Multiple products derived from two CCL4 loci: high incidence of a new polymorphism in HIV+ patients.

Human CCL4/macrophage inflammatory protein (MIP)-1beta and CCL3/MIP-1alpha are two highly related molecules that belong to a cluster of inflammatory CC chemokines located in chromosome 17. CCL4 and CCL3 were formed by duplication of a common ancestral gene, generating the SCYA4 and SCYA3 genes which, in turn, present a variable number of additional non-allelic copies (SCYA4L and SCYA3L1). In this study, we show that both CCL4 loci (SCYA4 and SCYA4L) are expressed and alternatively generate spliced variants lacking the second exon. In addition, we found that the SCYA4L locus is polymorphic and displays a second allelic variant (hereinafter SCYA4L2) with a nucleotide change in the intron 2 acceptor splice site compared with the one described originally (hereinafter SCYA4L1). Therefore, the pattern of SCYA4L2 transcripts is completely different from that of SCYA4L1, since SCYA4L2 uses several new acceptor splice sites and generates nine new mRNAs. Furthermore, we analyzed the contribution of each locus (SCYA4 and SCYA4L1/L2) to total CCL4 expression in human CD8 T cells by RT-amplified fragment length polymorphism and real-time PCR, and we found that L2 homozygous individuals (L2L2) only express half the levels of CCL4 compared with L1L1 individuals. The analysis of transcripts from the SCYA4L locus showed a lower level in L2 homozygous compared with L1 homozygous individuals (12% vs 52% of total CCL4 transcripts). A possible clinical relevance of these CCL4 allelic variants was suggested by the higher frequency of the L2 allele in a group of HIV(+) individuals (n = 175) when compared with controls (n = 220, 28.6% vs 16.6% (p = 0.00016)).

Regulated production and molecular diversity of human liver and activation-regulated chemokine/macrophage inflammatory protein-3 alpha from normal and transformed cells.

Liver and activation-regulated chemokine (LARC), also designated macrophage inflammatory protein-3alpha (MIP-3alpha), Exodus, or CCL20, is a C-C chemokine that attracts immature dendritic cells and memory T lymphocytes, both expressing CCR6. Depending on the cell type, this chemokine was found to be inducible by cytokines (IL-1beta) and by bacterial, viral, or plant products (including LPS, dsRNA, and PMA) as measured by a specific ELISA. Although coinduced with monocyte chemotactic protein-1 (MCP-1) and IL-8 by dsRNA, measles virus, and IL-1beta in diploid fibroblasts, leukocytes produced LARC/MIP-3alpha only in response to LPS. However, in myelomonocytic THP-1 cells LARC/MIP-3alpha was better induced by phorbol ester, whereas in HEp-2 epidermal carcinoma cells IL-1beta was the superior inducer. The production levels of LARC/MIP-3alpha (1-10 ng/ml) were, on the average, 10- to 100-fold lower than those of IL-8 and MCP-1, but were comparable to those of other less abundantly secreted chemokines. Natural LARC/MIP-3alpha protein isolated from stimulated leukocytes or tumor cell lines showed molecular diversity, in that NH(2)- and COOH-terminally truncated forms were purified and identified by amino acid sequence analysis and mass spectrometry. In contrast to other chemokines, including MCP-1 and IL-8, the natural processing did not affect the calcium-mobilizing capacity of LARC/MIP-3alpha through its receptor CCR6. Furthermore, truncated natural LARC/MIP-3alpha isoforms were equally chemotactic for lymphocytes as intact rLARC/MIP-3alpha. It is concluded that in addition to its role in homeostatic trafficking of leukocytes, LARC/MIP-3alpha can function as an inflammatory chemokine during host defense.

High-level secretion of biologically active recombinant human macrophage inflammatory protein-1alpha by the methylotrophic yeast Pichia pastoris.

The human CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) was produced at a high level in Pichia pastoris under transcriptional control of the highly inducible alcohol oxidase 1 promoter. To ensure proper folding and secretion of the recombinant polypeptide, the MIP-1alpha gene had been fused to the Saccharomyces cerevisiae alpha-factor prepropeptide. As was revealed by analysis of the cell culture supernatant of recombinant Pichia pastoris, MIP-1alpha was efficiently secreted. Immunoblot analysis of secreted proteins from recombinant clones using a polyclonal antibody directed against MIP-1alpha revealed an apparent molecular mass of 8 kDa for the recombinant polypeptide. Up to 70 mg of MIP-1alpha was purified from 1 liter of yeast culture supernatant by a single chromatography step. Biological activity of recombinant MIP-1alpha was shown in a chemotaxis assay. Here, the polypeptide specifically induced migration of U937 cells expressing the CCR1 (MIP-1alpha receptor). Also, in competition binding assays the recombinant MIP-1alpha displayed high affinity binding.

Structure and function of the glycosaminoglycan binding site of chemokine macrophage-inflammatory protein-1 beta.

The ability of chemokines to bind to glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix is thought to play a crucial role in chemokine function. We investigated the structural basis for chemokine binding to GAGs by using in vitro mutagenesis to identify amino acids of chemokine macrophage-inflammatory protein-1 beta (MIP-1 beta) that contribute to its interaction with the model GAG heparin. Among six basic residues that are organized into a single basic domain in the folded MIP-1 beta monomer, three (R18, K45, and R46) were found to contribute significantly to heparin binding. Of these, R46 was found to play a dominant role, and proved essential for the interaction of MIP-1 beta with both heparin and heparan sulfate in physiological salt. The results of this mutational analysis have implications for the structure of the MIP-1 beta-heparin complex, and a comparison of these results with those obtained by mutational analysis of the MIP-1 alpha-heparin interaction suggests a possible structural difference between the MIP-1 beta-heparin and MIP-1 alpha-heparin complexes. To determine whether GAG binding plays an important role in receptor binding and cellular activation by MIP-1 beta, the activities of wild-type MIP-1 beta and R46-substituted MIP-1 beta were compared in assays of T lymphocyte chemotaxis. The two proteins proved equipotent in this assay, arguing that interaction of MIP-1 beta with GAGs is not intrinsically required for functional interaction of MIP-1 beta with its receptor.

The LD78beta isoform of MIP-1alpha is the most potent CCR5 agonist and HIV-1-inhibiting chemokine.

LD78alpha and LD78beta are 2 highly related nonallelic genes that code for different isoforms of the human CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha). Two molecular forms of natural LD78beta (7.778 and 7.793 kDa) were identified from conditioned media of stimulated peripheral blood mononuclear cells. Although LD78alpha and LD78beta only differ in 3 amino acids, both LD78beta variants were 100-fold more potent chemoattractants for mouse lymphocytes than was LD78alpha. On the contrary, LD78beta was only 2-fold more efficient than LD78alpha in chemoattracting human lymphocytes and monocytes. Using CC chemokine receptor-transfected cells, both molecular forms of LD78beta proved to be much more potent than LD78alpha in inducing an intracellular calcium rise through CCR5. Compared with LD78alpha and RANTES, this preferential binding of LD78beta to CCR5 resulted in a 10- to 50-fold higher potency in inhibiting infection of peripheral blood mononuclear cells by CCR5-using (R5) HIV-1 strains. To date, LD78beta is the most potent chemokine for inhibiting HIV-1 infection, and can be considered as a potentially important drug candidate for the treatment of infection with R5 HIV-1 strains.

Identification of amino acids involved in the binding of hMIP-1 alpha to CC-CKR1, a MIP-1 alpha receptor found on neutrophils.

Human macrophage inflammatory protein-1alpha (hMIP-1alpha) and human macrophage inflammatory protein-1beta (hMIP-1beta) are chemokines involved in a diverse range of immunological effects. Both hMIP-1alpha and hMIP-1beta are involved in the activation of monocytes and THP-1 cells probably through a common receptor(s). However, only hMIP-1alpha can bind to neutrophils with high affinity, presumably through CC-CKR1 (CKR1). Since the structure of these two proteins is highly conserved, non-conserved amino acids must define the disparate binding patterns that these two proteins exhibit. Measurements of binding, chemotaxis and calcium influx conducted with hMIP-1alpha and hMIP-1beta chimeric proteins and mutants show that two amino acids (37K and 43L) are important in the binding and signaling of hMIP-1alpha through CKR1. Furthermore, we also show that mutations of the three charged amino acids at the C-terminus of hMIP-1alpha and hMIP-1beta (amino acids 61, 65 and 67), do not adversely affect the binding to THP-1 cells.

Identification of IL-16 as the lymphocyte chemotactic activity in the bronchoalveolar lavage fluid of histamine-challenged asthmatic patients.

OBJECTIVE: We have previously demonstrated that the earliest lymphocyte chemotactic factors present in bronchoalveolar lavage fluid (BALF) of subjects with atopic asthma after subsegmental antigen challenge are IL-16 and MIP-1alpha, of which IL-16 appears to contribute a majority of the chemotactic activity. Because IL-16 is released in vitro after histamine stimulation of CD8+ T cells and epithelial cells, we evaluated the potential role of histamine in the release of IL-16 into the airways of allergic asthmatics in vivo. METHODS: Eight allergic asthmatic subjects, six normal subjects, and six atopic nonasthmatic subjects were challenged with saline in the lingula and with serial concentrations of histamine (1 x 10(-7) to 5 x 10(-5) mol/L) in the right middle lobe followed by bronchoalveolar lavage (BAL) 15 minutes and 6 hours later. RESULTS: The BALF from saline- and histamine-challenged lobes of normal subjects and atopic nonasthmatic subjects contained no significant lymphocyte chemoattractant activity. In six of the eight atopic asthmatic subjects, the histamine-challenged but not saline-challenged segment contained IL-16 chemotactic activity but no other identifiable lymphocyte chemoattractant activities at 6 hours. CONCLUSIONS: IL-16 appears in the airways after histamine challenge and therefore could contribute to the earliest infiltration of CD4+ T cells and eosinophils observed after antigen challenge due to histamine release from mast cells.

Antigen-specific release of beta-chemokines by anti-HIV-1 cytotoxic T lymphocytes.

A major advance in understanding human immunodeficiency virus (HIV) biology was the discovery that the beta-chemokines MIP-1 alpha (macrophage inflammatory protein-1 alpha), MIP-1 beta (macrophage inflammatory protein-1 beta) and RANTES (regulated on activation, normal T-cell expressed and secreted) inhibit entry of HIV-1 into CD4+ cells by blocking the critical interaction between the CCR5 coreceptor and the V3 domain of the viral envelope glycoprotein gp120 [1,2]. CD8+ lymphocytes are a major source of beta-chemokines [3], but the stimulus for chemokine release has not been well defined. Here, we have shown that engagement of CD8+ cytotoxic T lymphocytes (CTLs) with HIV-1-encoded human leukocyte antigen (HLA) class I-restricted peptide antigens caused rapid and specific release of these beta-chemokines. This release paralleled cytolytic activity and could be attenuated by naturally occurring amino acid variation within the HLA class I-restricted peptide sequence. Epitope variants that bound to appropriate HLA class I molecules but failed to stimulate cytolytic activity in CTLs also failed to stimulate chemokine release. We conclude that signalling through the T-cell receptor (TCR) following binding of antigen results in beta-chemokine release from CTLs in addition to cytolytic activity, and that both responses can be abolished by epitope mutation. These results suggest that antigenic variation within HIV-1 might not only allow the host cell to escape lysis, but might also contribute to the propagation of infection by failing to activate beta-chemokine-mediated inhibition of HIV-1 entry.