Th1 effector CD4+ cells contribute to the pathogenesis of proliferative and crescentic glomerulonephritis, but whether effector Th17 cells also contribute is unknown. We compared the involvement of Th1 and Th17 cells in a mouse model of antigen-specific glomerulonephritis in which effector CD4+ cells are the only components of adaptive immunity that induce injury. We planted the antigen ovalbumin on the glomerular basement membrane of Rag1(-/-) mice using an ovalbumin-conjugated non-nephritogenic IgG1 monoclonal antibody against alpha3(IV) collagen. Subsequent injection of either Th1- or Th17-polarized ovalbumin-specific CD4+ effector cells induced proliferative glomerulonephritis. Mice injected with Th1 cells developed progressive albuminuria over 21 d, histologic injury including 5.5 +/- 0.9% crescent formation/segmental necrosis, elevated urinary nitrate, and increased renal NOS2, CCL2, and CCL5 mRNA. Mice injected with Th17 cells developed albuminuria by 3 d; compared with Th1-injected mice, their glomeruli contained more neutrophils and greater expression of renal CXCL1 mRNA. In conclusion, Th1 and Th17 effector cells can induce glomerular injury. Understanding how these two subsets mediate proliferative forms of glomerulonephritis may lead to targeted therapies.

Cocaine abuse hastens the neurodegeneration often associated with advanced HIV-1 infection. The mechanisms, in part, revolve around the neuroinflammatory processes mediated by the chemokine monocyte chemotactic protein-1 (MCP-1/CCL2). Understanding factors that modulate MCP-1 and, in turn, facilitate monocyte extravasation in the brain is thus of paramount importance. We now demonstrate that cocaine induces MCP-1 in rodent microglia through translocation of the sigma receptor to the lipid raft microdomains of the plasma membrane. Sequential activation of Src, mitogen-activated protein kinases (MAPKs), and phosphatidylinositol-3' kinase (PI3K)/Akt and nuclear factor kappaB (NF-kappaB) pathways resulted in increased MCP-1 expression. Furthermore, conditioned media from cocaine-exposed microglia increased monocyte transmigration, and thus was blocked by antagonists for CCR2 or sigma receptor. These findings were corroborated by demonstrating increased monocyte transmigration in mice exposed to cocaine, which was attenuated by pretreatment of mice with the sigma receptor antagonist. Interestingly, cocaine-mediated transmigratory effects were not observed in CCR2 knockout mice. We conclude that cocaine-mediated induction of MCP-1 accelerates monocyte extravasation across the endothelium. Understanding the regulation of MCP-1 expression and functional changes by cocaine/sigma receptor system may provide insights into the development of potential therapeutic targets for HIV-1-associated neurocognitive disorders.

The transcription factor Twist1 induces epithelial-mesenchymal transition and extracellular matrix degradation to promote tumor metastasis. Although Twist1 also plays a role in embryonic vascular development and tumor angiogenesis, the molecular mechanisms that underlie these processes are not as well understood. Here, we report a novel function for Twist1 in modifying the tumor microenvironment to promote progression. We found that expression of Twist1 in human mammary epithelial cells potently promoted angiogenesis. Surprisingly, Twist1 expression did not increase the secretion of the common proangiogenic factors VEGF and basic fibroblast growth factor but rather induced expression of the macrophage chemoattractant CCL2. Attenuation of endogenous Twist1 in vivo blocked macrophage recruitment and angiogenesis, whereas exogenous CCL2 rescued the ability of tumor cells lacking Twist1 to attract macrophages and promote angiogenesis. Macrophage recruitment also was essential for the ability of Twist1-expressing cells to elicit a strong angiogenic response. Together, our findings show that how Twist1 recruits stromal macrophages through CCL2 induction to promote angiogenesis and tumor progression. As Twist1 expression has been associated with poor survival in many human cancers, this finding suggests that anti-CCL2 therapy may offer a rational strategy to treat Twist1-positive metastatic cancers.

Chemokines and chemokine receptors comprise a large number of molecules implicated in a wide range of physiological and pathological functions. Numerous studies have demonstrated the roles of chemokines and chemokine receptors: 1) during development, by regulating hematopoiesis, cardiogenesis, and vascular and cerebellar development; 2) during tumor biology, by controlling cell proliferation, angiogenesis, and metastasis; and 3), especially during leukocyte migration, by acting on firm adhesion, locomotion, diapedesis, and chemotaxis. This review focuses on chemokine and chemokine receptor involvement in diverse neurological diseases and their therapeutic potentials. Because of its induction or upregulation during CNS pathologies, members of the chemokine system can be used as biological markers. CXCR4 and CXCL12, by the correlation between their expression and the glioblastoma tumor progression, could be a marker to grade this type of CNS tumor. CCR1, by virtue of specific expression in Abeta plaques, may be a marker for Alzheimer pathology. Downregulation of CCL2 in cerebrospinal fluid may be a candidate to characterize multiple sclerosis (MS), but needs additional investigation. Moreover, chemokines and chemokine receptors represent interesting therapeutic targets. Using chemokine receptor antagonists, several studies provided exciting findings for potential neurological disease treatment. Chemokine receptor antagonists reduce disease severity in animal models of MS. In glioblastoma, a CXCR4 antagonist (AMD3100) showed an inhibition of tumor growth. Inhibition of chemokine receptor signaling is not the only therapeutic strategy: for example, CXCR4-CXCL12 has anti-inflammatory properties and CX3CL1-CX3CR1 controls neurotoxicity. Thus, chemokine biology suggests several approaches for treating neurological disease.

Immune responses during infection with pandemic H1N1 2009 influenza A virus (2009-H1N1) are still poorly understood. Using an experimental infection model in ferrets, we examined the pathological features and characterized the host immune responses by using microarray analysis, during infection with 2009-H1N1 A/California/07/2009 and seasonal A/Brisbane/59/2007. Chemokines CCL2, CCL8, CXCL7 and CXCL10 along with the majority of interferon-stimulated genes were expressed early, correlated to lung pathology, and abruptly decreased expression on day 7 following infection of A/California/07/2009. Interestingly, the drop in innate immune gene expression was replaced by a significant increase of the adaptive immune genes for granzymes and immunoglobulins. Serum anti-influenza antibodies were first observed on day 7, commensurate with the viral clearance. We propose that lung pathology in humans occurs during the innate phase of host immunity and a delay or failure to switch to the adaptive phase may contribute to morbidity and mortality during severe 2009-H1N1 infections.

Disturbance of the tight junction (TJ) complexes between brain endothelial cells leads to increased paracellular permeability, allowing leukocyte entry into inflamed brain tissue and also contributing to edema formation. The current study dissects the mechanisms by which a chemokine, CCL2, induces TJ disassembly. It investigates the potential role of selective internalization of TJ transmembrane proteins (occludin and claudin-5) in increased permeability of the brain endothelial barrier in vitro. To map the internalization and intracellular fate of occludin and claudin-5, green fluorescent protein fusion proteins of these TJ proteins were generated and imaged by fluorescent microscopy with simultaneous measurement of transendothelial electrical resistance. During CCL2-induced reductions in transendothelial electrical resistance, claudin-5 and occludin became internalized via caveolae and further processed to early (EEA1+) and recycling (Rab4+) endosomes but not to late endosomes. Western blot analysis of fractions collected from a sucrose gradient showed the presence of claudin-5 and occludin in the same fractions that contained caveolin-1. For the first time, these results suggest an underlying molecular mechanism by which the pro-inflammatory chemokine CCL2 mediates brain endothelial barrier disruption during CNS inflammation.

Mice lacking CD38, an ectoenzyme that generates the calcium-mobilizing metabolite cADPR, make reduced T cell-dependent antibody responses. Despite the predicted role for CD38 in B cell activation, we find that CD38 regulates the migration of dendritic cell (DC) precursors from the blood to peripheral sites and controls the migration of mature DCs from sites of inflammation to lymph nodes. Thus, T cells are inefficiently primed in Cd38(-/-) mice, leading to poor humoral immune responses. We also show that CD38 and cADPR modulate calcium mobilization in chemokine-stimulated DCs and are required for the chemotaxis of immature and mature DCs to <em>CCL2</em>, CCL19, <em>CCL2</em>1, and CXCL12. Therefore, CD38 regulates adaptive immunity by controlling chemokine receptor signaling in DCs.

There has been recent evidence showing the correlation between neuroinflammation owing to the chemokine-cytokine network and neuropathic pain. Chemokines and cytokines are derived from several types of cells in the peripheral and central nervous systems following nerve injury, and are largely involved in the pathogenesis of neuropathic pain. The roles of typical inflammatory cytokines such as interleukin-1β have become the recent center of attention. There is growing evidence that inflammatory chemokines (CCL2, CCL3, and fractalkine) play pivotal roles in neuropathic pain. Further investigations concerning the functions of the chemokine-cytokine network-mediated regulation of neuroinflammation may lead to novel therapeutic strategies against intractable neuropathic pain.

We demonstrate that the secretome of mesenchymal stromal cells (MSCs) suppresses plasma cell (PC) immunoglobulin (Ig) production, induces plasmablast proliferation, and leads to interleukin-10-mediated blockade in vitro. We found that these effects are the result of MSC-derived CC chemokine ligands CCL2 and CCL7. More specifically, MSCs further processed these CC chemokines by the activity of matrix metalloproteinases (MMPs), leading to the generation of proteolytically processed antagonistic CCL2 variant. Neutralizing CCL2 or inhibiting MMP enzymatic activity abolished the PC-suppressive effect of MSCs. We also observed that MMP-processed CCL2 suppresses signal transducer and activator of transcription 3 (STAT3) activation in PC. As a result, the transcription factor PAX5 is induced, thus explaining the inhibition of Ig synthesis. The absence of inhibitory effects by MSC on the humoral response of CCR2(-/-) mice to xenoantigen suggests that MMP-cleaved CCL2/CCR2 interaction as well as downstream phosphatase activity is necessary for antagonistic effect. We tested syngeneic MSCs in hemophilic B6 mice with predeveloped antihuman factor VIII (hFVIII) antibodies and demonstrated a robust decrease in hFVIII-specific IgG levels. Thus, MSCs may play a role in modulating Ig production by PCs via MMP processing of CCL2 and may represent an appealing cell therapy approach for pathologic humoral responses.

Axonal degeneration is an important determinant of progressive neurological disability in multiple sclerosis (MS). Thus, therapeutic approaches promoting neuroprotection could aid the treatment of progressive MS. Here, we used what we believe is a novel water-soluble fullerene derivative (ABS-75) attached to an NMDA receptor antagonist, which combines antioxidant and anti-excitotoxic properties, to block axonal damage and reduce disease progression in a chronic progressive EAE model. Fullerene ABS-75 treatment initiated after disease onset reduced the clinical progression of chronic EAE in NOD mice immunized with myelin-oligodendrocyte glycoprotein (MOG). Reduced disease progression in ABS-75-treated mice was associated with reduced axonal loss and demyelination in the spinal cord. Fullerene ABS-75 halted oxidative injury, CD11b+ infiltration, and CCL2 expression in the spinal cord of mice without interfering with antigen-specific T cell responses. In vitro, fullerene ABS-75 protected neurons from oxidative and glutamate-induced injury and restored glutamine synthetase and glutamate transporter expression in astrocytes under inflammatory insult. Glutamine synthetase expression was also increased in the white matter of fullerene ABS-75-treated animals. Our data demonstrate the neuroprotective effect of treatment with a fullerene compound combined with a NMDA receptor antagonist, which may be useful in the treatment of progressive MS and other neurodegenerative diseases.

BACKGROUND

During the era of highly active antiretroviral therapy (HAART), the prevalence of HIV-associated central nervous system (CNS) disease has increased despite suppression of plasma viremia.

METHODS

In a simian immunodeficiency virus (SIV) model system in which all animals develop AIDS and 90% develop CNS disease by 3 months after inoculation, pigtailed macaques were treated with a regimen of tenofovir disoproxil fumarate, saquinavir, atazanavir, and an integrase inhibitor starting at 12 days after inoculation and were euthanized at approximately 175 days after inoculation.

RESULTS

Plasma and cerebrospinal fluid (CSF) viral loads declined rapidly after the initiation of HAART. Brain viral RNA was undetectable at necropsy, but viral DNA levels were not different from those in untreated SIV-infected macaques. CNS inflammation was significantly reduced, with decreased brain expression of major histocompatibility complex class II and glial fibrillary acidic protein and reduced levels of CSF CCL2 and interleukin 6. Brain from treated macaques had significantly lower levels of interferon beta, type 1 interferon-inducible gene myxovirus (influenza) resistance A, and indolamine 2,3-dioxygenase messenger RNA, suggesting that immune hyperactivation was suppressed, and fewer CD4(+) and CD8(+) T cells, suggesting that trafficking of T cells from peripheral blood was reduced. Brain levels of CD68 protein and tumor necrosis factor alpha and interferon gamma RNA were reduced but were not significantly lower, indicating continued CNS inflammation.

CONCLUSIONS

These data, generated in a rigorous, high-viral-load SIV-infected macaque model, showed that HAART provided benefits with respect to CNS viral replication and inflammation but that no change in the level of viral DNA and continued CNS inflammation occurred in some macaques.

Macrophage migration inhibitory factor (MIF) was originally identified for its ability to inhibit the random migration of macrophages in vitro. MIF is now recognized as an important mediator in a range of inflammatory disorders. We recently observed that the absence of MIF is associated with a reduction in leukocyte-endothelial cell interactions induced by a range of inflammatory mediators, suggesting that one mechanism whereby MIF acts during inflammatory responses is by promoting leukocyte recruitment. However, it is unknown whether MIF is capable of inducing leukocyte recruitment independently of additional inflammatory stimuli. In this study, we report that MIF is capable of inducing leukocyte adhesion and transmigration in postcapillary venules in vivo. Moreover, leukocytes recruited in response to MIF were predominantly CD68(+) cells of the monocyte/macrophage lineage. Abs against the monocyte-selective chemokine CCL2 (JE/MCP-1) and its receptor CCR2, but not CCL3 and CXCL2, significantly inhibited MIF-induced monocyte adhesion and transmigration. CCL2(-/-) mice displayed a similar reduction in MIF-induced recruitment indicating a critical role of CCL2 in the MIF-induced response. This hypothesis was supported by findings that MIF induced CCL2 release from primary microvascular endothelial cells. These data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues. This function may be critical to the ability of MIF to promote diseases such as atherosclerosis and rheumatoid arthritis, in which macrophages are key participants.

Both cancer-related inflammation and tumor-induced immune suppression are associated with expansion of myeloid cell subsets including myeloid-derived suppressor cells. However, little known regarding characteristics of myeloid cells in patients with bladder cancer. In this study, we analyzed myeloid cells from peripheral blood (PBMC) and tumor tissue that were collected from patients with superficial noninvasive and invasive urothelial carcinomas. Our results demonstrate that PBMC from bladder cancer patients contain two major CD11b myeloid cell subsets: granulocyte-type CD15(high) CD33(low) cells and monocyte-type CD15(low) CD33(high) cells. The number of circulating granulocytic but not monocytic myeloid cells in cancer patients was markedly increased when compared to healthy individuals. Both myeloid cell subsets from cancer patients were highly activated and produced substantial amounts of proinflammatory chemokines/cytokines including CCL2, CCL3, CCL4, G-CSF, IL-8 and IL-6. Granulocytic myeloid cells were able to inhibit in vitro T cell proliferation through induction of CD4(+) Foxp3(+) T regulatory cells. Analysis of bladder cancer tissues revealed that tumors were infiltrated with monocyte-macrophage CD11b(+) HLA-DR(+) and granulocytic CD11b(+) CD15(+) HLA-DR(-) myeloid cells. Collectively, this study identifies myeloid cell subsets in patients with bladder cancer. We demonstrate that these highly activated inflammatory myeloid cells represent a source of multiple chemokines/cytokines and may contribute to inflammation and immune dysfunction in bladder cancer.

Alternative activation of microglia/macrophages (M2a) by interleukin (IL)-4 is purported to support intrinsic growth and repair processes after CNS injury. Nonetheless, alternative activation of microglia is poorly understood in vivo, particularly in the context of inflammation, injury, and aging. Here, we show that aged mice (18-19 months) had reduced functional recovery after spinal cord injury (SCI) associated with impaired induction of IL-4 receptor α (IL-4Rα) on microglia. The failure to successfully promote an IL-4/IL-4Rα response in aged mice resulted in attenuated arginase (M2a associated), IL-1β, and chemokine ligand 2 (CCL2) expression, and diminished recruitment of IL-4Rα(+) macrophages to the injured spinal cord. Furthermore, the link between reduced IL-4Rα expression and reduced arginase, IL-1β, and CCL2 expression was confirmed using adult IL-4Rα knock-out (IL-4Rα(KO)) mice. To better understand IL-4Rα-mediated regulation of active microglia, a series of studies was completed in mice that were peripherally injected with lipopolysaccharide and later provided IL-4 by intracerebroventricular infusion. These immune-based studies demonstrate that inflammatory-induced IL-4Rα upregulation on microglia was required for the induction of arginase by IL-4. In addition, IL-4-mediated reprogramming of active microglia enhanced neurite growth ex vivo and increased inflammatory gene expression (i.e., IL-1β and CCL2) and the corresponding recruitment of CCR2(+)/IL-4Rα(+)/arginase(+) myeloid cells in vivo. IL-4 reprogrammed active microglia to a unique and previously unreported phenotype (arginase(+)/IL-1β(+)) that augmented neurite growth and enhanced recruitment of peripheral IL-4Rα(+) myeloid cells to the CNS. Moreover, this key signaling cascade was impaired with age corresponding with reduced functional recovery after SCI.

Islets from patients with type 2 diabetes exhibit β cell dysfunction, amyloid deposition, macrophage infiltration, and increased expression of proinflammatory cytokines and chemokines. We sought to determine whether human islet amyloid polypeptide (hIAPP), the main component of islet amyloid, might contribute to islet inflammation by recruiting and activating macrophages. Early aggregates of hIAPP, but not nonamyloidogenic rodent islet amyloid polypeptide, caused release of CCL2 and CXCL1 by islets and induced secretion of TNF-α, IL-1α, IL-1β, CCL2, CCL3, CXCL1, CXCL2, and CXCL10 by C57BL/6 bone marrow-derived macrophages. hIAPP-induced TNF-α secretion was markedly diminished in MyD88-, but not TLR2- or TLR4-deficient macrophages, and in cells treated with the IL-1R antagonist (IL-1Ra) anakinra. To determine the significance of IL-1 signaling in hIAPP-induced pancreatic islet dysfunction, islets from wild-type or hIAPP-expressing transgenic mice were transplanted into diabetic NOD/SCID recipients implanted with mini-osmotic pumps containing IL-1Ra (50 mg/kg/d) or saline. IL-1Ra significantly improved the impairment in glucose tolerance observed in recipients of transgenic grafts 8 wk following transplantation. Islet grafts expressing hIAPP contained amyloid deposits in close association with F4/80-expressing macrophages. Transgenic grafts contained 50% more macrophages than wild-type grafts, an effect that was inhibited by IL-1Ra. Our results suggest that hIAPP-induced islet chemokine secretion promotes macrophage recruitment and that IL-1R/MyD88, but not TLR2 or TLR4 signaling is required for maximal macrophage responsiveness to prefibrillar hIAPP. These data raise the possibility that islet amyloid-induced inflammation contributes to β cell dysfunction in type 2 diabetes and islet transplantation.

Studies in IL-6-deficient (IL-6(-/-)) mice highlight that IL-6 contributes to arthritis progression. However, the molecular mechanism controlling its activity in vivo remains unclear. Using an experimental arthritis model in IL-6(-/-) mice, we have established a critical role for the soluble IL-6R in joint inflammation. Although intra-articular administration of IL-6 itself was insufficient to reconstitute arthritis within these mice, a soluble IL-6R-IL-6 fusion protein (HYPER-IL-6) restored disease activity. Histopathological assessment of joint sections demonstrated that HYPER-IL-6 increased arthritis severity and controlled intrasynovial mononuclear leukocyte recruitment through the CC-chemokine CCL2. Activation of synovial fibroblasts by soluble IL-6R and IL-6 emphasized that these cells may represent the source of CCL2 in vivo. Specific blockade of soluble IL-6R signaling in wild-type mice using soluble gp130 ameliorated disease. Consequently, soluble IL-6R-mediated signaling represents a promising therapeutic target for the treatment of rheumatoid arthritis.

BACKGROUND

Respiratory syncytial virus (RSV) bronchiolitis in infants is characterized by a massive neutrophilic infiltrate into the airways. Chemokines direct migration of leukocytes and contribute to the pathogenesis of RSV disease. However, little is known about pulmonary chemokine responses to RSV in humans. Our aim was to characterize the production of chemokines in the lungs of infants with RSV bronchiolitis and how this production changes over time.

METHODS

Chemokine mRNA and the concentration of chemokines were measured in nonbronchoscopic bronchoalveolar lavage (BAL) samples from infants with RSV bronchiolitis and from control infants. In infants with RSV bronchiolitis, changes in the concentrations of chemokines during the 7 days after intubation and between the days of intubation and extubation were examined.

RESULTS

The production of chemokines within the lower respiratory tract was shown in all patients with RSV bronchiolitis. CXC chemokines (particularly CXCL10/interferon-inducible protein 10 and CXCL8/interleukin-8) were found to be the most abundant, but CC chemokines (CCL2/monocyte chemotactic protein 1 and CCL3/macrophage inflammatory protein-1 alpha) were also present. Concentrations of some of these chemokines remained elevated over the course of the illness, whereas others decreased steadily. No differences in the concentrations were found between the days of intubation and extubation.

CONCLUSIONS

CXC chemokines predominate within the RSV-infected lung. Much of this response comes from inflammatory cells within the lower respiratory tract. Chemokine response patterns vary over time, possibly indicating different cellular sources for individual chemokines in the RSV-infected lung.

Macrophages constitute a major proinflammatory component during chronic liver diseases and are considered a key factor in promoting hepatic fibrosis. However, there is increasing evidence that distinct monocyte and macrophage subsets exert critical functions in regression from organ fibrosis as well. Experimental mouse models of fibrosis regression have identified "restorative" macrophages as Ly-6C (Ly6C, Gr1) low-expressing, monocyte-derived cells. We investigated molecular pathways balancing proinflammatory and restorative macrophages during fibrosis regression as well as pharmacologically augmenting beneficial macrophage functionality in fibrosis resolution. Therefore, we employed a Spiegelmer-based inhibitor of the chemokine, C-C motif chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1), termed mNOX-E36, in the regression phase of two murine models of toxic (CCl4 ) and metabolic (methionine-choline-deficient diet) liver fibrosis. Although inflammation rapidly declined after cessation of injury, we observed a transient influx of Ly-6C(+) infiltrating monocytes (iMΦ), which are characterized by typical macrophage morphology, up-regulated expression of CCR2, and the pro-inflammatory cytokine, tumor necrosis factor (TNF), in injured liver. By inhibiting the early influx of Ly-6C(+) iMΦ by the CCL2 inhibitor, mNOX-E36, the intrahepatic macrophage equilibration shifted toward the "restorative" Ly-6C(-) subset of iMΦ. Consequently, fibrosis resolution was significantly accelerated upon mNOX-E36 administration in both models. Blocking transient recruitment of infiltrating Ly-6C(+) monocytes, but not direct effects of the inhibitor on the remaining macrophages, resulted in reduced intrahepatic levels of proinflammatory cytokines.

CONCLUSIONS

Transient CCL2-dependent recruitment of infiltrating Ly-6C(+) monocytes during fibrosis regression counteracts scar resolution by perpetuating inflammatory reactions through release of proinflammatory cytokines such as TNF. Pharmacological inhibition of Ly-6C(+) monocyte recruitment using the CCL2-inhibitor, mNOX-E36, accelerates regression from toxic and metabolic liver fibrosis in two independent experimental models.

IFN-alpha/beta-mediated functions promote production of MIP-1alpha (or CCL3) by mediating the recruitment of MIP-1alpha-producing macrophages to the liver during early infection with murine CMV. These responses are essential for induction of NK cell inflammation and IFN-gamma delivery to support effective control of local infection. Nevertheless, it remains to be established if additional chemokine functions are regulated by IFN-alpha/beta and/or play intermediary roles in supporting macrophage trafficking. The chemokine MCP-1 (or CCL2) plays a distinctive role in the recruitment of macrophages by predominantly stimulating the CCR2 chemokine receptor. Here, we examine the roles of MCP-1 and CCR2 during murine CMV infection in liver. MCP-1 production preceded that of MIP-1alpha during infection and was dependent on IFN-alpha/beta effects for induction. Resident F4/80(+) liver leukocytes were identified as primary IFN-alpha/beta responders and major producers of MCP-1. Moreover, MCP-1 deficiency was associated with a dramatic reduction in the accumulation of macrophages and NK cells, as well as decreased production of MIP-1alpha and IFN-gamma in liver. These responses were also markedly impaired in mice with a targeted disruption of CCR2. Furthermore, MCP-1- and CCR2-deficient mice exhibited increased viral titers and elevated expression of the liver enzyme alanine aminotransferase in serum. These mice also had widespread virus-induced liver pathology and succumbed to infection. Collectively, these results establish MCP-1 and CCR2 interactions as factors promoting early liver inflammatory responses and define a mechanism for innate cytokines in regulation of chemokine functions critical for effective localized antiviral defenses.

BACKGROUND

An increase in the number of mononuclear phagocytes in lung biopsies from patients with idiopathic pulmonary fibrosis (IPF) worsens prognosis. Chemokines that recruit mononuclear phagocytes, such as CC chemokine ligand 2 (CCL2), are elevated in bronchoalveolar lavage (BAL) fluid (BALF) from patients with IPF. However, little attention is given to the role of the mononuclear phagocyte survival and recruitment factor, macrophage colony-stimulating factor (M-CSF), in pulmonary fibrosis.

OBJECTIVE

To investigate the role of mononuclear phagocytes and M-CSF in pulmonary fibrosis.

METHODS

Wild-type, M-CSF-/-, or CCL2-/- mice received intraperitoneal bleomycin. Lung inflammation and fibrosis were measured by immunohistochemistry, ELISA, collagen assay, BAL differentials, real-time polymerase chain reaction, and Western blot analysis. Human and mouse macrophages were stimulated with M-CSF for CCL2 expression. BALF from patients with IPF was examined for M-CSF and CCL2.

RESULTS

M-CSF-/- and CCL2-/- mice had less lung fibrosis, mononuclear phagocyte recruitment, collagen deposition, and connective tissue growth factor (CTGF) expression after bleomycin administration than wild-type littermates. Human and mouse macrophages stimulated with M-CSF had increased CCL2 production, and intratracheal administration of M-CSF in mice induced CCL2 production in BALF. Finally, BALF from patients with IPF contained significantly more M-CSF and CCL2 than BALF from normal volunteers. Elevated levels of M-CSF were associated with elevated CCL2 in BALF and the diagnosis of IPF.

CONCLUSIONS

These data suggest that M-CSF contributes to the pathogenesis of pulmonary fibrosis in mice and in patients with IPF through the involvement of mononuclear phagocytes and CCL2 production.

BACKGROUND

Lungs of adult mice exposed to hyperoxia as newborns are simplified and exhibit reduced function much like that observed in people who had bronchopulmonary dysplasia (BPD) as infants. Because survivors of BPD also show increased risk for symptomatic respiratory infections, we investigated how neonatal hyperoxia affected the response of adult mice infected with influenza A virus infection.

OBJECTIVE

To determine whether neonatal hyperoxia increased the severity of influenza A virus infection in adult mice.

METHODS

Adult female mice exposed to room air or hyperoxia between Postnatal Days 1 and 4 were infected with a sublethal dose of influenza A virus.

RESULTS

The number of macrophages, neutrophils, and lymphocytes observed in airways of infected mice that had been exposed to hyperoxia as neonates was significantly greater than in infected siblings that had been exposed to room air. Enhanced inflammation correlated with increased levels of monocyte chemotactic protein-1 (CCL2) in lavage fluid, whereas infection-associated changes in IFN-gamma, IL-1beta, IL-6, tumor necrosis factor-alpha, KC, granulocyte-macrophage colony-stimulating factor, and macrophage inflammatory protein-1alpha, and production of virus-specific antibodies, were largely unaffected. Increased mortality of mice exposed to neonatal hyperoxia occurred by Day 14 of infection, and was associated with persistent inflammation and fibrosis.

CONCLUSIONS

These data suggest that the disruptive effect of hyperoxia on neonatal lung development also reprograms key innate immunoregulatory pathways in the lung, which may contribute to exacerbated pathology and poorer resistance to respiratory viral infections typically seen in people who had BPD.

CD40 ligand (CD40L), identified as a costimulatory molecule expressed on T cells, is also expressed and functional on platelets. We investigated the thrombotic and inflammatory contributions of platelet CD40L in atherosclerosis. Although CD40L-deficient (Cd40l(-/-)) platelets exhibited impaired platelet aggregation and thrombus stability, the effects of platelet CD40L on inflammatory processes in atherosclerosis were more remarkable. Repeated injections of activated Cd40l(-/-) platelets into Apoe(-/-) mice strongly decreased both platelet and leukocyte adhesion to the endothelium and decreased plasma CCL2 levels compared with wild-type platelets. Moreover, Cd40l(-/-) platelets failed to form proinflammatory platelet-leukocyte aggregates. Expression of CD40L on platelets was required for platelet-induced atherosclerosis as injection of Cd40l(-/-) platelets in contrast to Cd40l(+/+) platelets did not promote lesion formation. Remarkably, injection of Cd40l(+/+), but not Cd40l(-/-), platelets transiently decreased the amount of regulatory T cells (Tregs) in blood and spleen. Depletion of Tregs in mice injected with activated Cd40l(-/-) platelets abrogated the athero-protective effect, indicating that CD40L on platelets mediates the reduction of Tregs leading to accelerated atherosclerosis. We conclude that platelet CD40L plays a pivotal role in atherosclerosis, not only by affecting platelet-platelet interactions but especially by activating leukocytes, thereby increasing platelet-leukocyte and leukocyte-endothelium interactions.

Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) inhibit anti-tumor immune responses and facilitate tumor growth. Precursors for these immune cell populations migrate to the tumor site in response to tumor secretion of chemokines, such as monocyte chemoattractant protein-1 (MCP-1/CCL2), which was originally purified and identified from human gliomas. In syngeneic mouse GL261 glioma and human U87 glioma xenograft models, we evaluated the efficacy of systemic CCL2 blockade by monoclonal antibodies (mAb) targeting mouse and/or human CCL2. Intraperitoneal (i.p.) administration of anti-mouse CCL2 mAb as monotherapy (2 mg/kg/dose, twice a week) significantly, albeit modestly, prolonged the survival of C57BL/6 mice bearing intracranial GL261 glioma (P = 0.0033), which was concomitant with a decrease in TAMs and MDSCs in the tumor microenvironment. Similarly, survival was modestly prolonged in severe combined immunodeficiency mice bearing intracranial human U87 glioma xenografts treated with both anti-human CCL2 mAb and anti-mouse CCL2 antibodies (2 mg/kg/dose for each, twice a week) compared to mice treated with control IgG (P = 0.0159). Furthermore, i.p. administration of anti-mouse CCL2 antibody in combination with temozolomide (TMZ) significantly prolonged the survival of C57BL/6 mice bearing GL261 glioma with 8 of 10 treated mice surviving longer than 70 days, while only 3 of 10 mice treated with TMZ and isotype IgG survived longer than 70 days (P = 0.0359). These observations provide support for development of mAb-based CCL2 blockade strategies in combination with the current standard TMZ-based chemotherapy for treatment of malignant gliomas.