CXCR3 ligands are augmented during the pathogenesis of pulmonary sarcoidosis.

We and other investigators have hypothesised that the CXC chemokine receptor (CXCR)3/CXCR3 ligand biological axis is involved in the formation of sarcoid lung granulomas; however, significant discrepancies in the current literature remain. In an effort to clarify previous conflicting findings, we performed the largest observational study to date of interferon-inducible ELR(-) (lacking the sequence glutamic acid-leucine-arginine) CXC chemokines in sarcoid bronchoalveolar fluid (BALF). BALF chemokine levels from sarcoid patients (n = 72) and healthy controls (n = 8) were measured with the ELISA method. Immunohistochemical staining was performed for CXCR3 and its ligands. BALF CXC chemokine ligand (CXCL)10 levels from sarcoid patients were not significantly increased compared with controls. BALF CXCL11 levels from sarcoid patients demonstrated a trend towards elevation; subgroup analysis by stage showed significant BALF CXCL11 elevation in stage I sarcoid patients compared with controls. BALF CXCL9 levels were elevated from sarcoid patients compared with controls. CXC11, CXCL9 and CXCR3 were expressed from epithelioid histiocytes, multinucleated giant cells and other inflammatory cells forming sarcoid lung granulomas. Our data suggest that CXCL9 and CXCL11 are important mediators in recruiting CXCR3-expressing cells. Importantly, we have made the novel observation that both lymphocytes and cells of monocyte linage express CXCR3 and are involved in the formation of sarcoid lung granulomas.

Altered levels of CC chemokines during pulmonary CMV predict BOS and mortality post-lung transplantation.

Pulmonary CMV infection (CMVI) and disease (CMVD) is associated with reduced long-term survival post-lung transplantation, however, the specific biologic mechanisms remain unclear. We have demonstrated a role of CC chemokines during lung allograft dysfunction. Based on these findings, we hypothesized that pulmonary CMV upregulates the expression of multiple CC chemokines that leads to allograft dysfunction and decreased long-term survival. We performed a nested case control study in lung transplant recipients to investigate alterations in CC chemokine biology during pulmonary CMV. Levels of CC chemokines were measured in bronchoalveolar lavage fluid (BALF) from recipients with CMVI (n = 33), CMVD (n = 6), and in healthy lung transplant controls (n = 33). We found a trend toward increased levels of MIP-1alpha/CCL3 during pulmonary CMVI. Levels of MCP-1/CCL2 and RANTES/CCL5 were significantly elevated during pulmonary CMV. Interestingly, elevated levels of CCL3 in BALF were protective with regards to survival. Importantly, elevated levels of CCL2 in BALF predicted the development of BOS, while elevated levels of CCL5 in BALF predicted an increase in mortality post-lung transplant. Altered levels of specific CC chemokines during pulmonary CMV are associated with future clinical outcomes. These results suggest a possible utility of BALF CC chemokines as biomarkers for guiding risk assessment during pulmonary CMV post-lung transplantation.

Fimbrial lectins influence the chemokine repertoire in the urinary tract mucosa.

The defense against mucosal infections relies on chemokines that recruit inflammatory cells to the mucosa. This study examined if the chemokine response to uro-pathogenic Escherichia coli is influenced by fimbrial expression. The CXC (CXCL1, CXCL5, CXCL8, CXCL9, CXCL10) and CC chemokines (CCL2, CCL3, CCL5) were quantified after in vitro infection of uro-epithelial cells with a fimbriated E. coli pyelonephritis isolate, or with P or type 1 fimbriated transformants of an avirulent E. coli K-12 strain. The response profile was shown to vary with the fimbrial type. Type 1 fimbriated E. coli elicited mainly CXCL1 and CXCL8, whereas P fimbriated E. coli stimulated CCL2 and CCL5 and class II were more potent chemokine inducers than class III P fimbriae. Chemokines were also quantified in urine samples from 73 patients with febrile urinary tract infection, and analyzed as a function of disease severity and fimbrial expression by the strain infecting each patient. A complex CXC and CC chemokine response was detected in patient urine, with a significant influence of the fimbrial type. The results show that virulence factors like fimbriae may modify the mucosal chemokine response. This mechanism may allow the host to adjust the inflammatory cell infiltrate to fit the infecting strain.

CCL19 reduces tumour burden in a model of advanced lung cancer.

Epstein-Barr virus-induced molecule 1 ligand chemokine (CCL19) is a CC chemokine that chemoattracts both dendritic cells (DC) and T lymphocytes. We evaluated the antitumour efficacy of CCL19 in a murine model of spontaneous bronchoalveolar cell carcinoma. These transgenic mice (CC-10 TAg) express the SV40 large T antigen under the Clara Cell promoter, develop bilateral, multifocal, pulmonary carcinomas and die at 4 months owing to progressive pulmonary tumour burden. To mimic therapy in late-stage disease, 3-month-old transgenic mice were treated with recombinant CCL19 (0.5 microg dose(-1)) by intranodal (axillary lymph node region) injection three times per week for 4 weeks. CCL19 treatment led to a marked reduction in tumour burden with extensive mononuclear infiltration of the tumours compared to diluent treated controls. Flow cytometric analyses showed significant increases in CD4 and CD8T cell subsets as well as DC in the lungs of CCL19-treated mice. Lung tissue cytokine profiles showed a shift towards immune stimulatory molecules with a decrease in the immunosuppressive cytokine TGF-beta. Our findings show that CCL19 may serve as a potential immune stimulator and provide a strong rationale for the evaluation of CCL19 in cancer immunotherapy.

Chemokine CCL2 and chemokine receptor CCR2 in early active multiple sclerosis.

The chemokine monocyte chemoattractant protein (MCP)-1/CCL2 and its receptor CCR2 have been strongly implicated in disease pathogenesis in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS), whereas data on the CCL2-CCR2 axis are scarce in MS. We studied the expression of CCR2 on leukocytes in blood and cerebrospinal fluid (CSF) from patients with monosymptomatic optic neuritis and MS, and the concentration of CCL2 in the CSF from these patients. Results were compared with the results in non-inflammatory neurological controls and were correlated with other parameters (magnetic resonance imaging and CSF data). Our findings suggest a limited role for CCL2/CCR2 in early active MS.

Chemokines as therapeutic targets in non-small cell lung cancer.

Angiogenesis, defined as the generation of new blood vessels from pre-existing vessels, is one of life's essential processes. Inflammation and angiogenesis, while distinct and separable, are closely related processes. One of the hallmarks of chronic inflammation is granulation tissue, a prominent feature of which is neovascularization. Whenever tissue constituents proliferate, repair, or hypertrophy, such change must be accompanied by a proportional increase in capillary blood supply to assure delivery of nutrients, and removal of metabolic waste. This absolute dependence suggests two characteristics of angiogenesis. First, under normal conditions the process must be tightly controlled. Second, in the absence of such strict control, abnormal physiology, or disease is likely to result. The role of angiogenesis in solid tumor growth has attracted a great deal of attention as a potential therapeutic target. Lung cancer is a particularly devastating disease in industrialized countries. The majority of patients with lung cancer are faced with very poor therapeutic options, and gaining insight to the mechanism of angiogenesis in this disease has obvious implications for the design of therapeutic agents. Research in our laboratories has demonstrated that chemokines (chemotactic cytokines) are pivotal determinants of the angiogenic activity of non-small cell lung cancer (NSCLC). This review will focus on the evidence supporting the central role of these molecules in lung cancer angiogenesis, and focus on potential novel means of targeting this family of angiogenic regulators.

ENA-78 is an important angiogenic factor in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and often fatal disorder. Fibroplasia and deposition of extracellular matrix are dependent, in part, on angiogenesis and vascular remodeling. We obtained open lung biopsies from patients undergoing thoracic surgery for reasons other than interstitial lung disease (control) (n = 78) and from patients with IPF (n = 91). We found that levels of epithelial neutrophil-activating peptide 78 (ENA-78) were greater from tissue specimens of IPF patients, as compared with control subjects. When ENA-78 was depleted from IPF tissue specimens, tissue-derived angiogenic activity was markedly reduced. Immunolocalization of ENA-78 demonstrated that hyperplastic Type II pneumocytes and macrophages were the predominant cellular sources of ENA-78. These findings support the notion that ENA-78 may be an important additional factor that regulates angiogenic activity in IPF.

IFN-gamma-inducible protein-10 (CXCL10) is hepatoprotective during acute liver injury through the induction of CXCR2 on hepatocytes.

IFN-gamma-inducible protein-10 (IP-10/CXCL10) is a non-ELR-CXC chemokine that is present during various forms of acute and chronic liver injury. The purpose of this study was to explore the role of IP-10 during acute liver injury induced by acetaminophen (APAP). After a 400 mg/kg APAP challenge in fasted CD-1 mice, immunoreactive levels of IP-10 were dramatically elevated in the serum within 8 h. CXCR3, the receptor for IP-10, was up-regulated in the liver. Mice that received an i.v. injection of rIP-10 10 h after APAP challenge exhibited a dramatic reduction in alanine aminotransferase 8 h later. Histologic analysis confirmed that the delayed IP-10 therapy dramatically improved the appearance of the liver when examined 48 h after APAP. The therapeutic effect of IP-10 was associated with a marked increase in CXCR2 expression on hepatocytes. Neutralization of CXCR2 during IP-10 therapy resulted in an abrogation of the hepatoprotective effect of IP-10. Furthermore, IP-10 treatment of cultured hepatocytes stimulated a CXCR2-dependent proliferative response. In conclusion, IP-10 has a hepatoregenerative effect in a murine model of acute liver injury that is dependent on its up-regulation of CXCR2 on hepatocytes.

The regulation of interleukin-8 by hypoxia in human macrophages--a potential role in the pathogenesis of the acute respiratory distress syndrome (ARDS).

BACKGROUND: The acute respiratory distress syndrome (ARDS) represents a form of severe acute inflammatory lung disease. We have previously demonstrated significantly raised interleukin-8 (IL-8) levels in the lungs of at-risk patients that progress to ARDS, and identified the alveolar macrophage as an important source of this chemokine. We wished to extend this study in a well-defined group of patients with major trauma, and to investigate potential mechanisms for rapid intrapulmonary IL-8 generation. MATERIALS AND METHODS: Patients with major trauma underwent bronchoalveolar lavage (BAL) and IL-8 levels were measured in BAL fluid by ELISA. Human macrophages were derived from peripheral blood monocytes from healthy volunteers. Rabbit alveolar macrophages were obtained from ex-vivo lavage of healthy rabbit lungs. Macrophages were culture under normoxic or hypoxic (PO2 26 mmHg) conditions. IL-8 and other proinflammatory mediator expression was measured by ELISA, northern blotting or multi-probe RNase protection assay. RESULTS: In patients with major trauma, IL-8 levels were significantly higher in patients that progressed to ARDS compared to those that did not (n = 56, P = 0.0001). High IL-8 levels negatively correlated with PaO2/FiO2 (r = -0.56, P < 0.001). In human monocyte derived macrophages hypoxia rapidly upregulated IL-8 protein (within 2 hours) and mRNA expression (within 30 mins). Acute hypoxia also increased rabbit alveolar macrophage IL-8 expression. Hypoxia increased DNA binding activity of AP-1 and C/EBP but not NF-kappaB. Hypoxia induced HIF-1 expression, but cobaltous ions and desferrioxamine did not mimic hypoxic IL-8 induction. Hypoxia downregulated a range of other proinflammatory mediators, including MCP-1 and TNF-alpha. Both the pattern of cytokine expression and transcription factor activation by hypoxia was different to that seen with endotoxin. CONCLUSIONS: Rapidly raised intrapulmonary IL-8 levels are associated with ARDS progression in patients with major trauma. Acute hypoxia, a clinically relevant stimulus, rapidly and selectively upregulates IL-8 in macrophages associated with a novel pattern of transcription factor activation. Acute hypoxia may represent one of potentially several proinflammatory stimuli responsible for rapid intrapulmonary IL-8 generation in patients at-risk of ARDS.

Monokine induced by IFN-gamma is a dominant factor directing T cells into murine cardiac allografts during acute rejection.

The use of chemokine antagonism as a strategy to inhibit leukocyte trafficking into inflammatory sites requires identification of the dominant chemokines mediating recruitment. The chemokine(s) directing T cells into cardiac allografts during acute rejection remain(s) unidentified. The role of the CXC chemokines IFN-gamma inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig) in acute rejection of A/J (H-2(a)) cardiac grafts by C57BL/6 (H-2(b)) recipients was tested. Intra-allograft expression of Mig was observed at day 2 posttransplant and increased to the time of rejection at day 7 posttransplant. IP-10 mRNA and protein production were 2.5- to 8-fold lower than Mig. Whereas allografts were rejected at day 7-9 in control recipients, treatment with rabbit antiserum to Mig, but not to IP-10, prolonged allograft survival up to day 19 posttransplant. At day 7 posttransplant, allografts from Mig antiserum-treated recipients had marked reduction in T cell infiltration. At the time of rejection in Mig antiserum-treated recipients (i.e., days 17-19), intra-allograft expression of macrophage-inflammatory protein-1alpha, -1beta, and their ligand CCR5 was high, whereas expression of CXCR3, the Mig receptor, was virtually absent. Mig was produced by the allograft endothelium as well as by recipient allograft-infiltrating macrophages and neutrophils, indicating the synergistic interactions between innate and adaptive immune compartments during acute rejection. Collectively, these results indicate that Mig is a dominant recruiting factor for alloantigen-primed T cells into cardiac allografts during acute rejection. Although Mig antagonism delays acute heart allograft rejection, the results also suggest that the alloimmune response circumvents Mig antagonism through alternative mechanisms.

Overexpression of CXC chemokines by an adrenocortical carcinoma: a novel clinical syndrome.

A patient with adrenocortical carcinoma presented with fever, leukocytosis, and increased acute phase reactants. The tumor was infiltrated with neutrophils. Immunohistochemical staining of the tumor showed positive signal for epithelial neutrophil-activating protein-78, an angiogenic and chemotactic CXC chemokine. Conditioned medium from tumor-derived cells (RL-251) showed high concentration of IL-8, epithelial neutrophil-activating protein-78, Gro alpha, and Gro gamma, angiogenic CXC chemokines with a potential role in tumorigenesis. An adrenal cancer/severe combined immunodeficiency mouse chimera was developed. Mice grew tumors rapidly, and circulating levels of IL-8 and epithelial neutrophil-activating protein-78 were detected. In contrast, animals transplanted with NCI-H295 cells, a nonchemokine-secreting cell line, grew tumors more slowly and did not have detectable chemokine levels. Similar to the patient, mice with RL-251 tumors developed marked leukocytosis and neutrophilia, and their tumors were infiltrated with neutrophils. Mice were passively immunized with epithelial neutrophil-activating protein-78 antisera. A marked decrease in tumor growth was observed. Potential for chemokine production by other adrenocortical tumors was investigated by RT-PCR in archival material. Six of seven adrenal carcinomas and one of three adenomas had cDNA for IL-8; six of seven carcinomas and the three adenomas had cDNA for epithelial neutrophil-activating protein-78. We concluded that the clinical presentation of this case resulted from increased tumor production of chemotactic chemokines. Through their angiogenic and chemotactic properties these chemokines may play an important role in adrenal tumorigenesis.

Critical role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival after lung transplantation. Acute rejection, its main risk factor, is characterized by perivascular/bronchiolar leukocyte infiltration. BOS is characterized by persistent peribronchiolar leukocyte recruitment leading to airway fibrosis and obliteration. The specific mechanism(s) by which these leukocytes are recruited are unknown. Because MCP-1, acting through its receptor CCR2, is a potent mononuclear cell chemoattractant, we hypothesized that expression of this chemokine during an allogeneic-response promotes persistent recruitment of leukocytes and, ultimately, rejection. We found that elevated levels of biologically active MCP-1 in human bronchial lavage fluid (BALF) were associated with the continuum from acute to chronic allograft rejection. Translational studies in a murine model of BOS demonstrated increased MCP-1 expression paralleling mononuclear cell recruitment and CCR2 expression. Loss of MCP-1/CCR2 signaling, as seen in CCR2(-/-) mice or in WT mice treated with neutralizing antibodies to MCP-1, significantly reduced recruitment of mononuclear phagocytes following tracheal transplantation and led to attenuation of BOS. Lymphocyte infiltration was not reduced under these conditions. We suggest that MCP-1/CCR2 signaling plays an important role in recruitment of mononuclear phagocytes, a pivotal event in the pathogenesis of BOS.

Interleukin-8 stimulates human immunodeficiency virus type 1 replication and is a potential new target for antiretroviral therapy.

Production of the C-X-C chemokines interleukin-8 (IL-8) and growth-regulated oncogene alpha (GRO-alpha) in macrophages is stimulated by exposure to human immunodeficiency virus type 1 (HIV-1). We have demonstrated previously that GRO-alpha then stimulates HIV-1 replication in both T lymphocytes and macrophages. Here we demonstrate that IL-8 also stimulates HIV-1 replication in macrophages and T lymphocytes. We further show that increased levels of IL-8 are present in the lymphoid tissue of patients with AIDS. In addition, we demonstrate that compounds which inhibit the actions of IL-8 and GRO-alpha via their receptors, CXCR1 and CXCR2, also inhibit HIV-1 replication in both T lymphocytes and macrophages, indicating potential therapeutic uses for these compounds in HIV-1 infection and AIDS.

CXC chemokine receptor 2 contributes to host defense in murine urinary tract infection.

CXC chemokines have been implicated in the recruitment of neutrophils to sites of infection. To determine the role of CXC chemokines in the host response to urinary tract infection (UTI), female mice were treated with an antibody against the major CXC chemokine receptor in the mouse, CXCR2, before intravesical inoculation with Escherichia coli. Anti-CXCR2 prevented the influx of neutrophils in urine and kidneys. The absence of a neutrophil response only temporarily impaired the clearance of bacteria from the urinary tract, as indicated by 100- and 1000-fold more E. coli colony-forming units in urine and kidneys of anti-CXCR2-treated mice at 24 h, but not at 48 h, after the infection. UTI induced increases in the renal concentrations of the CXCR2 ligands macrophage inflammatory protein-2 and KC, which were not influenced by anti-CXCR2 administration. CXC chemokines play an important role in the development of a local inflammatory response to UTI.

Cutting edge: IFN-inducible ELR- CXC chemokines display defensin-like antimicrobial activity.

Recent reports highlighted the chemotactic activities of antimicrobial peptide defensins whose structure, charge, and size resemble chemokines. By assaying representative members of the four known families of chemokines we explored the obverse: whether some chemokines exert antimicrobial activity. In a radial diffusion assay, only recombinant monokine induced by IFN-gamma (MIG/CXCL9), IFN-gamma-inducible protein of 10 kDa (IP-10/CXCL10), and IFN-inducible T cell alpha chemoattractant (I-TAC/CXCL11), members of the IFN-gamma-inducible tripeptide motif Glu-Leu-Arg (ELR)(-) CXC chemokines, were antimicrobial against Escherichia coli and Listeria monocytogenes. Similar to human defensins, antimicrobial activities of the chemokines were inhibited by 50 and 100 mM NaCl. The concentration of MIG/CXCL9 and IP-10/CXCL10 released from IFN-gamma-stimulated PBMC in 24 h were, respectively, 35- and 28-fold higher than from unstimulated cells. Additionally, the amounts of chemokines released per monocyte suggest that, in tissues with mononuclear cell infiltration, IFN-gamma-inducible chemokines may reach concentrations necessary for microbicidal activity. IFN-gamma-inducible chemokines may directly inactivate microbes before attracting other host defense cells to the area of infection.

IL-12 attenuates bleomycin-induced pulmonary fibrosis.

Interleukin (IL)-12 is a potent inducer of interferon (IFN)-gamma. We postulated that IL-12 would attenuate bleomycin-induced pulmonary fibrosis. To test this hypothesis, we administered IL-12 or murine serum albumin to bleomycin-treated mice by daily intraperitoneal injection until day 12. Mice treated with IL-12 demonstrated decreased hydroxyproline levels compared with control treated mice. Furthermore, administration of IL-12 led to a time-dependent increase in both lung and bronchoalveolar lavage fluid IFN-gamma. The antifibrotic effect of IL-12 could be attenuated with simultaneous administration of neutralizing anti-IFN-gamma antibodies. These findings support the notion that IL-12 attenuates bleomycin-induced pulmonary fibrosis via modulation of IFN-gamma production.

Human immunodeficiency virus type 1 (HIV-1)-induced GRO-alpha production stimulates HIV-1 replication in macrophages and T lymphocytes.

We examined the early effects of infection by CCR5-using (R5 human immunodeficiency virus [HIV]) and CXCR4-using (X4 HIV) strains of HIV type 1 (HIV-1) on chemokine production by primary human monocyte-derived macrophages (MDM). While R5 HIV, but not X4 HIV, replicated in MDM, we found that the production of the C-X-C chemokine growth-regulated oncogene alpha (GRO-alpha) was markedly stimulated by X4 HIV and, to a much lesser extent, by R5 HIV. HIV-1 gp120 engagement of CXCR4 initiated the stimulation of GRO-alpha production, an effect blocked by antibodies to CXCR4. GRO-alpha then fed back and stimulated HIV-1 replication in both MDM and lymphocytes, and antibodies that neutralize GRO-alpha or CXCR2 (the receptor for GRO-alpha) markedly reduced viral replication in MDM and peripheral blood mononuclear cells. Therefore, activation of MDM by HIV-1 gp120 engagement of CXCR4 initiates an autocrine-paracrine loop that may be important in disease progression after the emergence of X4 HIV.

Interleukin-8 administration enhances venous thrombosis resolution in a rat model.

BACKGROUND: Therapy for deep vein thrombosis (DVT) resolution in those patients in whom a complication or contraindication to anticoagulation occurs is limited. As prior work suggests that thrombus maturation involves early influx of neutrophils (PMN) and neovascularization, we hypothesized that administering the proinflammatory/proangiogenic chemokine interleukin (IL)-8 might accelerate thrombus resolution. MATERIALS AND METHODS: An established rodent model of DVT (inferior vena cava [IVC] ligation) was used whereby daily intravenous recombinant human IL-8 (1 microg) or vehicle control was administered, with sacrifice at 4 and 8 days. Prior to sacrifice and at harvest, duplex ultrasound of the DVT and femoral venous pressure measurements were performed. Thrombi were analyzed by immunohistochemical techniques for PMN, monocytes, and neovascularization; for chemokines, by enzyme-linked immunoassay; and fibrosis, by hydroxyproline assay and trichrome staining. RESULTS: IL-8 accelerated thrombus dissolution 4 days after IVC ligation, with 6-fold increased thrombus blood flow by duplex ultrasound and a 23% increased absolute femoral venous pressure compared with controls (both P < 0.05). These findings may be partially explained by the fact that animals receiving IL-8, as compared with controls, had 2.5-fold greater thrombus neovascularization (with a trend continuing to 8 days) and increased PMN at 4 days. Thrombus vascular endothelial growth factor was significantly reduced at 8 days postligation, while monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha were not altered by IL-8 administration. At 8 days post-IVC-ligation, fibrosis was 12-fold greater with IL-8 treatment compared with controls. CONCLUSIONS: A proinflammatory/proangiogenic thrombus milieu, as conferred by IL-8, enhances thrombus resolution and underscores the important relationship between neovascularity and inflammation.