Pregnancy complications are frequent in long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency

Hemofiltrate C-C chemokine (HCC)-1 is a recently cloned C-C chemokine that is structurally similar to macrophage inflammatory protein (MIP)-1alpha. Unlike most chemokines, it is constitutively secreted by tissues and is present at high concentrations in normal human plasma. Also atypical for chemokines, HCC-1 is reported not to be chemotactic for leukocytes. In this paper, we have investigated the chemokine receptor usage and downstream signaling pathways of HCC-1. Cross-desensitization experiments using THP-1 cells suggested that HCC-1 and MIP-1alpha activated the same receptor. Experiments using a panel of cloned chemokine receptors revealed that HCC-1 specifically activated C-C chemokine receptor (CCR)1, but not closely related receptors, including CCR5. HCC-1 competed with MIP-1alpha for binding to CCR1-transfected cells, but with a markedly reduced affinity (IC50 = 93 nM versus 1.3 nM for MIP-1alpha). Similarly, HCC-1 was less potent than MIP-1alpha in inducing inhibition of adenylyl cyclase in CCR1-transfected cells. HCC-1 induced chemotaxis of freshly isolated human monocytes, THP-1 cells, and CCR1-transfected cells, and the optimal concentration for cell migration (100 nM) was approximately 100-fold lower than that of MIP-1alpha (1 nM). These data demonstrate that HCC-1 is a chemoattractant and identify CCR1 as a functional HCC-1 receptor on human monocytes.     

Genetic susceptibility testing: ethical and social quandaries

We have isolated a novel human C-C chemokine, MIP-1 delta from a human fetal spleen cDNA library. The human MIP-1 delta cDNA has an unusually long 400-bp 5-prime untranslated region and a predicted 113-amino acid protein of 10 kDa. The coding sequence contains a signal peptide of 21 amino acids, indicating that the mature protein has 92 amino acids (8 kDa). Recombinant human MIP-1 delta produced by transfected human embryonic kidney 293 cells produced an 8-kDa protein, which confirmed the presence of a signal peptide. Compared with other human C-C chemokines, human MIP-1 delta shows the highest homology with human HCC-1, CK beta-8, murine C10, and CCF18 (MIP-1 gamma). The human MIP-1 delta gene is localized on chromosome 17 where most of the C-C chemokine superfamily is located. Human MIP-1 delta is expressed in T and B lymphocytes, NK cells, monocytes, and monocyte-derived dendritic cells, but not in bone marrow-derived dendritic cells. Its expression can be induced by other proinflammatory cytokines in monocytes and dendritic cells. Human MIP-1 delta is chemotactic for T cells and monocytes, but not for neutrophils, eosinophils, or B cells. Human MIP-1 delta induced calcium flux in human CCR1-transfected cells.     

Predisposition to essential hypertension and development of diabetic nephropathy in IDDM patients

The immunosuppressive and antiinflammatory cytokine interleukin (IL) 10 selectively upregulates the expression of the CC chemokine receptors CCR5, 2, and 1 in human monocytes by prolonging their mRNA half-life. IL-10-stimulated monocytes display an increased number of cell surface receptors for, and better chemotactic responsiveness to, relevant agonists than do control cells. In addition, IL-10-stimulated monocytes are more efficiently infected by HIV BaL. This effect was associated to the enhancement of viral entry through CCR5. These data add support to an emerging paradigm in which pro- and antiinflammatory molecules exert reciprocal and opposing influence on chemokine agonist production and receptor expression.     

Identification of RANTES receptors on human monocytic cells: competition for binding and desensitization by homologous chemotactic cytokines

RANTES (regulated on activation, normal T expressed and secreted) is a member of the chemotactic cytokine (chemokine) beta subfamily. High affinity receptors for RANTES have been identified on a human monocytic leukemia cell line THP-1, which responded to RANTES in chemotaxis and calcium mobilization assays. Steady-state binding data analyses revealed approximately 700 binding sites/cell on THP-1 cells with a Kd value of 400 pM, comparable to that expressed on human peripheral blood monocytes. The RANTES binding to monocytic cells was competed for by monocyte chemotactic and activating factor (MCAF) and macrophage inflammatory protein 1 (MIP-1) alpha, two other chemokine beta cytokines. Although MCAF and MIP-1 alpha competed for RANTES binding to monocytes with apparent lower affinity (with estimated Kd of 6 and 1.6, nM respectively) both of these cytokines effectively desensitized the calcium mobilization induced by RANTES. The chemotactic response of THP-1 cells to RANTES was also markedly inhibited by preincubation with MCAF or MIP-1 alpha. In contrast, RANTES did not desensitize the THP-1 calcium mobilization and chemotaxis in response to MCAF or MIP-1 alpha. These results, together with our previous observations that RANTES did not compete for MCAF or MIP-1 alpha binding on monocytic cells, indicate the expression of promiscuous receptors on monocytes that recognize one or more cytokines within the chemokine beta family.     

Expression of monocyte chemotactic protein-3 in human monocytes exposed to the mycobacterial cell wall component lipoarabinomannan

Monocyte chemotactic protein-3 (MCP-3) is a C-C chemokine which interacts with the CCR1, CCR2 (MCP-1) and CCR3 receptors and has a distinct spectrum of action. The present study was designed to assess whether mycobacterial components were able to induce expression and production of MCP-3 in human monocytes. Mycobacterial lipoarabinomannan (LAM) induced expression of MCP-3 mRNA in human peripheral blood mononuclear cells. The non-mannose-capped version of lipoarabinomannan (AraLAM) was considerably more potent than the mannose-capped version ManLAM or the simpler version phosphatidylinositol mannoside (PLM). Among mononuclear cells, monocytes were responsible for LAM-induced MCP-3 mRNA expression. Whole mycobacteria (Mycobacterium bovis BCG) strongly induced MCP-3 expression. Pretreatment with actinomycin D abolished LAM-induced MCP-3 expression, whereas cycloheximide only partially reduced the expression. LAM-induced MCP-3 expression was associated with the production of immunoreactive PTX3. Interleukin 10 (IL-10) and IL-13 inhibited the induction of MCP-3 by LAM. Thus mycobacterial cell wall components induced expression of MCP-3 in human monocytes. MCP-3, a chemokine active on mononuclear phagocytes, NK cells, T cells and dendritic cells, may be relevant to the induction and expression of immunity against mycobacteria.     

Treatment of vaginal infections: candidiasis, bacterial vaginosis, and trichomoniasis

Chemokines contribute to the inflammatory response by selective attraction of various leukocytic cell types. Human GCP-2 was originally identified by amino acid sequence analysis as a CXC chemokine co-produced with IL-8 by osteosarcoma cells. Furthermore, the complete coding domain of human GCP-2 was disclosed by means of RT-PCR. Similarly, mouse GCP-2 was isolated from fibroblastoid and epithelial cells and completely identified by sequence analysis. Human and mouse GCP-2 share 61% identical amino acids. Both chemokines occur as multiple NH2-terminally truncated forms. The shorter forms of mouse, but not those of human, GCP-2 showed a higher neutrophil chemotactic potency and gelatinase B releasing capacity. Mouse GCP-2 was a more potent neutrophil activator than human GCP-2, natural mouse KC, and MIP-2. Human GCP-2 was not chemotactic for monocytes, lymphocytes, or eosinophils. Quantitative studies of mRNA expression in diploid fibroblasts revealed GCP-2 induction by IL-1beta. Human GCP-2 induced [Ca2+]i increase in neutrophils, which was reciprocally desensitized by IL-8, GROalpha, and ENA-78. Human GCP-2 induced [Ca2+]i increases and chemotactic responses in both CXCR1- and CXCR2-transfected cells. Finally, GCP-2 provoked neutrophil accumulation and plasma extravasation in rabbit skin. In humans, GCP-2 complements the activity of IL-8 as neutrophil chemoattractant and activator but it constitutes a major neutrophil chemokine in the mouse. GCP-2 induces neutrophil chemotaxis and activation but it might also contribute to detrimental tissue damage in sepsis, acute respiratory distress syndrome, acute hypersensitivity reactions, and autoimmune diseases. It might also influence the invasive capacity of GCP-2-secreting tumor cells.     

Evidence for the presence of a kininogen-like species in a case of total deficiency of low and high molecular weight kininogens

EBI1-ligand chemokine (ELC) is a CC chemokine constitutively expressed in various lymphoid tissues and a high-affinity functional ligand for EBI1/CCR7, a seven transmembrane G-protein-coupled receptor originally identified as an Epstein-Barr virus (EBV)-inducible gene. Here we examined chemotactic activity of ELC on peripheral blood leukocytes. ELC attracted both CD4+ and CD8+ T cells, particularly efficiently after activation with IL-2 or with phytohemagglutinin (PHA) plus IL-2, as well as CD19+ B cells, but not CD16+ NK cells, CD14+ monocytes or neutrophils. Among CD3+ T cells, ELC attracted both CD45RO- naive and CD45RO+ memory subsets. ELC also induced vigorous calcium mobilization in T cells stimulated with IL-2 with an ED50 of 3 nM. ELC fused with the secreted form of alkaline phosphatase (ELC-SEAP) specifically bound to lymphocytes and this binding was blocked only by ELC among 10 CC chemokines so far tested. Notably, lymphocytes stimulated with IL-2 or T cells expanded by PHA plus IL-2 showed much higher levels of binding than fresh lymphocytes. Consistently, CCR7 mRNA was detected in CD4+ and CD8+ T cells as well as B cells, but not in NK cells, monocytes or neutrophils, and was dramatically increased in T cells upon treatment with IL-2 or with PHA plus IL-2. Like ELC mRNA, CCR7 mRNA was expressed in various lymphoid tissues. By in situ hybridization, ELC and CCR7 mRNA were detected in the parafollicular and inner cortical regions of a lymph node, and in the parafollicular regions of an appendix. Collectively, ELC and CCR7 may be involved in the trafficking of a broad spectrum of lymphocytes, especially activated T cells, into and within various lymphoid tissues.     

Human Mig chemokine: biochemical and functional characterization

Mig is a chemokine of the CXC subfamily that was discovered by differential screening of a cDNA library prepared from lymphokine-activated macrophages. The mig gene is inducible in macrophages and in other cells in response to interferon (IFN)-gamma. We have transfected Chinese hamster ovary (CHO) cells with cDNA encoding human Mig and we have derived CHO cell lines from which we have purified recombinant human Mig (rHuMig). rHuMig induced the transient elevation of [Ca2+]i in human tumor-infiltrating T lymphocytes (TIL) and in cultured, activated human peripheral blood-derived lymphocytes. No responses were seen in human neutrophils, monocytes, or Epstein-Barr virus-transformed B lymphoblastoid cell lines. rHuMig was chemotactic for TIL by a modified Boyden chamber assay but rHuMig was not chemotactic for neutrophils or monocytes. The CHO cell lines, IFN-gamma-treated human peripheral-blood monocytes, and IFN-gamma-treated cells of the human monocytic cell line THP-1 all secreted multiple and identical HuMig species as revealed by SDS-PAGE. Using the CHO-derived rHuMig, we have shown that the species' heterogeneity is due to proteolytic cleavage at basic carboxy-terminal residues, and that the proteolysis occurs before and not after rHuMig secretion by the CHO cells. The major species of secreted rHuMig ranged from 78 to 103 amino acids in length, the latter corresponding to the full-length secreted protein predicted from the HuMig cDNA. Carboxy-terminal-truncated forms of rHuMig were of lower specific activity compared to full-length rHuMig in the calcium flux assay, and the truncated species did not block the activity of the full-length species. It is likely that HuMig plays a role in T cell trafficking and perhaps in other aspects of the physiology of activated T cells.     

Enhanced anti-HIV-1 activity and altered chemotactic potency of NH2-terminally processed macrophage-derived chemokine (MDC) imply an additional MDC receptor

Posttranslational processing of chemokines increases (IL-8) or decreases (monocyte chemotactic protein-1) their chemotactic potency. Macrophage-derived chemokine (MDC) attracts monocytes, dendritic cells, activated lymphocytes, and NK cells and has reportedly anti-HIV-1 activity. Here we report that truncation of MDC by deletion of two NH2-terminal residues resulted in impaired binding to CC chemokine receptor (CCR)4, the only identified MDC receptor so far. Truncated MDC(3-69) failed to desensitize calcium mobilization by MDC(1-69) or thymus- and activation-regulated chemokine (TARC), another CCR4 ligand. MDC(3-69) lacked HUT-78 T cell chemotactic activity but retained its capacity to attract monocytes and to desensitize chemotaxis. Compared with MDC(1-69), MDC(3-69) had weak but enhanced antiviral activity against M- and T-tropic HIV-1 strains. Furthermore, both MDC forms failed to signal through the orphan receptors Bonzo/STRL33 and BOB/GPR15 and to desensitize RANTES and stromal cell-derived factor (SDF)-1 responses in CCR5-transfected and CXC chemokine receptor (CXCR)4-transfected cells, respectively. These findings suggest that MDC recognizes another, yet unidentified, receptor. We conclude that minimal NH2-terminal truncation of MDC differentially affects its various immunologic functions.     

Alternative macrophage activation-associated CC-chemokine-1, a novel structural homologue of macrophage inflammatory protein-1 alpha with a Th2-associated expression pattern

We have cloned a novel human CC-chemokine, alternative macrophage activation-associated CC-chemokine (AMAC)-1. The isolated cDNA clone (803 bp) shows a single open reading frame of 267-bp coding for 89 amino acid residues; mature AMAC-1 protein is predicted to consist of 69 amino acids with a m.w. of 7855. Sequence alignment and 3D-modeling show the typical structural characteristics of CC-chemokines with special features in the receptor-activating domain. AMAC-1 is most closely related to MIP-1 alpha with a cDNA and protein sequence homology of 55% and 59%, respectively. However, the expression pattern of AMAC-1 is directly opposite to that of MIP-1 alpha. While MIP-1 alpha is induced by classical macrophage mediators such as LPS and is inhibited by IL-4 and glucocorticoids, AMAC-1 is specifically induced in macrophages by alternative macrophage mediators such as IL-4, IL-13, and IL-10. Expression of AMAC-1 is inhibited by IFN-gamma while glucocorticoids exert a slightly positive synergistic effect in combination with IL-4. Peripheral blood monocytes do not express AMAC-1; time course experiments show that monocyte-to-macrophage differentiation is a prerequisite for AMAC-1 expression. Expression of AMAC-1 by granulocyte-macrophage CSF/IL-4-induced, monocyte-derived dendritic cells is complex; in mature adherent dendritic cells, however, only minor AMAC-1 mRNA expression was found. In vivo, AMAC-1 is expressed by alveolar macrophages from healthy persons, smokers, and asthmatic patients. In conclusion, AMAC-1 is a novel CC-chemokine whose expression is induced in alternatively activated macrophages by Th2-associated cytokines; thus, AMAC-1 may be involved in the APC-dependent T cell development in inflammatory and immune reactions.     

An inter district quality partnership: the experience of a large rural health service

The interleukin-2 receptor (IL-2R) gamma chain (gamma c chain) is shared by IL-4R, IL-7R, IL-9R, and IL-15R and plays an important role in regulation of the immune system. However, its regulation in monocytic cell lines has not been well clarified. We examined the expression and regulation of the IL-2R alpha, IL-2R beta, gamma c chain, IL-4R and IL-7R mRNA in a human monoblastic leukemia cell line, THP-1. Unstimulated THP-1 cells constitutively expressed a low level of gamma c chain and IL-4R mRNA. Phorbol myristate acetate (PMA) induced macrophage-like differentiation and up-regulated the gamma c chain mRNA expression in THP-1 cells. This effect of PMA was suppressed by the protein kinase inhibitors H-7 and staurosporine. PMA did not affect the expression of the other IL-R mRNAs examined. 1 alpha, 25(OH)2D3 and interferon-gamma also induced differentiation of THP-1 cells, but these reagents did not affect the expression of the IL-R mRNAs in THP-1 cells. These findings suggest that the expression of the gamma c chain mRNA is regulated by the PMA-dependent pathway and is not associated with that of the other IL-R mRNAs.     

Posttranslational modifications affect the activity of the human monocyte chemotactic proteins MCP-1 and MCP-2: identification of MCP-2(6-76) as a natural chemokine inhibitor

Chemokines are important mediators in infection and inflammation. The monocyte chemotactic proteins (MCPs) form a subclass of structurally related C-C chemokines. MCPs select specific target cells due to binding to a distinct set of chemokine receptors. Recombinant and synthetic MCP-1 variants have been shown to function as chemokine antagonists. In this study, posttranslationally modified immunoreactive MCP-1 and MCP-2 were isolated from mononuclear cells. Natural forms of MCP-1 and MCP-2 were biochemically identified by Edman degradation and mass spectrometry and functionally characterized in chemotaxis and Ca2+-mobilization assays. Glycosylated MCP-1 (12 and 13.5 kDa) was found to be two- to threefold less chemotactic for monocytes and THP-1 cells than nonglycosylated MCP-1 (10 kDa). Natural, NH2-terminally truncated MCP-1(5-76) and MCP-1(6-76) were practically devoid of bioactivity, whereas COOH-terminally processed MCP-1(1-69) fully retained its chemotactic and Ca2+-inducing capacity. The capability of naturally modified MCP-1 forms to desensitize the Ca2+ response induced by intact MCP-1 in THP-1 cells correlated with their agonistic potency. In contrast, naturally modified MCP-2(6-76) was devoid of activity, but could completely block the chemotactic effect of intact MCP-2 as well as that of MCP-1, MCP-3, and RANTES. Carboxyl-terminally processed MCP-2(1-74) did retain its chemotactic potency. Although comparable as a chemoattractant, natural intact MCP-2 was found to be 10-fold less potent than MCP-1 in inducing an intracellular Ca2+ increase. It can be concluded that under physiologic or pathologic conditions, posttranslational modification affects chemokine potency and that natural MCP-2(6-76) is a functional C-C chemokine inhibitor that might be useful as an inhibitor of inflammation.     

Cell-associated human retinal pigment epithelium interleukin-8 and monocyte chemotactic protein-1: immunochemical and in-situ hybridization analyses

Human retinal pigment epithelial (RPE) cells secrete chemokines, interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in response to pro-inflammatory cytokines. In this study we (1) examined the efficiency of human RPE IL-8 and MCP-1 secretion, (2) determined the amount of neutrophil and monocyte chemotactic activity in human RPE cell conditioned media and cell extracts that is attributable to IL-8 and MCP-1, respectively, and (3) assessed the sensitivity of immunohistochemistry and in situ hybridization for detecting chemokine production by cytokine-stimulated human RPE cells. Conditioned media and extracts from human RPE cells stimulated with various physiologic concentrations of interleukin-1 beta (IL-1 beta) (0.2-20 ng ml-1), tumor necrosis factor (TNF-alpha) (0.2-20 ng ml-1) or interferon-gamma (IFN-gamma) (10-1000 U ml-1) were examined to compare secreted and cell associated levels of IL-8 and MCP-1 at various time points up to 24 hr. ELISA demonstrated that IL-8 and MCP-1 are both efficiently secreted by pro-inflammatory cytokine treated human RPE cells. Substantial dose- and time-dependent RPE secretion of IL-8 was observed following stimulation with IL-1 beta or TNF-alpha, but cell associated IL-8 was detectable only after high dose (20 ng ml-1) IL-1 beta stimulation and comprised less than 1% of the total IL-8 induced. Dose- and time-dependent RPE cell MCP-1 secretion was also observed following IL-1 beta > TNF-alpha > IFN-gamma stimulation, with an average of 4% of the total MCP-1 retained within RPE. Bioassays demonstrated neutrophil and monocyte chemotactic activity in conditioned media from stimulated RPE cells, but not in human RPE cell extracts. Inhibition of conditioned media-induced chemotaxis by specific anti-IL-8 or anti-MCP-1 antibodies demonstrated that IL-8 and MCP-1 were responsible for the majority of HRPE-derived neutrophil (> 60%) and monocyte (53-57%) chemotactic activity, respectively. Using in situ hybridization IL-8 mRNA was readily detected within IL-1 beta > TNF-alpha stimulated RPE cells and MCP-1 mRNA easily visualized within IL-1 beta > TNF-alpha > or IFN-gamma stimulated cells. Immunohistochemistry to detect IL-8 was positive only in RPE cells exposed to high dose IL-1 beta (20 ng ml-1) for 8 or 24 hr and was weak. Immunohistochemical staining for MCP-1 in RPE cells was more intense and was visualized within RPE cells stimulated with IL-beta, TNF-alpha, or IFN-gamma. This study demonstrates that: (1) RPE cells efficiently secrete IL-8 and MCP-1 upon stimulation with pro-inflammatory cytokines; (2) secreted IL-8 and MCP-1 account for the majority of human RPE neutrophil and monocyte chemotactic activity; (3) in situ hybridization readily detects IL-8 and MCP-1 mRNA in cytokine stimulated RPE cells; and (4) immunohistochemistry demonstrates cell-associated MCP-1 in cytokine stimulated RPE cells, but only minimal cell-associated IL-8.