Production Of MIP-1alpha Research Articles

Chronic alcohol intoxication primes kupffer cells and endothelial cells for enhanced CC-chemokine production and concomitantly suppresses phagocytosis and chemotaxis

Chemokines are involved in the pathogenesis of alcoholic hepatitis and are considered to contribute to the migration of leukocytes into the liver during chronic ethanol intoxication. This work tests the hypothesis that chronic ethanol consumption selectively enhances chemokine release by Kupffer cells and hepatic sinusoidal endothelial cells and migration of inflammatory cells into the liver. Furthermore, enhanced hepatic chemokine secretion may induce an autocrine effect on the ability of Kupffer cells and endothelial cells to chemotax and ingest microbial particles. Male Wistar rats were fed with ethanol in agar block and water for 32 weeks, and were allowed free access to solid food. Results show that after 32 weeks of feeding, leukocyte infiltration and steatosis were observed in the livers of ethanol-fed rats. The majority of the infiltrated cells were CD8+ cells. Serum ALT, endotoxin, MIP-1alpha, MCP-1 and RANTES, (but not CINC and MIP-2) were also increased in the ethanol-fed rats than in the pair-fed group. Isolated Kupffer cells from ethanol-fed rats were primed for enhanced MIP-1alpha, MCP-1, and RANTES production in vitro, while the endothelial cells were primed for enhanced MIP-1alpha release only. Chronic alcohol intoxication was also associated with increased basal H2O2 formation, enhanced nuclear translocation and binding of NF-kappaB, AP-1 and MNP-1 in Kupffer Cells. Chronic ethanol feeding significantly enhanced MNP-1 binding, but not those of NF-kappaB and AP-1 in endothelial cells. Concomitantly, chemokine-induced chemotaxis, E.coli phagocytosis and f-met-leu-phe-induced superoxide anion production by Kupffer cells were downregulated in the ethanol-fed group. Taken together these data demonstrate that prolonged alcohol consumption may compromise the host to hepatitis as a result of increased chemokine production and at the same time may suppress the innate immune function of hepatic non-parenchymal cells.

Age-Related Alterations in the Inflammatory Response to Dermal Injury

Previous studies have documented that the ability to heal wounds declines with age. Although many factors contribute to this age-associated deficit, one variable that has not been carefully examined is leukocyte recruitment and function in wounds. This investigation compares the inflammatory response in excisional wounds of young (age 8 wk) and aged (age 22 mo) mice. In the early inflammatory response, neutrophil content of wounds was similar for both aged and young mice. In contrast, macrophage levels were 56% higher in aged versus young mice (81 +/- 20 vs 52 +/- 13 cells per mm2). In the later inflammatory response, wounds of aged mice exhibited a delay in T cell infiltration, with maximum T cell levels at day 10 in aged mice versus day 7 in young mice. Despite this delay, the eventual peak concentration of T cells was 23% higher in the wounds of aged mice (152 +/- 11 cells per mm2 vs 124 +/- 21cells per mm2). The observed alterations in inflammatory cell content suggested that chemokine production might be altered with age. An elevation of monocyte chemoattractant protein (MCP-1) levels was observed in wounds of aged mice. RNase protection studies, however, revealed that the production of most chemokines, including MIP-2, MIP-1alpha, MIP-1beta, and eotaxin, tended to decline with age. Because optimal wound healing requires both appropriate macrophage infiltration and phagocytic activity, phagocytosis was examined. Compared to young mice, wound macrophages from aged mice exhibited a 37%-43% reduction in phagocytic capacity. Taken together, the data demonstrate age-related shifts in both macrophage and T cell infiltration into wounds, alterations in chemokine content, and a concurrent decline in wound macrophage phagocytic function. These alterations may contribute to the delayed repair response of aging.

Activation of microglia and astrocytes by CpG oligodeoxynucleotides.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate cells of the immune system to secrete a variety of cytokines and chemokines. This function can be carried out by microglia and astrocytes in the CNS. To evaluate the effect of CpG ODN on microglia and astrocytes, purified cells were isolated and cultured in vitro. CpG ODN rapidly up-regulated their production of IL-1beta, IL-6, IL-12, TNFalpha, MIP-1alpha and/or MIP-1beta. In vivo, systemically administered CpG ODN up-regulated the expression of mRNA encoding cytokines and chemokines in normal mouse brain. These findings suggest that CpG ODN can directly activate immune cells of the CNS.

HIV-1 infection of placental cord blood monocyte-derived dendritic cells.

Dendritic cells (DC), the most potent antigen-presenting cells (APC), have been implicated as the initial targets of HIV infection in skin and mucosal surfaces. DC can be generated in vitro from blood-isolated CD14(+) monocytes or CD34(+) hematopoietic progenitor cells in the presence of various cytokines. In this study, we investigated whether monocytes obtained from placental cord blood are capable of differentiation into dendritic cells when cultured with a combination of cytokines - granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor necrosis factor-alpha (TNF-alpha). We then examined HIV infection, HIV receptor (CD4, CCR5) expression, and beta-chemokine [macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha, MIP-1beta)] production by placental cord monocyte-derived dendritic cells (MDDC) as compared to that of autologous cord monocyte-derived macrophages (MDM). Monocytes isolated from placental cord blood differentiate into DC after 7 days in culture with the mixture of cytokines, as demonstrated by development of characteristic DC morphology, loss of CD14 expression, and gain of CD83, a marker for mature DC. Mature cord MDDC had significantly lower susceptibility to M-tropic ADA (CCR5-dependent) envelope-pseudotyped HIV infection in comparison to autologous placental cord MDM, whereas there was no significant difference in virus replication in cord MDDC and MDM infected with murine leukemia virus envelope-pseudotyped HIV (HIV receptor-independent). This limited susceptibility of cord MDDC to M-tropic HIV infection may be due to lower expression of CD4 and CCR5 on the cell membrane and higher production of MIP-1alpha and MIP-1beta. These data provide important information toward our understanding of the biological properties of cord MDDC in relation to HIV infection.

Enhanced production of CC-chemokines (RANTES, MCP-1, MIP-1α, MIP-1β, and eotaxin) in patients with atopic dermatitis

CC-chemokines are potent molecules that direct the migration of leukocytes to inflammatory foci. To determine their role in inflammation associated with atopic dermatitis (AD), we determined serum levels and spontaneous production of CC-chemokines by peripheral blood mononuclear cells (PBMC) in AD patients using an ELISA. Serum levels of RANTES, MCP-1, MIP-1beta, and eotaxin were increased in AD patients (n = 52) compared with normal controls (n = 22). Serum levels of RANTES, MCP-1, and MIP-1beta were increased in AD patients with severe disease (n = 19) compared with normal controls (n = 22). Spontaneous production of RANTES, MCP-1, MIP-1alpha and MIP-1beta by PBMC was augmented in AD patients (n = 39) and in patients with severe AD (n = 14) compared with normal controls (n = 20). Serum RANTES levels correlated with total serum IgE levels, eosinophil numbers, and serum lactate dehydrogenase levels. Our results suggest that augmented production of CC-chemokines correlates with inflammation associated with AD.

Activation of beta-chemokines and CCR5 in persons infected with human immunodeficiency virus type 1 and tuberculosis.

Tuberculosis (TB) in human immunodeficiency virus type 1 (HIV-1)-infected persons is associated with progression of HIV-1 disease. The expression of macrophage inflammatory protein (MIP)-1alpha and CCR5 was assessed in HIV-1-infected patients with pulmonary TB (HIV-1/PTB) and without PTB (HIV-1/C), PTB patients not infected with HIV-1 (PTB), and control subjects. Mycobacterium tuberculosis (MTB)-induced MIP-1alpha production was lower in peripheral blood mononuclear cells (PBMC) of HIV-1/PTB patients than in those of PTB patients (P< .05) and was lower in PBMC of HIV-1/C patients than in those of control subjects (P< .005). However, MIP-1alpha production was higher in PBMC of HIV/PTB patients than in those of HIV-1/C patients (P< .01). The pattern of MTB-induced RANTES production was similar to that of MIP-1alpha. However, MTB induced greater expression of mRNA for CCR5 in PBMC of HIV-1/PTB patients than in those of HIV-1/C patients (P< .04). Furthermore, the MTB-induced HIV p24 antigen level in PBMC of HIV-1/PTB patients with a CD4 cell count <500 cells/microL was higher (P< .05) than that in HIV-1/C patients. Thus, perturbations in chemokine pathways in HIV-1/PTB patients may accelerate HIV-1 disease.

Role of CD8+ cell-produced anti-viral factors in protective immunity in HIV-2-exposed but seronegative macaques resistant to intrarectal SIVsm challenge.

The cell-mediated immune response is likely to be important in controlling HIV/SIV infection. There is evidence that beta-chemokines and other, as yet unknown, anti-viral factors play a role in host defence against HIV infection. We reported previously that HIV-2 exposed but seronegative cynomolgus macaques developed SIV-specific cytotoxic T lymphocytes and were resistant to mucosal SIV challenge. The aim of this study was to examine CD8+ cell-dependent production of beta-chemokines and other anti-viral factors in these macaques. The animals, selected from among 17 monkeys enrolled in two separate experiments, were either treated with an anti-viral drug or immunized passively with HIV-2 antibody-positive serum. Three of these monkeys were protected against repeated HIV-2 challenge and were also able to control SIV infection 3 years later. Control samples were obtained from four macaques that became SIV infected and from 39 naïve animals. The three resistant monkeys showed significantly higher production of RANTES and MIP-1alpha than the 39 naïve animals. In addition, SIV infection was suppressed by CD8+ cell culture supernatants of these monkeys. However, antibodies to chemokines only partially neutralized CD8+ cell-mediated SIV suppression indicating that the anti-viral activity observed in these monkeys was the result of combined action of several inhibitory factors.

Higher macrophage inflammatory protein (MIP)-1alpha and MIP-1beta levels from CD8+ T cells are associated with asymptomatic HIV-1 infection.

To test the hypothesis that beta-chemokine levels may be relevant to the control of HIV in vivo, we compared RANTES, MIP-1alpha, and MIP-1beta production from purified CD8(+) T cells from 81 HIV-infected subjects and from 28 uninfected donors. Asymptomatic HIV(+) subjects produced significantly higher levels of MIP-1alpha and MIP-1beta, but not RANTES, than uninfected donors or patients that progressed to AIDS. In contrast, beta chemokines in plasma were either nondetectable or showed no correlation with clinical status. The high beta-chemokine-mediated anti-HIV activity was against the macrophage tropic isolate HIV-1(BAL), with no demonstrable effect on the replication of the T-cell tropic HIV-1(IIIB). These findings suggest that constitutive beta-chemokine production may play an important role in the outcome of HIV-1 infection.

Nitric Oxide Regulates MIP-1α Expression in Primary Macrophages and T Lymphocytes: Implications for Anti-HIV-1 Response

Chemokines and chemokine receptors have been shown to play a critical role in HIV infection. Chemokine receptors have been identified as coreceptors for viral entry into susceptible target cells, and several members of the beta chemokine subfamily of cytokines, MIP-1alpha, MIP-1beta, and RANTES, have been identified as the major human immunodeficiency virus (HIV)-suppressive factors produced by activated CD8+ T lymphocytes. In macrophages, HIV-1 infection itself was shown to upregulate the production of MIP-1alpha and MIP-1beta. In the present study, we address the mechanisms by which HIV-1 infection regulates beta chemokine responses in macrophages and lymphocytes. To address whether nitric oxide (NO), generated as a consequence of HIV-1 infection, regulates beta chemokine responses in monocyte/macrophages and/or macrophage-depleted peripheral blood mononuclear cells (PBMCs) these two cell populations were isolated from HIV seronegative donors, placed in culture, and infected with HIV-1 in either the presence or absence of exogenous activators (e.g. lipopolysaccharide, phytohemagglutinin), inhibitors of nitric oxide synthase (NOS), or chemical donors of NO. Cultures were analyzed for beta chemokine responses by ELISA and RNase protection. LPS-induced MIP-1alpha release is enhanced in HIV-1-infected, as compared to uninfected, monocyte/macrophage cultures, and this enhancing effect is partially blocked by the addition of inhibitors of NOS, and can be reproduced by chemical generators of NO even in the absence of HIV-1 infection. A similar strategy was used to demonstrate a role for NO in HIV-1-mediated induction of MIP-1alpha in unstimulated macrophage cultures. NOS inhibitors also decreased MIP-1alpha and MIP-1beta production by phytohemagglutinin-stimulated monocyte-depleted PBMC cultures. These results indicate that NO amplifies MIP-1alpha responses in activated macrophages and lymphocytes, and suggests that this pleiotropic molecule might function as an enhancing signal that regulates secretion of beta chemokines during HIV-1 infection. These findings reveal a novel mechanism by which NO might regulate the anti-HIV activity of immune cells.

Human osteoblast‐like cells and osteosarcoma cell lines synthesize macrophage inhibitory protein 1α in response to interleukin 1β and tumour necrosis factor α stimulation in vitro

Recent investigations have demonstrated that macrophage inhibitory protein 1alpha (MIP-1alpha) plays a critical role in haematopoiesis. In part, MIP-1alpha limits the differentiation of early haematopoietic cells, thereby ensuring that sufficient quantities of blood precursors are available to meet haematopoietic demands. MIP-1alpha is produced by cells of the marrow microenvironment (marrow stromal cells) in response to a variety of stimuli, including interleukin 1beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha). Our recent investigations demonstrated that normal human osteoblast-like cells (HOBs) maintain the early phenotype of haematopoietic precursors, like other members of the bone marrow stroma. Although the precise molecular mechanisms for these observations have not been determined, the production of MIP-1alpha remains one such possibility. In the present study, we investigated whether cells of the osteoblast lineage under basal, IL-1beta and/or TNF-alpha stimulation produce MIP-1alpha. We observed that IL-1beta and TNF-alpha stimulated HOBs and human osteosarcoma cells to rapidly express MIP-1alpha mRNA and to secrete large quantities of the protein. MIP-1alpha mRNA and protein was not, however, detected under basal conditions. Perhaps more importantly, enriched human CD34+ bone marrow cells in co-culture may be capable of stimulating the expression of MIP-1alpha mRNA by HOBs in vitro. These findings suggest that human osteoblast-like cells may produce MIP-1alpha in vivo to support haematopoiesis at sites where osteoblasts and haematopoietic cells are closely associated.

Chemokine responses and accumulation of inflammatory cells in the lungs of mice infected with highly virulent Cryptococcus neoformans: effects of interleukin-12.

We examined the mechanisms involved in the development of lung lesions after infection with Cryptococcus neoformans by comparing the histopathological findings and chemokine responses in the lungs of mice infected with C. neoformans and assessed the effect of interleukin (IL) 12 which protects mice from lethal infection. In mice infected intratracheally with a highly virulent strain of C. neoformans, the yeast cells multiplied quickly in the alveolar spaces but only a poor cellular inflammatory response was observed throughout the course of infection. Very little or no production of chemokines, including MCP-1, RANTES, MIP-1alpha, MIP-1beta and IP-10, was detected at the mRNA level using RT-PCR as well as at a protein level in MCP-1, RANTES and MIP-1alpha. In contrast, intraperitoneal administration of IL-12 induced the synthesis of these chemokines and a marked cellular inflammatory response involving histiocytes and lymphocytes in infected mice. Our findings were confirmed by flow cytometry of intraparenchymal leukocytes obtained from lung homogenates which showed IL-12-induced accumulation of inflammatory cells consisting mostly of macrophages and CD4+ alphabeta T cells. On the other hand, C-X-C chemokines including MIP-2 and KC, which attract neutrophils, were produced in infected and PBS-treated mice but treatment with IL-12 showed a marginal effect on their level, and neutrophil accumulation was similar in PBS- and IL-12-treated mice infected with C. neoforman. Our results demonstrate a close correlation between chemokine levels and development of lung lesions, and suggest that the induction of chemokine synthesis may be one of the mechanisms of IL-12-induced protection against cryptococcal infection.

Sustained suppression of plasma HIV RNA is associated with an increase in the production of mitogen-induced MIP-1alpha and MIP-1beta.

The chemokine receptor CCR5 has been shown to be a major coreceptor for HIV-1. The chemokines that bind to this receptor (MIP-1alpha, MIP-1beta, and RANTES) are potent inhibitors of HIV replication and may play an important role in the pathophysiology of HIV disease. We investigated the effect of potent antiretroviral therapy (ritonavir and saquinavir) on the production of MIP-1alpha, MIP-1beta, and RANTES in 19 HIV-infected patients who had sustained decreases in plasma HIV RNA levels (<200 copies/ml). Chemokine concentrations were measured in serum, plasma, and PHA-stimulated PBMCs at baseline and 24 and 48 weeks after initiating therapy. MIP-1alpha, MIP-1beta, and RANTES levels in serum and plasma did not significantly change in the 48-week period. In contrast, MIP-1alpha and MIP-1beta secreted by PHA-stimulated PBMCs increased at 24 weeks, with this increase sustained at 48 weeks, whereas no significant change was observed in PHA-induced RANTES production. A significant positive correlation was found between the changes in PHA-induced chemokine production and baseline CD4+ T cell counts. These data demonstrate that sustained suppression of viral replication by potent antiretroviral therapy has a potentially beneficial effect on chemokine production and early initiation of this therapy appears to confer a more favorable chemokine profile.

EXPOSURE TO URBAN AIR PARTICULATES ALTERS THE MACROPHAGE-MEDIATED INFLAMMATORY RESPONSE TO RESPIRATORY VIRAL INFECTION

Epidemiology studies associate increased pulmonary morbidity with episodes of high particulate air pollution (size range 0.1-10 mum diameter, PM10). Pneumonia, often viral in origin, is increased following episodes of high PM10 pollution. Therefore, this study was undertaken to investigate how PM10 alters airway inflammatory responses to respiratory syncytial virus (RSV), a frequent cause of viral pneumonia in infants and the elderly. Supernatants of unexposed and PM10-exposed alveolar macrophage (AM) cultured with uninfected or RSV-infected airway epithelial cells were assessed for a number of chemokines responsible for inflammatory responses in the lung. AM exposure to PM10 in the absence of infection resulted in a significant increase in interleukin (IL)-8 and macrophage inflammatory protein (MIP)-1 apha production but not in MIP-1 beta or monocyte chemotactic protein (MCP)-1. AM responded to RSV infection by the production of IL-8, MIP-1 alpha, MIP-1 beta, and MCP-1, while RANTES was derived solely from the RSV-infected bronchial epithelial cell line BEAS-2B. In the presence of PM10, the AM response to RSV was blunted. RSV-induced MCP-1 was significantly decreased, and the levels of MIP-1 and IL-8 were lower than expected from a combined response to PM10 and RSV. Furthermore, AM analyzed for uptake of virus showed a 50% decrease in viral antigen when exposed to PM10. RSV-induced production of RANTES by epithelial cells was decreased in the presence of AM but not affected by PM10 exposure. Taken together, these results suggest that AM-regulated inflammatory responses to viral infection are altered by exposure to PM10 in a manner that may result in increased spread of infection and thus may increase viral pneumonia-related hospital admissions.

Granulocyte/macrophage-colony-stimulating factor released by adenovirally transduced CT26 cells leads to the local expression of macrophage inflammatory protein 1alpha and accumulation of dendritic cells at vaccination sites in vivo.

Antigen presenting cells (APC) play an essential role in the generation of tumor-specific immune responses. Dendritic cells are the most potent of APC, capable of activating both antigen-specific CD4+ and CD8+ T cells. Previously, we have described how vaccination of mice with irradiated tumor cells producing granulocyte/macrophage-colony-stimulating factor (GM-CSF) induces tumor-specific immunity capable of protecting mice from a subsequent tumor challenge. The present study extends these findings to examine the types of APC infiltrating vaccination sites and the chemokines responsible for their recruitment. GM-CSF released from genetically engineered tumor cells led to the local accumulation of dendritic cells in and around the vaccination site. Quantification revealed a significant ten-fold increase in the number of dendritic cells infiltrating GM-CSF-producing as opposed to beta-galactosidase-producing (control) vaccination sites. Reverse transcription/polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical analysis of vaccination sites revealed that MIP-1alpha may be responsible for dendritic cell infiltration into GM-CSF-producing tissues. These findings suggest that GM-CSF may indirectly recruit dendritic cells into vaccination sites through the local production of MIP-1alpha.

Secretion of beta-chemokines by bronchoalveolar lavage cells during primary infection of macaques inoculated with attenuated nef-deleted or pathogenic simian immunodeficiency virus strain mac251.

Primary infection of macaques with simian immunodeficiency virus (SIV) as a model of human immunodeficiency virus (HIV) infection represents a unique opportunity to investigate early lentivirus-host interactions. In order to gain insight into immunopathogenic events taking place in the lung during lentiviral infection, we analysed lymphocyte expansion in the lung and chemokine secretion by mononuclear cells obtained by bronchoalveolar lavage (BALMCs) during primary infection by a pathogenic and a non-pathogenic SIV. Two groups of cynomolgus macaques were inoculated intravenously with a fully pathogenic isolate of SIVmac251 or with an attenuated, nef-deleted, molecular clone of SIVmac251. Spontaneous MIP-1alpha, MIP-1beta and RANTES production was assessed by ELISA in supernatants of short-term cultured BALMCs. Kinetics of haematological, virological and immunological parameters were investigated simultaneously. All 11 inoculated animals became infected. Monkeys inoculated with the nef-deleted SIV clone exhibited a significantly reduced plasma virus load and a less pronounced accumulation of lymphocytes in the lung compared to monkeys infected with the pathogenic SIVmac251 isolate. Compared to pre-infection levels, we observed an increase in the levels of RANTES, MIP1-alpha and MIP1-beta production in the two groups of monkeys, by the time of peak viraemia. Strikingly, a greater enhancement of RANTES and MIP-1alpha production was detected in monkeys infected with the attenuated virus. Given the potential influence of beta-chemokines on the immune response and virus replication, such results suggest that RANTES, MIP1-alpha and MIP1-beta could contribute to the singular features of the immune response elicited during infection of macaques with an attenuated SIV.

Immunization with Human Immunodeficiency Virus Type 1 rgp120W61Din QS21/MPL Adjuvant Primes T Cell Proliferation and C‐C Chemokine Production to Multiple Epitopes within Variable and Conserved Domains of gp120W61D

Human immunodeficiency virus type 1 (HIV-1) gp120W61D-specific T cell lines (TCL) were generated from an HIV-1-seronegative volunteer who received rgp120W61D in QS21/MPL adjuvant with emulsion. TCL were challenged with pools of consecutive, overlapping peptides spanning the gp120W61D sequence and then with the individual peptides of the immunostimulatory pool. T cell epitopes were found within both variable and conserved domains, and there was no evidence of a single immunodominant epitope. The two most frequently recognized peptides were located in the C1 domain and in the C-terminal region of the V3 loop. Several TCL were shown to recognize multiple peptides from nonoverlapping regions. Peptides from both conserved and variable domains were capable of inducing MIP-1alpha, MIP-1beta, and RANTES production. When tested against the equivalent peptide from the HIV-1IIIB sequence, however, TCL were able to tolerate only minor conserved changes in the amino acid sequence.

Cytokine-induced production of macrophage inflammatory protein-1alpha (MIP-1alpha) in cultured human astrocytes.

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a member of a superfamily of inflammatory cytokines termed chemokines, and it has been implicated in the pathogenesis of several human diseases with inflammatory components. It has been known that MIP-1alpha plays a role in recruiting and activating mononuclear phagocytes in the central nervous system (CNS), and that astrocytes and microglia are sources of this chemokine. However, details of the regulation of MIP-1alpha production by these glial cells are not known. In the present study, expression of MIP-1alpha was determined in purified cultures of human astrocyte. MIP-1alpha mRNA levels in human astrocyte cell preparations were determined by reverse transcription polymerase chain reaction (RT-PCR) and amount of MIP-1alpha protein secreted into culture supernatants by human astrocytes was assayed by enzyme-linked immunosorbent assay (ELISA). Under the unstimulated conditions, human astrocytes did not express MIP-1alpha message or protein, indicating that human astrocytes do not constitutively carry MIP-1alpha message. Following treatment with interleukin-1beta (IL-1beta), human astrocytes demonstrated increased message and protein expression for MIP-1alpha, while other immune modulators such as interferon-gamma (IFN)-gamma, tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), lipopolysaccharide, or phorbol ester (a protein kinase C activator) did not induce MIP-1alpha expression in human astrocytes.

Induction of macrophage inflammatory protein-1alpha and vascular endothelial growth factor during inflammatory neovascularization in the mouse cornea.

We predicted that the appearance of macrophages in inflammatory areas is necessary for angiogenic responses in various inflammatory diseases. Using a mouse inflammatory corneal model in which model mouse corneas were cauterized with silver nitrate, we examined the infiltration of macrophages immunohistochemically and the total area of neovascularization quantitively. The expression of macrophage inflammatory protein-1alpha (MIP-1alpha) and vascular endothelial growth factor (VEGF) levels were also examined. A day after cauterization, short capillaries began to develop into the corneal stroma, and after 4 or 5 days the neovascularization became maximal and then began to regress. The number of macrophages within the cauterized cornea increased to a maximum at day 3 and began to decrease at day 5. The number of infiltrated macrophages reached maximum at day 3. Both MIP-1alpha and VEGF protein levels increased markedly immediately after the chemical cauterization, and production of MIP-1alpha (85.8 pg/4 corneas) and VEGF (206.5 pg/4 corneas) was maximal at 1 day and 0.5 day after cauterization, respectively. MIP-1alpha and VEGF mRNA levels also increased at 0.5 day after cauterization. In situ hybridization showed that MIP-1alpha was localized in corneal epithelial cells, and VEGF was localized in corneal epithelial cells and infiltrating inflammatory cells. MIP-1alpha and VEGF may have an important role in recruiting macrophages and neovascularization.

Differential T cell responses to Mycobacterium tuberculosis ESAT6 in tuberculosis patients and healthy donors.

Vaccination against and diagnosis of tuberculosis are still insufficient. Proteins secreted by Mycobacterium tuberculosis induce strong immune responses in tuberculosis and constitute prime candidates for development of novel vaccines against tuberculosis as well as for immunodiagnostic assays. We investigated the role of the secreted proteins MPT63, MPT64 and ESAT6 from M. tuberculosis in healthy individuals and tuberculosis patients. None of the secreted proteins stimulated peripheral blood mononuclear cells from healthy donors. In contrast, CD4+ T cells from many tuberculosis patients were stimulated in an MHC class II-restricted fashion by ESAT6, but not by MPT63 or MPT64. T cell reactivities of tuberculosis patients were focused on the N-terminal region of ESAT6. The ESAT6 T cell epitopes were presented by different HLA-DR phenotypes. Cell cultures responding to either ESAT6 or synthetic peptides thereof showed mRNA transcripts for macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic protein (MCP)-1 or IL-8 and production of IFN-gamma and MIP-1alpha. Our results suggest that the secreted M. tuberculosis proteins MPT63, MPT64 or ESAT6 do not stimulate unprimed T cells, and that ESAT6 may be a potential candidate antigen for detection of clinical disease.

Characterization of the response of human bone marrow endothelial cells to in vitro irradiation.

Endothelial cell dysfunction is a classic consequence of radiation damage. Bone marrow endothelial cells (BMEC) are a critical component of the stroma in the regulation of haemopoiesis. In animal models, radiation-induced injury of BMEC has been described and a role for BMEC in haemopoietic regeneration after irradiation has been suggested. However, functions of BMEC involved in the haemopoietic regeneration have not been assessed. Therefore we studied the functional response of human BMEC to irradiation using the transformed human BMEC line (TrHBMEC) irradiated with 2. 5 or 10Gy. Our results showed a time- and a dose-dependent increase in damage to irradiated TrHBMEC measured by a decreased number of adherent cells which correlated with increased apoptosis and augmented release of soluble ICAM-1 and von Willebrand factor. 2 Gy irradiated TrHBMEC expressed more ICAM-1 on their surface than non-irradiated cells, whereas no change in VCAM-1, E-selectin and PECAM-1 expression was observed. An increased production of G-CSF, GM-CSF, IL-8, IL-6, IL-1alpha, IL-11, MIP-1alpha and SCF and no production of LIF, TNF-alpha, TPO and IL-3 by 2 Gy irradiated TrHBMEC was observed. The haemopoietic supportive function of TrHBMEC was not altered after a 2 Gy exposure. These results suggest that although radiation induces endothelial cell damage, irradiated cells still support the proliferation and the differentiation of CD34+ haemopoietic cells.