The orphan tyrosine kinase receptor, ROR2, mediates Wnt5A signaling in metastatic melanoma.

Tyrosine kinase receptors represent targets of great interest for cancer therapy. Here we show, for the first time, the importance of the orphan tyrosine kinase receptor, ROR2, in melanoma progression. Using melanoma tissue microarrays, we show that ROR2 is expressed predominantly in metastatic melanoma. As ROR2 has been shown to specifically interact with the non-canonical Wnt ligand, Wnt5A, this corroborates our earlier data implicating Wnt5A as a mediator of melanoma metastasis. We show here that increases in Wnt5A cause increases in ROR2 expression, as well as the PKC-dependent, clathrin-mediated internalization of ROR2. WNT5A knockdown by siRNA decreases ROR2 expression, but silencing of ROR2 has no effect on WNT5A levels. ROR2 knockdown does, however, result in a decrease in signaling downstream of Wnt5A. Using in vitro and in vivo metastasis assays, we show that ROR2 is necessary for the Wnt5A-mediated metastasis of melanoma cells. These data imply that ROR2 may represent a novel target for melanoma therapy.

Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility.

Serial analysis of gene expression followed by pathway analysis implicated the tight junction protein claudin-1 (CLDN1) in melanoma progression. Tight junction proteins regulate the paracellular transport of molecules, but staining of a tissue microarray revealed that claudin-1 was overexpressed in melanoma, and aberrantly expressed in the cytoplasm of malignant cells, suggesting a role other than transport. Indeed, melanoma cells in culture demonstrate no tight junction function. It has been shown that protein kinase C (PKC) can affect expression of claudin-1 in rat choroid plexus cells, and we observed a correlation between levels of activated PKC and claudin expression in our melanoma cells. To determine if PKC could affect the expression of CLDN1 in human melanoma, cells lacking endogenous claudin-1 were treated with 200 nM phorbol myristic acid (PMA). PKC activation by PMA caused an increase in CLDN1 transcription in 30 min, and an increase in claudin-1 protein by 12 h. Inhibition of PKC signaling in cells with high claudin-1 expression resulted in decreased claudin-1 expression. CLDN1 appears to contribute to melanoma cell invasion, as transient transfection of melanoma cells with CLDN1 increased metalloproteinase 2 (MMP-2) secretion and activation, and subsequently, motility of melanoma cells as demonstrated by wound-healing assays. Conversely, knockdown of CLDN1 by siRNA resulted in the inhibition of motility, as well as decreases in MMP-2 secretion and activation. These data implicate claudin-1 in melanoma progression.

Effects of knee injection on skeletal muscle metabolism and contractile force in rats.

OBJECTIVE: We tested the hypothesis that intrusion of the knee joint capsule alters quadriceps muscle metabolism and function independently from the damage induced to knee cartilage. METHODS: Adult rats were separated into four groups: intraarticular injections of saline (SAL; n=9); intraarticular injections of papain, a model for osteoarthritis (PIA; n=7); sham injections (SHAM; n=8); and controls (CTL; n=5). 31P magnetic resonance spectroscopy (31P-MRS) was performed after 2 weeks. Spectra were obtained from the left quadriceps: two at baseline, eight during electrical stimulation with simultaneous measurement of contractile force, and 15 during recovery. 31P-MRS data were presented as the ratio of inorganic phosphate (Pi) to phosphocreatine (PCr), concentrations of PCr [PCr], intramuscular pH, and the rates and time constants of PCr breakdown during stimulation and PCr recovery. Intramuscular cytokine concentrations were measured within the quadriceps. Histologic slides of the knees were scored for severity of cartilage damage. RESULTS: The interventional groups produced values of Pi/PCr ratio, [PCr], contractile force and pH that were significantly different from CTL. These changes in muscle function were accompanied by higher concentrations of interleukin-1 observed with PIA and SAL. We did not observe any effect of cartilage damage on muscle function or metabolism. CONCLUSIONS: Knee joint intrusion alters quadriceps muscle metabolism with accelerated depletion of energy stores and fatigue during stimulation. This study demonstrates that needle intrusion into the knee joint results in muscle dysfunction, independently from the extent of cartilage damage.

Pharmacologic profiling of transcriptional targets deciphers promoter logic.

The blueprint for cellular diversity and response to environmental change is encoded in the cis-acting regulatory sequences of most genes. Deciphering this 'cis-regulatory code' requires multivariate data sets that examine how these regions coordinate transcription in response to diverse environmental stimuli and therapeutic treatments. We describe a transcriptional approach that profiles the activation of multiple transcriptional targets against combinatorial arrays of therapeutic and signal transducing agents. Application of this approach demonstrates how cis-element composition and promoter context combine to influence transcription downstream of mitogen-induced signaling networks. Computational dissection of these transcriptional profiles in activated T cells uncovers a novel regulatory synergy between IGF-1 and CD28 costimulation that modulates NF-kappaB and AP1 pathways through signaling cascades sensitive to cyclosporin A and wortmannin. This approach provides a broader view of the hierarchical signal integration governing gene expression and will facilitate a practical design of combinatorial therapeutic strategies for exploiting critical control points in transcriptional regulation.

Identification of genes differentially expressed in T cells following stimulation with the chemokines CXCL12 and CXCL10.

BACKGROUND: Chemokines are involved in many biological activities ranging from leukocyte differentiation to neuronal morphogenesis. Despite numerous reports describing chemokine function, little is known about the molecular changes induced by cytokines. METHODS: We have isolated and identified by differential display analysis 182 differentially expressed cDNAs from CXCR3-transfected Jurkat T cells following treatment with CXCL12 or CXCL10. These chemokine-modulated genes were further verified using quantitative RT-PCR and Western blot analysis. RESULTS: One hundred and forty-six of the cDNAs were successfully cloned, sequenced, and identified by BLAST. Following removal of redundant and non-informative clones, seventeen mRNAs were found to be differentially expressed post treatment with either chemokine ligand with several representing known genes with established functions. Twenty-one genes were upregulated in these transfected Jurkat cells following both CXCL12 and CXCL10, four genes displayed a discordant response and seven genes were downregulated upon treatment with either chemokine. Identified genes include geminin (GEM), thioredoxin (TXN), DEAD/H box polypeptide 1 (DDX1), growth hormone inducible transmembrane protein (GHITM), and transcription elongation regulator 1 (TCERG1). Subsequent analysis of several of these genes using semi-quantitative PCR and western blot analysis confirmed their differential expression post ligand treatment. CONCLUSIONS: Together, these results provide insight into chemokine-induced gene activation and identify potentially novel functions for known genes in chemokine biology.

Monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 are involved in both excitotoxin-induced neurodegeneration and regeneration.

Intrahippocamal injections of kainic acid (KA) significantly increase the expression of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) in the ipsilateral hippocampus at 2-4 h and 21-45 days post-administration, suggesting the possible involvement of these chemokines in both neurodegenerative and regenerative processes. To examine the possible role of these chemokines on neuronal cell death, hippocampal neurons were incubated with either MCP-1 or MIP-2 in vitro and examined to assess the effects on neuronal cell viability. These treatments resulted in significant neuronal apoptosis that could be abrogated by prior treatment with the caspase-1 inhibitor, Z-VAD-FMK, the caspase-3 inhibitor, Z-DEVD-FMK, the Galphai inhibitor, pertussis toxin, or the MAO-B inhibitor, (-)deprenyl. Furthermore, this chemokine apoptotic effect could also be observed in vivo as intrahippocampal injections of MCP-1 or MIP-2 resulted in the apoptosis of hippocampal neurons, thus supporting a direct role of these chemokines in neuronal death. In contrast, immunohistological analysis of kainic acid lesions on days 21-45 revealed significant expression of MCP-1 and MIP-2 associated with reactive astrocytes and macrophages, respectively, with no apoptotic populations being observed. These results suggested that these chemokines might also mediate distinct biological effects on local microenvironmental cell populations at various stages post truama and during cellular repair. To address this possibility, astrocyte were cultured in the presence or absence of these chemokines and examined by microarray analysis for effects on astrocytes gene expression. A number of genes encoding proteins associated with inflammation, cellular signaling, differentiation, and repair were directly modulated by chemokine treatment. More specifically, the RNA and protein expression of the neurotrophic factor, basic fibroblast growth factor (bFGF), was found to be significantly increased upon culture with MCP-1 and MIP-2. Conditioned media derived from chemokine-stimulated astrocytes also facilitated bFGF-dependent neuronal cell differentiation and promoted survival of H19-7 neurons in vitro, suggesting a possible role for chemokine-activated astrocytes as a source of trophic support. Taken together, these data support possible autocrine and paracrine roles for MCP-1 and MIP-2 in both the "death and life" of hippocampal neurons following CNS injury.

Regulation of mast cell migration by T and T cytokines: identification of tumour necrosis factor-alpha and interleukin-4 as mast cell chemotaxins.

Mast cells act as central effector and regulatory cells in many inflammatory disorders, including T helper 1 (T(H1))-mediated inflammations such as autoimmunity and T(H2)-mediated inflammations such as allergy and parasite infections. One characteristic for mast cell-mediated inflammations is the accumulation of mast cells in the inflamed tissue. The factors regulating mast cell recruitment in these inflammations are still not fully characterized. We have investigated the potency of T(H1)- and T(H2)-secreted cytokines to mediate mast cell migration. Supernatants from six different T(H1) and T(H2) clones were tested for mast cell-chemotactic activity using the human mast cell line (HMC-1) as a responder cell. All six clones produced factors that induced mast cell migration. Using blocking antibodies to a broad range of cytokines, we found that anti-tumour necrosis factor-alpha (anti-TNF-alpha) reduced the migration of mast cells to supernatants from T(H1) clones. In contrast, the main mast cell chemoattractants secreted by T(H2) clones were found to be interleukin-4 (IL-4) and IL-8. The potency of these cytokines to act as mast cell chemoattractants was confirmed by using recombinant IL-4, IL-8 and TNF-alpha. Our results suggest that TNF-alpha can be involved in the recruitment of mast cells in T(H1)-mediated inflammations, whereas IL-4 and IL-8 might play a similar role in T(H2)-mediated inflammations.

Gene expression profile of herpesvirus-infected T cells obtained using immunomicroarrays: induction of proinflammatory mechanisms.

Herpesvirus infections can frequently lead to acute inflammation, yet the mechanisms regulating this event remain poorly understood. In order to determine some of the immunological mechanisms regulated by human herpesvirus infections, we studied the gene expression profile of lymphocytes infected with human herpesvirus 6 (HHV-6) by using a novel immunomicroarray. Our nylon-based immunomicroarray contained more than 1,150 immune response-related genes and was highly consistent between experiments. Experimentally, we found that independently of the HHV-6 strain used to infect T cells, multiple proinflammatory genes were increased and anti-inflammatory genes were decreased at the mRNA and protein levels. HHV-6 strains A and B increased expression of the genes for interleukin-18 (IL-18), the IL-2 receptor, members of the tumor necrosis factor alpha superfamily receptors, mitogen-activated protein kinase, and Janus kinase signaling proteins. As reported previously, CD4 protein levels were also increased significantly. Specific type 2 cytokines, including IL-10, its receptor, and IL-14, were downregulated by HHV-6 infection and, interestingly, amyloid precursor proteins and type 1 and 2 presenilins. Thus, T cells respond to HHV-6 infection by inducing a type 1 immune response that may play a significant role in the development and progression of diseases associated with HHV-6, including pediatric, hematologic, transplant, and neurologic disorders.

Human recombinant interferon-inducible protein-10: intact disulfide bridges are not required for inhibition of hematopoietic progenitors and chemotaxis of T lymphocytes and monocytes.

Human recombinant interferon-inducible protein-10 (rIP-10), a C-X-C chemokine, inhibits proliferation of human hematopoietic progenitors responsive to co-stimulation by recombinant steel factor (rSLF), is chemotactic for human monocytes and T-lymphocytes, and promotes T-lymphocyte adhesion to endothelial cells. Because chemokines have four conserved cysteines forming two intramolecular disulfide bridges, we decided to investigate their contribution in the biological activity of rIP-10. Since amino acid residues 22-98 of the sequence predicted by the cDNA constitute the naturally occurring IP-10, they were cloned after an initiating methionine into expression vector pET-3d. Subsequently rIP-10 was purified by enzymatic cell lysis, solubilization of refractile bodies with guanidine hydrochloride, renaturation by dialysis against dilute acetic acid, and sequential ion-exchange and reverse-phase high-performance liquid chromatography. Purified rIP-10 was reduced with 20 mM dithiothreitol, and chemically modified with 100 mM iodoacetamide (IAA), or S-methyl-methanethiosulfonate (MMTS), or N-methylmaleimide (NMM). Radiolabeling experiments demonstrated that 95% of the rIP-10 thiols were modified, and this was confirmed with SDS-PAGE. The biological activity of modified rIP-10 was determined in vitro by inhibition of rSLF-responsive human bone marrow hematopoietic progenitor proliferation and by chemotaxis assays using human T-lymphocytes and monocytes. In both assay systems, the biological activity was evident at rIP-10 concentrations of 20-100 ng/ml. The activity was preserved after modification of rIP-10 by IAA or MMTS, but was abolished after modification by NMM. We conclude that disulfide bridges are not essential for the biological activity of rIP-10.

RANTES potentiates antigen-specific mucosal immune responses.

RANTES is produced by lymphoid and epithelial cells of the mucosa in response to various external stimuli and is chemotactic for lymphocytes. The role of RANTES in adaptive mucosal immunity has not been studied. To better elucidate the role of this chemokine, we have characterized the effects of RANTES on mucosal and systemic immune responses to nasally coadministered OVA. RANTES enhanced Ag-specific serum Ab responses, inducing predominately anti-OVA IgG2a and IgG3 followed by IgG1 and IgG2b subclass Ab responses. RANTES also increased Ag-specific Ab titers in mucosal secretions and these Ab responses were associated with increased numbers of Ab-forming cells, derived from mucosal and systemic compartments. Splenic and mucosally derived CD4(+) T cells of RANTES-treated mice displayed higher Ag-specific proliferative responses and IFN-gamma, IL-2, IL-5, and IL-6 production than control groups receiving OVA alone. In vitro, RANTES up-regulated the expression of CD28, CD40 ligand, and IL-12R by Ag-activated primary T cells from DO11.10 (OVA-specific TCR-transgenic) mice and by resting T cells in a dose-dependent fashion. These studies suggest that RANTES can enhance mucosal and systemic humoral Ab responses through help provided by Th1- and select Th2-type cytokines as well as through the induction of costimulatory molecule and cytokine receptor expression on T lymphocytes. These effects could serve as a link between the initial innate signals of the host and the adaptive immune system.

Aged mice exhibit greater mortality concomitant to increased brain and plasma TNF-alpha levels following intracerebroventricular injection of lipopolysaccharide.

BACKGROUND: Age-related defects in the development of peripheral inflammatory responses have been observed in rodents and humans. OBJECTIVE: We examined the effects of age on a centrally injected endotoxin-induced cytokine production and cellular activation in mice. METHODS: Male C57BL/6J (B6) mice, C3H/HeN mice, and C3H/HeJ mice received an intracerebroventricular injection of lipopolysaccharide (LPS) and were sacrificed at various times (2, 4, 8 h) thereafter. ELISA for IL-1beta, IL-6, IL-12, and TNF-alpha were conducted on forebrain tissue homogenates as well as plasma samples, and lectin staining to detect activated microglia was prepared for selected brain slices. RESULTS: Intracerebroventricular injection of LPS in B6 mice produced an age-associated increase in mortality which was paralleled with a significant increase in brain and plasma levels of TNF-alpha. AntiTNF-alpha- and IL-6-immunoreactive cells possessed macrophagelike morphologies and were observed along the LPS injection tract and scattered throughout the hilus of the dorsal hippocampus and cerebral cortices. This LPS-mediated response was found to be specific in that the LPS-hyporesponsive mouse strain (C3H/HeJ) failed to demonstrate significant brain or plasma levels of TNF-alpha after LPS administration compared to C3H/HeN mice. CONCLUSION: These results suggest that the age-related increases in TNF-alpha production and mortality following the intracerebroventricular administration of LPS may be due to an increased endotoxin hypersensitivity of brain microglia/macrophages within aged animals.

Demonstration that platelet-activating factor is capable of activating mast cells and inducing a chemotactic response.

Platelet-activating factor (PAF) is generated in a variety of inflammatory conditions in which mast cells accumulate. However, little is known about the ability of PAF to influence mast cell function directly. In this study we examine the ability of PAF to activate mast cells and regulate mast cell chemotaxis. PAF was found to induce intracellular calcium mobilization and chemotactic responses in both murine and human mast cells. PAF induced transient increases in intracellular Ca2+ concentrations with a 50% effective dose of 1 nM and induced significant migratory responses at PAF concentrations of 1 nM to 1 microM in the human leukaemia mast cell line (HMC-1). Using signal transduction inhibitors, both PAF-induced calcium mobilization and migration of mast cells were shown to require activation of pertussis toxin-sensitive G proteins. PAF-induced calcium and chemotactic responses were cross-desensitized by C5a. Together, these data demonstrate that PAF is capable of activating distinct signalling pathways in mast cells associated with calcium mobilization and cell migration; and that PAF may thus contribute to the regulation of mast cell responses and hyperplasia at sites of inflammation.

Role for CD40-CD40 ligand interactions in the immune response to solid tumours.

CD40-mediated interactions play an important role in the response to infections, transplantation, and cancer by affecting the development, activation, proliferation and differentiation of a variety of immune cells. In the current study we examined the role of CD40-mediated interactions in immune responses to bladder, pancreatic and breast carcinomas as well as melanoma cell lines using soluble human CD40L (rhCD40L) or anti-CD40 mAb in vitro. CD40 expression was readily detected in a large proportion of the cell lines and was augmented but not induced de novo by treatment with IFNgamma. Treatment of CD40-positive cell lines with rhCD40L or anti-CD40mAb enhanced cell surface expression of ICAM-1 and FAS and stimulated the production of IL-6, IL-8, GROalpha, GM-CSF and TNFalpha but not IL-4, IL-10, TGFbeta, MCP-1, RANTES, MIP-1beta, or IP-10. In addition, incubation of CD40+ tumour cell lines with immobilised rhCD40L or anti-CD40 mAb in vitro resulted in significant inhibition of proliferation and a corresponding decrease in viability. This CD40-mediated inhibition of cell growth was due, at least in part, to alterations in cell cycle and the induction of apoptosis. Transfection of CD40-negative tumour cell lines with the cDNA for CD40 conferred responsiveness to rhCD40L and anti-CD40 antibody. Finally, the presence of CD40 on the surface of carcinoma lines was found to be an important factor in the generation of tumour-specific T cell responses.

Early increased chemokine expression and production in murine allogeneic skin grafts is mediated by natural killer cells.

BACKGROUND: Increased expression of chemokine mRNA is observed in allogeneic but not syngeneic skin grafts 3-4 days after transplantation. The recipient cells mediating this early inflammatory response in allografts remain unidentified. METHODS: Isogeneic and allogeneic skin grafts were transplanted to euthymic and athymic nude mice. mRNA expression and protein production of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and the murine homolog of Gro(alpha), i.e. KC, from graft homogenates retrieved 3-4 days posttransplantation was tested by Northern blot hybridization and ELISA. To deplete NK cells, recipients were treated with antiasialo GM1 (ASGM1) antisera or with anti-NK1.1 mAb before transplantation. RESULTS: Expression of KC, MIP-1alpha, and MIP-1beta mRNA was equivalent in C57BL/6 allogeneic skin grafts and BALB/c isografts at day 2 posttransplant. At day 3 posttransplant, chemokine mRNA levels decreased in isografts but were maintained at high levels in the allografts. Increased early chemokine mRNA was also observed in C57BL/6, but not BALB/c++ grafts on BALB/c athymi(nu/nu) recipients. Treatment of allograft recipients with ASGM1 or with anti-NK1.1 antibody eliminated NK cells from the spleen and allograft infiltrating cell populations and decreased early chemokine mRNA levels in allografts 60-70%. Analyses of allograft homogenates indicated increased levels of KC, MIP-1alpha, and MIP-1beta protein at day 4 posttransplant that were decreased in recipients depleted of NK cells. Early chemokine mRNA levels were equivalent in isogeneic and semiallogeneic F1 grafts. CONCLUSIONS: Early chemokine mRNA expression and protein production in allogeneic skin grafts is amplified by recipient natural killer (NK) cells. These results indicate a novel function for infiltrating NK cells in mediating early increased intra-allograft chemokine production and inflammation during the initiation of acute rejection.

Treatment with tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor increases epidermal Langerhans' cell numbers in cancer patients.

Dendritic cells (DCs) initiate primary and stimulate secondary T-cell responses. We conducted a phase I trial of tumor necrosis factor (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with cancer to increase DCs in peripheral blood or skin based on in vitro data that showed that CD34(+) hematopoietic precursors require these cytokines to mature into functional antigen-presenting DCs. Eleven patients were treated for 7 days with GM-CSF, 125 microg/m(2) twice daily as subcutaneous injections, and TNF-alpha as a continuous infusion at dose levels of 25, 50, or 100 microg/m(2)/day. The maximum tolerated dose of TNF-alpha was 50 microg/m(2)/day with this dose of GM-CSF; dose-limiting toxicities occurred in both patients treated with 100 microg/m(2)/day. One became thrombocytopenic and the other had transient confusion. Epidermal Langerhans' cells were quantitated by S100 staining of skin biopsies and DC precursors in peripheral blood by colony-forming unit dendritic (CFU-dendritic) assays. S100-positive cells in the epidermis doubled after treatment (2.55 S100(+) cells/high-power field before treatment to 6.05 after treatment, p = 0.029). CFU-dendritic in peripheral blood increased after treatment in 3 colorectal cancer patients but not in 3 patients with melanoma. CD11c(+) or CD123(+), HLA-DR(bright), lineage-negative dendritic cell precursors were not increased in peripheral blood mononuclear cells. This trial demonstrates that treatment with TNF-alpha and GM-CSF can increase the number of DCs in the skin and the number of dendritic cell precursors in the blood of some patients with cancer. This approach may increase the efficacy of vaccination to tumor antigens in cancer patients.

Immunomodulatory role of C10 chemokine in a murine model of allergic bronchopulmonary aspergillosis.

The immunomodulatory role of the chemokine C10 was explored in allergic airway responses during experimental allergic bronchopulmonary aspergillosis (ABPA). The intratracheal delivery of Asperigillus fumigatus Ag into A. fumigatus-sensitized mice resulted in significantly increased levels of C10 within the bronchoalveolar lavage, and these levels peaked at 48 h after A. fumigatus challenge. In addition, C10 levels in BAL samples were greater than 5-fold higher than levels of other chemokines such as monocyte-chemoattractant protein-1, eotaxin, and macrophage-inflammatory protein-1alpha. From in vitro studies, it was evident that major pulmonary sources of C10 may have included alveolar macrophages, lung fibroblasts, and vascular smooth muscle cells. Experimental ABPA was associated with severe peribronchial eosinophilia, bronchial hyperresponsiveness, and augmented IL-13 and IgE levels. The immunoneutralization of C10 with polyclonal anti-C10 antiserum 2 h before the intratracheal A. fumigatus challenge significantly reduced the airway inflammation and hyperresponsiveness in this model of ABPA, but had no effect on IL-10 nor IgE levels. Taken together, these data suggest that C10 has a unique role in the progression of experimental ABPA.

Cocaine enhances brain endothelial adhesion molecules and leukocyte migration.

Leukocyte infiltration of cerebral vessels in cocaine-associated vasculopathy suggests that cocaine may enhance leukocyte migration. We have investigated cocaine's effects on leukocyte adhesion in human brain microvascular endothelial cell (BMVEC) cultures and monocyte migration in an in vitro blood-brain barrier (BBB) model constructed with BMVEC and astrocytes. Cocaine (10(-5) to 10(-9) M) enhanced adhesion of monocytes and neutrophils to BMVEC. In the BBB model, cocaine (10(-4) to 10(-8) M) enhanced monocyte transmigration. Cocaine increased expression of endothelial adhesion molecules, intercellular adhesion molecule-1 (ICAM-1, CD54), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (ELAM-1) on BMVEC. The peak effect on ICAM-1 expression was between 6 and 18 h after treatment. ICAM-1 was increased by cocaine in BMVEC, but not in human umbilical vein endothelial cells, and the enhancement was greater in a coculture of BMVEC with monocytes. ICAM-1 expression was enhanced by a transcriptional mechanism. Polymyxin B inhibited up-regulation of adhesion molecules by LPS but not by cocaine. In LPS-activated BMVEC/monocyte coculture, cocaine increased secretion of tumor necrosis factor-alpha and interleukin-6. Taken together, these findings indicate that cocaine enhances leukocyte migration across the cerebral vessel wall, in particular under inflammatory conditions, but the effects are variable in different individuals. Cocaine's effects are exerted through a cascade of augmented expression of inflammatory cytokines and endothelial adhesion molecules. These could underlie the cerebrovascular complications of cocaine abuse.

Mast cell migratory response to interleukin-8 is mediated through interaction with chemokine receptor CXCR2/Interleukin-8RB.

To explore the role of chemokines in mast cell chemotaxis and accumulation at sites of inflammation, we first investigated the response of human mast cells to 18 different chemokines by induction of intracellular calcium mobilization in the human mast cell line, HMC-1. Only a subgroup of CXC chemokines defined by the conserved sequence motif glutamic acid-leucine-arginine (ELR) tripeptide motif, which included interleukin-8 (IL-8), growth-regulated oncogene alpha (GROalpha), neutrophil-activating peptide-2 (NAP-2), and epithelial cell-derived neutrophil activating peptide-78 (ENA-78), induced calcium flux in the cells. These observations suggested that the receptor CXCR2 (IL-8RB) should be expressed on the surface of these cells. Using the RNAse protection assay, CXCR2 mRNA, but not CXCR1 (IL-8RA) mRNA expression was detected in HMC-1 cells. Flow cytometry analysis documented the surface expression of CXCR2. A binding analysis performed with 125I-IL-8 determined that there were approximately 3,600 high affinity IL-8 binding sites per HMC-1 cell, with a calculated kd of 1.2 to 2 nmol/L. The activity of this receptor was further explored using IL-8, which was found to induce dose-dependent chemotactic and haptotactic responses in both HMC-1 cells and in vitro cultured human cord blood-derived mast cells. These results show the expression of functional CXCR2 receptors on the surface of human mast cells, which may play an important role in mast cell recruitment during the genesis of an inflammatory response.

Inhibition of human breast carcinoma growth by a soluble recombinant human CD40 ligand.

CD40 is present on B cells, monocytes, dendritic cells, and endothelial cells, as well as a variety of neoplastic cell types, including carcinomas. CD40 stimulation by an antibody has previously been demonstrated to induce activation-induced cell death in aggressive histology human B-cell lymphoma cell lines. Therefore, we wanted to assess the effects of a recombinant soluble human CD40 ligand (srhCD40L) on human breast carcinoma cell lines. Human breast carcinoma cell lines were examined for CD40 expression by flow cytometry. CD40 expression could be detected on several human breast cancer cell lines and this could be augmented with interferon-gamma. The cell lines were then incubated with a srhCD40L to assess effects on in vitro growth. srhCD40L significantly inhibited the proliferation of the CD40(+) human breast cancer cell lines. This inhibition could also be augmented with interferon-gamma. Viability was also affected and this was shown to be due to increased apoptosis of the cell lines in response to the ligand. Treatment of tumor-bearing mice was then performed to assess the in vivo efficacy of the ligand. Treatment of tumor-bearing SCID mice with the ligand resulted in significant increases in survival. Thus, CD40 stimulation by its ligand directly inhibits human breast carcinoma cells in vitro and in vivo. These results suggest that srhCD40L may be of clinical use to inhibit human breast carcinoma growth.