid leukemia.

In the initiation process of chronic myeloid leukemia (CML), a small number of transformed leukemia-initiating cells (LICs) coexist with a large number of normal hematopoietic cells, gradually increasing thereafter and eventually predominating in the hematopoietic space. However, the interaction between LICs and normal hematopoietic cells at the early phase has not been clearly delineated because of the lack of a suitable experimental model. In this study, we succeeded in causing a marked leukocytosis resembling CML from restricted foci of LICs in the normal hematopoietic system by direct transplantation of BCR-ABL gene-transduced LICs into the bone marrow (BM) cavity of nonirradiated mice. Herein, we observed that BCR-ABL(+)lineage(-)c-kit(-) immature leukemia cells produced high levels of an inflammatory chemokine, MIP-1α/CCL3, which promoted the development of CML. Conversely, ablation of the CCL3 gene in LICs dramatically inhibited the development of CML and concomitantly reduced recurrence after the cessation of a short-term tyrosine kinase inhibitor treatment. Finally, normal hematopoietic stem/progenitor cells can directly impede the maintenance of LICs in BM in the absence of CCL3 signal.

Leukocyte-specific CCL3 deficiency inhibits atherosclerotic lesion development by affecting neutrophil accumulation.

OBJECTIVE: Despite common disbelief that neutrophils are involved in atherosclerosis, evidence is accumulating for a causal role of neutrophils in atherosclerosis. CC chemokine ligand (CCL)3 is an inflammatory chemokine and its expression is significantly increased during atherosclerotic lesion formation in mice. It has recently been shown that under conditions of inflammation neutrophils can migrate along a CCL3 gradient. In this study, we aimed to elucidate the role of leukocyte-derived CCL3 in atherogenesis. METHODS AND RESULTS: Irradiated low density lipoprotein receptor(-/-) mice, reconstituted with CCL3(-/-) or littermate bone marrow showed markedly reduced CCL3 response to lipopolysaccharide treatment, establishing the critical relevance of leukocytes as source of CCL3. Hematopoietic deficiency of CCL3 significantly reduced aortic sinus lesion formation by 31% after 12 weeks of western-type diet. Interestingly, whereas plaque macrophage, collagen, and vascular smooth muscle cell content were unchanged, neutrophil adhesion to and presence in plaques was significantly attenuated in CCL3(-/-) chimeras. These mice had reduced circulating neutrophil numbers, which could be ascribed to an increased neutrophil turnover and CCL3(-/-) neutrophils were shown to be less responsive toward the neutrophil chemoattractant CXC chemokine ligand 1. CONCLUSIONS: Our data indicate that under conditions of acute inflammation leukocyte-derived CCL3 can induce neutrophil chemotaxis toward the atherosclerotic plaque, thereby accelerating lesion formation.

Neuronal and epithelial cell rescue resolves chronic systemic inflammation in the lipid storage disorder Niemann-Pick C.

Chronic systemic inflammation is thought to be a major contributor to metabolic and neurodegenerative diseases. Since inflammatory components are shared among different disorders, targeting inflammation is an attractive option for mitigating disease. To test the significance of inflammation in the lipid storage disorder (LSD) Niemann-Pick C (NPC), we deleted the macrophage inflammatory gene Mip1a/Ccl3 from NPC diseased mice. Deletion of Ccl3 had been reported to delay neuronal loss in Sandhoff LSD mice by inhibiting macrophage infiltration. For NPC mice, in contrast, deleting Ccl3 did not retard neurodegeneration and worsened the clinical outcome. Depletion of visceral tissue macrophages also did not alter central nervous system (CNS) pathology and instead increased liver injury, suggesting a limited macrophage infiltration response into the CNS and a beneficial role of macrophage activity in visceral tissue. Prevention of neuron loss or liver injury, even at late stages in the disease, was achieved through specific rescue of NPC disease in neurons or in liver epithelial cells, respectively. Local epithelial cell correction was also sufficient to reduce the macrophage-associated pathology in lung tissue. These results demonstrate that elevated inflammation and macrophage activity does not necessarily contribute to neurodegeneration and tissue injury, and LSD defects in immune cells may not preclude an appropriate inflammatory response. We conclude that inflammation remains secondary to neuronal and epithelial cell dysfunction and does not irreversibly contribute to the pathogenic cascade in NPC disease. Without further exploration of possible beneficial roles of inflammatory mediators, targeting inflammation may not be therapeutically effective at ameliorating disease severity.

Impact of macrophage inflammatory protein-1α deficiency on atherosclerotic lesion formation, hepatic steatosis, and adipose tissue expansion.

Macrophage inflammatory protein-1α (CCL3) plays a well-known role in infectious and viral diseases; however, its contribution to atherosclerotic lesion formation and lipid metabolism has not been determined. Low density lipoprotein receptor deficient (LDLR(-/-)) mice were transplanted with bone marrow from CCL3(-/-) or C57BL/6 wild type donors. After 6 and 12 weeks on western diet (WD), recipients of CCL3(-/-) marrow demonstrated lower plasma cholesterol and triglyceride concentrations compared to recipients of C57BL/6 marrow. Atherosclerotic lesion area was significantly lower in female CCL3(-/-) recipients after 6 weeks and in male CCL3(-/-) recipients after 12 weeks of WD feeding (P<0.05). Surprisingly, male CCL3(-/-) recipients had a 50% decrease in adipose tissue mass after WD-feeding, and plasma insulin, and leptin levels were also significantly lower. These results were specific to CCL3, as LDLR(-/-) recipients of monocyte chemoattractant protein(-/-) (CCL2) marrow were not protected from the metabolic consequences of high fat feeding. Despite these improvements in LDLR(-/-) recipients of CCL3(-/-) marrow in the bone marrow transplantation (BMT) model, double knockout mice, globally deficient in both proteins, did not have decreased body weight, plasma lipids, or atherosclerosis compared with LDLR(-/-) controls. Finally, there were no differences in myeloid progenitors or leukocyte populations, indicating that changes in body weight and plasma lipids in CCL3(-/-) recipients was not due to differences in hematopoiesis. Taken together, these data implicate a role for CCL3 in lipid metabolism in hyperlipidemic mice following hematopoietic reconstitution.

Host-derived MCP-1 and MIP-1α regulate protective anti-tumor immunity to localized and metastatic B16 melanoma.

Leukocytic infiltration into malignant melanoma lesions is tightly regulated by chemokines. To assess the role of the CC chemokines monocyte chemotactic protein-1 (MCP-1/chemokine ligand 2) and macrophage inflammatory protein-1α (MIP-1α/chemokine ligand 3) in this process, s.c. primary and metastatic B16 F10 melanoma tumor growth levels were examined in mice lacking MCP-1 or MIP-1α. Primary s.c. B16 F10 melanoma growth was augmented by loss of MCP-1 or MIP-1α. Similarly, lung metastasis was enhanced by the deficiency of MCP-1 or MIP-1α. Enhanced tumor outgrowth was associated with decreased percentages of infiltrating CD4(+) T cells, CD8(+) T cells, and natural killer cells. In the absence of MCP-1 or MIP-1α, melanoma outgrowth was correlated with reduced local expression of interferon-γ, IL-6, tumor necrosis factor-α, and transforming growth factor-ß. Among these cytokines, reduced expression levels of interferon-γ and tumor necrosis factor-α on leukocytes from the spleen were associated with the development of lung metastasis in chemokine-deficient mice. The local s.c. administration of these four cytokines significantly augmented another chemokine's expression and suppressed primary melanoma growth in mice deficient for MCP-1 or MIP-1α. The s.c. injection of MCP-1 or MIP-1α significantly inhibited the primary tumor growth in wild-type mice. These results indicate that host-derived MCP-1 and MIP-1α regulate protective anti-tumor immunity to B16 F10 melanoma by promoting lymphocyte infiltration into the tumor and subsequent cytokine production.

CD1d-restricted IFN-γ-secreting NKT cells promote immune complex-induced acute lung injury by regulating macrophage-inflammatory protein-1α production and activation of macrophages and dendritic cells.

Immune complex-induced acute lung injury (IC-ALI) has been implicated in various pulmonary disease states. However, the role of NKT cells in IC-ALI remains unknown. Therefore, we explored NKT cell functions in IC-ALI using chicken egg albumin and anti-chicken egg albumin IgG. The bronchoalveolar lavage fluid of CD1d(-/-) and Jα18(-/-) mice contained few Ly6G(+)CD11b(+) granulocytes, whereas levels in B6 mice were greater and were increased further by α-galactosyl ceramide. IFN-γ and MIP-1α production in the lungs was greater in B6 than CD1d(-/-) mice. Adoptive transfer of wild type (WT) but not IFN-γ-, MIP-1α-, or FcγR-deficient NKT cells into CD1d(-/-) mice caused recruitment of inflammatory cells to the lungs. Moreover, adoptive transfer of IFN-γR-deficient NKT cells enhanced MIP-1α production and cell recruitment in the lungs of CD1d(-/-) or CD1d(-/-)IFN-γ(-/-) mice, but to a lesser extent than WT NKT cells. This suggests that IFN-γ-producing NKT cells enhance MIP-1α production in both an autocrine and a paracrine manner. IFN-γ-deficient NKT cells induced less IL-1ß and TNF-α production by alveolar macrophages and dendritic cells in CD1d(-/-) mice than did WT NKT cells. Taken together, these data suggest that CD1d-restricted IFN-γ-producing NKT cells promote IC-ALI by producing MIP-1α and enhancing proinflammatory cytokine production by alveolar macrophages and dendritic cells.

Fetally derived CCL3 is not essential for the migration of maternal cells across the blood-placental barrier in the mouse.

In mammals with a hemochorial placenta (e.g., primates and rodents), the maternal and fetal bloodstreams are separated by the blood-placenta barrier. However, a few maternal cells in the general circulation pass through the barrier during normal pregnancy. So far, the transfer mechanism has not been investigated. In this study, we established a chemokine (C-C motif) ligand 3 (CCL3)-deficient mouse model to examine the effect of fetus-derived chemokine(s) on the migration of maternal cells through the blood-placenta barrier. Using this model, we obtained CCL3-positive and -negative littermates from a mother expressing both CCL3 and green fluorescent protein (GFP). The numbers of GFP positive maternal cells in the lung, liver, spleen and heart of CCL3-positive and -negative fetuses were compared. A few GFP-positive cells were detected in the lung and liver of both types of fetus. These results indicate that maternal cells can migrate through the blood-placenta barrier even in the absence of fetal CCL3.

Intrahepatic IL-10 maintains NKG2A+Ly49- liver NK cells in a functionally hyporesponsive state.

The tolerogenic nature of the liver allows daily exposure to gut-derived foreign Ags without causing inflammation, but it may facilitate persistent infection in the liver. NK cells play a central role in innate immunity, as well as in shaping the adaptive immune response. We hypothesized that the naive mouse liver maintains intrahepatic NK cells in a functionally hyporesponsive state. Compared with splenic NK cells, liver NK cells displayed a dampened IFN-gamma response to IL-12/IL-18 stimulation. Importantly, the liver contains a significant population of functionally hyporesponsive NK cells that express high levels of the inhibitory receptor NKG2A and lack expression of MHC class I-binding Ly49 receptors. Adoptively transferred splenic NK cells that migrate to the liver displayed phenotypic and functional changes, suggesting that the liver environment modifies NK cell receptor expression and functional responsiveness. Notably, IL-10 is present at high levels within the liver, and in vivo blockade of IL-10R resulted in a decreased percentage of intrahepatic NKG2A(+)Ly49(-) NK cells. These data suggest that the liver environment regulates NK cell receptor expression and that IL-10 contributes to the regulation of liver NK cells, in part, by maintaining a greater percentage of the hyporesponsive NKG2A(+)Ly49(-) NK cells in the liver.

Matrix metalloproteinase-8 inactivates macrophage inflammatory protein-1 alpha to reduce acute lung inflammation and injury in mice.

To determine the role of matrix metalloproteinase-8 (MMP-8) in acute lung injury (ALI), we delivered LPS or bleomycin by the intratracheal route to MMP-8(-/-) mice versus wild-type (WT) mice or subjected the mice to hyperoxia (95% O(2)) and measured lung inflammation and injury at intervals. MMP-8(-/-) mice with ALI had greater increases in lung polymorphonuclear neutrophils (PMNs) and macrophage counts, measures of alveolar capillary barrier injury, lung elastance, and mortality than WT mice with ALI. Bronchoalveolar lavage fluid (BALF) from LPS-treated MMP-8(-/-) mice had more MIP-1alpha than BALF from LPS-treated WT mice, but similar levels of other pro- and anti-inflammatory mediators. MIP-1alpha(-/-) mice with ALI had less acute lung inflammation and injury than WT mice with ALI, confirming that MIP-1alpha promotes acute lung inflammation and injury in mice. Genetically deleting MIP-1alpha in MMP-8(-/-) mice reduced the increased lung inflammation and injury and mortality in MMP-8(-/-) mice with ALI. Soluble MMP-8 cleaved and inactivated MIP-1alpha in vitro, but membrane-bound MMP-8 on activated PMNs had greater MIP-1alpha-degrading activity than soluble MMP-8. High levels of membrane-bound MMP-8 were detected on lung PMNs from LPS-treated WT mice, but soluble, active MMP-8 was not detected in BALF samples. Thus, MMP-8 has novel roles in restraining lung inflammation and in limiting alveolar capillary barrier injury during ALI in mice by inactivating MIP-1alpha. In addition, membrane-bound MMP-8 on activated lung PMNs is likely to be the key bioactive form of the enzyme that limits lung inflammation and alveolar capillary barrier injury during ALI.

The CCL3/macrophage inflammatory protein-1alpha-binding protein evasin-1 protects from graft-versus-host disease but does not modify graft-versus-leukemia in mice.

CCL3 is a protein of the CC chemokine family known to be important for T cell recruitment in inflammatory diseases. The aim of the current study was to evaluate the effects and putative mechanism of action of evasin-1, a novel CCL3-binding protein, in the pathogenesis of acute graft-versus-host disease (GVHD). GVHD was induced by the transplantation of splenocytes from C57BL/6J to B6D2F1 mice. Treatment of recipient mice with evasin-1 prevented mortality associated with GVHD. This was correlated with reduced weight loss and clinical disease severity. Analysis of the small intestine showed that evasin-1 treatment reduced the histopathological score and decreased levels of IFN-gamma and CCL5. Mechanistically, evasin-1 treatment reduced the number of CD4(+) and CD8(+) T cells infiltrating the small intestine, as assessed by immunohistochemistry, and the adhesion of leukocytes to intestinal venules of recipient mice, as assessed by intravital microscopy. Evasin-1 was also able to decrease liver damage, as seen by reduction of inflammatory infiltrate and IFN-gamma levels. Treatment with evasin-1 did not interfere with graft-versus-leukemia. Altogether, our studies demonstrate that CCL3 plays a major role in mediating GVHD, but not graft-versus-leukemia in mice and suggest that blockade of CCL3 with evasin-1 has potential therapeutic application in patients undergoing bone marrow transplantation.