RANTES and macrophage inflammatory protein 1 alpha induce the migration and activation of normal human eosinophil granulocytes

The cellular infiltrates of certain inflammatory processes found in parasitic infection or in allergic diseases consist predominantly of eosinophilic granulocytes, often in association with activated T cells. This suggests the existence of chemotactic agonists specific for eosinophils and lymphocyte subsets devoid of neutrophil-activating properties. We therefore examined four members of the intercrine/chemokine superfamily of cytokines (monocyte chemotactic peptide 1 [MCP-1], RANTES, macrophage inflammatory protein 1 alpha [MIP-1 alpha], and MIP-1 beta), which do not activate neutrophils, for their ability to affect different eosinophil effector functions. RANTES strongly attracted normal human eosinophils by a chemotactic rather than a chemokinetic mechanism with a similar efficacy as the most potent chemotactic myeloid cell agonist, C5a. MIP-1 alpha also induced eosinophil migration, however, with lower efficacy. RANTES and MIP-1 alpha induced eosinophil cationic protein release in cytochalasin B-treated eosinophils, but did not promote leukotriene C4 formation by eosinophils, even after preincubation with interleukin 3 (IL-3), in contrast to other chemotactic agonists such as C5a and formyl-methionyl-leucyl-phenylalanine (FMLP). RANTES, but not MIP-1 alpha, induced a biphasic chemiluminescence response, however, of lower magnitude than C5a. RANTES and MIP-1 alpha both promoted identical transient changes in intracellular free calcium concentration ([Ca2+]i), with kinetics similar to those induced by chemotactic peptides known to interact with G protein-coupled receptors. No cross-desensitization towards other peptide agonists (e.g., C5a, IL-8, FMLP) was observed, suggesting the presence of specific receptors. Despite its weaker eosinophil-activating properties, MIP-1 alpha was at least 10 times more potent on a molar basis than RANTES at inducing [Ca2+]i changes. Interestingly, RANTES deactivated the MIP-1 alpha-induced [Ca2+]i changes, while the RANTES response was preserved after MIP-1 alpha stimulation. MCP-1, a potent monocyte chemoattractant and basophil agonist, as well as MIP-1 beta, a peptide with pronounced homology to MIP-1 alpha, did not activate the eosinophil functions tested. Our results indicate that RANTES and MIP-1 alpha are crucial mediators of inflammatory processes in which eosinophils predominate.     

Type I interferons regulate inflammatory cell trafficking and macrophage inflammatory protein 1alpha delivery to the liver

Macrophage inflammatory protein 1alpha (MIP-1alpha, CCL3) is critical for liver NK cell inflammation and delivery of IFN-gamma to mediate downstream protective responses against murine cytomegalovirus (MCMV) infections. This system was used to evaluate the upstream contribution of the type 1 IFNs, IFN-alpha/beta, in promotion of MIP-1alpha production. Mice deficient in IFN-alpha/beta functions, as a result of mutation in the receptor for these cytokines (IFN-alpha/betaR(-)), were profoundly deficient in MIP-1alpha expression and accumulation of NK cells and macrophages in the liver and had increased sensitivity to MCMV infection. The cytokines themselves were responsible for the immunoregulatory effects, since administration of recombinant IFN-alpha (rIFN-alpha) to immunocompetent mice also induced these changes. IFN-alpha/beta was required for NK cell accumulation during infection, and MIP-1alpha was required for NK cell accumulation in response to administered rIFN-alpha. In vivo trafficking assays demonstrated a requirement for IFN-alpha/betaR signaling for leukocyte localization in, and delivery of MIP-1alpha-producing macrophages to, the liver. These results extend characterization of the cytokine and chemokine cascade required for protection against viral infections in tissues by defining IFN-alpha/beta-dependent mechanisms promoting MIP-1alpha production and the resulting hepatic accumulation of NK cells.     

Human macrophage inflammatory protein alpha (MIP-1 alpha) and MIP-1 beta chemokines attract distinct populations of lymphocytes

Lymphocyte trafficking is an essential process in immune and inflammatory functions which can be thought to contain at least two main components: adhesion and migration. Whereas adhesion molecules such as the selections are known to mediate the homing of leukocytes from the blood to the endothelium, the chemoattractant substances responsible for the migration of specific subsets of lymphocytes to sites of infection or inflammation are largely unknown. Here we show that two molecules in the chemokine (for chemoattractant cytokine) superfamily, human macrophage inflammatory protein 1 alpha (MIP-1 alpha) and MIP-1 beta, do not share identical attractant activities for lymphocyte subpopulations. When analyzed in vitro in microchemotaxis experiments, HuMIP-1 beta tends to attract CD4+ T lymphocytes, with some preference for T cells of the naive (CD45RA) phenotype. HuMIP-1 alpha, when tested in parallel with HuMIP-1 beta, is a more potent lymphocyte chemoattractant with a broader range of concentration- dependent chemoattractant specificities. HuMIP-1 alpha at a concentration of 100 pg/ml attracts B cells and cytotoxic T cells, whereas at higher concentrations (10 ng/ml), the migration of these cells appears diminished, and the migration of CD4+ T cells is enhanced. Thus, in this assay system, HuMIP-1 alpha and -1 beta have differential attractant activities for subsets of immune effector cells, with HuMIP-1 alpha having greater effects than HuMIP-1 beta, particularly on B cells.     

The role of macrophage inflammatory protein 1 alpha in Schistosoma mansoni egg-induced granulomatous inflammation

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is a 6-8-kD, lipopolysaccharide-inducible monocyte and neutrophil chemotactic protein that may be important in acute and chronic inflammation. The present study determined the sequential production, source, and in vivo contribution of murine MIP-1 alpha in synchronized Schistosoma mansoni egg pulmonary granuloma formation. Granulomas were examined under conditions of primary, secondary vigorous, and secondary immunomodulated immunity. Secreted MIP-1 alpha was measured in 24-h supernatants from intact granulomas (700/ml) cultured with or without soluble egg antigen (SEA). Primary granulomas isolated from naive mice over a 16-d period showed low spontaneous MIP-1 alpha production (< 1 ng/ml). However, when primary granulomas were challenged with SEA, significant MIP-1 alpha production was observed beginning at day 4 and peaking at day 16. Intact vigorous (isolated from 8-wk-infected mice) and modulated (isolated from 20-wk-infected mice) secondary pulmonary granulomas demonstrated comparable spontaneous MIP-1 alpha production. Addition of SEA to vigorous stage granulomas augmented expression of MIP-1 alpha at all time points, whereas stimulated modulated stage granulomas did not increase production. The latter observation is likely related to endogenous immunoregulatory mechanisms reported for modulated stage animals. Immunohistochemical localization of MIP-1 alpha in granuloma sections and cytocentrifuge preparations from vigorous lesions localized MIP-1 alpha production to macrophages within granulomas. Treatment of mice with rabbit anti-mouse MIP-1 alpha antibodies significantly decreased 8-d primary granuloma formation (> 40%) when compared with control mice. Anti-MIP-1 alpha sera also decreased vigorous (> 20%), but not modulated granuloma formation. These findings demonstrate that MIP-1 alpha contributes to cellular recruitment during schistosome egg granuloma formation.     

Formation of eosinophilic and monocytic intradermal inflammatory sites in the dog by injection of human RANTES but not human monocyte chemoattractant protein 1, human macrophage inflammatory protein 1 alpha, or human interleukin 8

Equilibrium binding studies on canine mononuclear and granulocytic cells allow the identification of a single high affinity receptor for the human C-C chemokine RANTES (dissociation constant, 14 +/- 8 pM), that, in contrast to the human RANTES receptor, has no affinity for human macrophage inflammatory protein 1 alpha (hMIP-1 alpha). A single intradermal injection of hRANTES in dog resulted in eosinophil- and macrophage-rich inflammatory sites within 4 h. Cell infiltration peaked at 16-24 h after hRANTES injection. There was histological evidence of intravascular activation of eosinophils at 4 h, although eosinophils in the vasculature and interstitium contained apparently intact granules. Monocytes were the predominant cells adherent to venular endothelium at 16-24 h. Human MIP-1 alpha elicited no response in canine dermis, whereas monocyte chemoattractant protein 1 caused mild perivascular cuffing with monocytes. In contrast, human interleukin 8 induced a neutrophilic dermal infiltrate that was maximal by 4 h after challenge. This provides the first direct evidence in vivo that RANTES has significant proinflammatory activity and, in addition, could be a mediator in atopic pathologies characterized by eosinophilic and monocytic inflammatory responses.     

RANTES and macrophage inflammatory protein 1 alpha selectively enhance immunoglobulin (IgE) and IgG4 production by human B cells

We studied the effects of various chemokines including neutrophil- activating peptide 2 (NAP-2), beta-thromboglobulin (beta-TG), platelet factor 4 (PF-4), melanoma growth stimulating activity (GRO), gamma interferon-induced protein (IP-10), regulated on activation, normal T expressed and secreted (RANTES), macrophage inflammatory protein 1 alpha (MIP-1 alpha), MIP-1 beta, and monocyte chemotactic protein 1 (MCP-1) on Immunoglobulin (IgE) and IgG4 production by human B cells. None of these chemokines with or without interleukin (IL-4), anti-CD40 or -CD58 monoclonal antibody (mAb), induced IgE and IgG4 production by B cells from nonatopic donors. However, RANTES and MIP-1 alpha selectively enhanced IgE and IgG4 production induced by IL-4 plus anti- CD40 or -CD58 mAb without affecting production of IgM, IgG1, IgG2, IgG3, IgA1, or IgA2, whereas other chemokines failed to do so. Enhancement of IgE and IgG4 production by RANTES and MIP-1 alpha was specifically blocked by anti-RANTES mAb and anti-MIP-1 alpha antibody (Ab), respectively, whereas anti-IL-5 mAb, anti-IL-6 mAb, anti-IL-10 Ab, anti-IL-13 Ab, and anti-tumor necrosis factor-alpha mAb failed to do so. Purified surface IgE positive (slgE4) and slgG4+ B cells generated either in vitro or in vivo spontaneously produced IgE and IgG4, respectively, whereas sIgE- and sIgG4- B cells failed to do so. RANTES and MIP-1 alpha enhanced spontaneous IgE and IgG4 production in slgE+ and slgG4- B cells, respectively, whereas neither RANTES nor MIP- 1 alpha did so in sIgE- or sIgG4- B cells. Purified sIgE4+ and sIgG4+, but not sIgE- or sIgG4- B cells, generated in vitro and in vivo expressed receptors for RANTES and MIP-1 alpha, whereas they failed to express receptors for other chemokines. These findings indicate that RANTES and MIP-1 alpha enhance IgE and IgG4 production by directly stimulating sIgE+ and sIgG4+ B cells.     

Identification and characterization of a rat macrophage inflammatory protein-1alpha receptor

A macrophage inflammatory protein-1alpha (MIP-1alpha) receptor has been cloned from rat (Rattus norvegicus) genomic DNA by PCR using oligonucleotides based on the mouse CCR1 DNA sequence and expressed in HEK293 cells to enable its characterization. The receptor was cloned three times in independent experiments to generate a consensus sequence. The rat ccrl receptor amino acid sequence is 92% identical to mouse and 80% identical to human CCR1. A consensus clone was transfected into HEK293 cells using the expression vector pIRES, and stable receptor expressing cell lines were isolated. In competitive receptor binding assays using iodinated human MIP-1alpha, rat ccrl binds hMIP-1alpha, hMIP-1beta, and hMCP-3, but not hRANTES or human interleukin-8 (hIL-8). We have been unable to demonstrate calcium mobilization by rat ccrl in HEK293 cells using human chemokines as ligands. Therefore, we have adopted lowercase nomenclature for the receptor until signaling is observed. This receptor and cell line may be of use in the preclinical development of CCR1 antagonists.     

Resolution of the two components of macrophage inflammatory protein 1, and cloning and characterization of one of those components, macrophage inflammatory protein 1 beta

A number of macrophage-derived mediators have been implicated in the vascular changes of inflammation. We recently reported the isolation of a novel monokine, macrophage inflammatory protein 1 (MIP-1), which causes local inflammatory responses in vivo, and induces superoxide production by neutrophils in vitro. Purified native MIP-1 comprises two peptides with very similar physical characteristics. We report here the resolution of MIP-1 into component peptides by SDS-hydroxylapatite chromatography, and compare the NH2-terminal sequences of the two peptides, now referred to as MIP-1 alpha and MIP-1 beta. A synthetic oligonucleotide probe pool corresponding to the NH2-terminal amino acid sequence of MIP-1 beta was used to isolate a cDNA clone containing its coding sequence. The sequence codes for a 109 amino acid-long polypeptide, of which 69 amino acids correspond to the mature product. Comparison of this MIP-1 beta cDNA with our previously cloned MIP-1 alpha sequence reveals that the MIP-1 peptides, members of a growing family of potential inflammatory mediators, are distinct but highly homologous (58.9% sequence identity) products of different genes.     

Cloning and characterization of cDNAs for murine macrophage inflammatory protein 2 and its human homologues

A cDNA clone of murine macrophage inflammatory protein 2 (MIP-2) has been isolated from a library prepared from lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and the nucleotide sequence determined. This cDNA was used to clone cDNAs for human homologues of MIP-2 from a library prepared from phorbol myristate acetate-treated and LPS-stimulated U937 cells. Two homologues were isolated and sequenced. Human MIP-2 alpha and MIP-2 beta are highly homologous to each other and to a previously isolated gene, human gro/melanoma growth-stimulating activity (MGSA). These three human genes, MIP-2 alpha, MIP-2 beta, and gro/MGSA, constitute a sub-family within the cytokine family represented by platelet factor 4 and interleukin 8.     

Human eosinophils can express the cytokines tumor necrosis factor-alpha and macrophage inflammatory protein-1 alpha.

By in situ hybridization, 44-100% of the blood eosinophils from five patients with hypereosinophilia and four normal subjects exhibited intense hybridization signals for TNF-alpha mRNA. TNF-alpha protein was detectable by immunohistochemistry in blood eosinophils of hypereosinophilic subjects, and purified blood eosinophils from three atopic donors exhibited cycloheximide-inhibitable spontaneous release of TNF-alpha in vitro. Many blood eosinophils (39-91%) from hypereosinophilic donors exhibited intense labeling for macrophage inflammatory protein-1 alpha (MIP-1 alpha) mRNA, whereas eosinophils of normal donors demonstrated only weak or undetectable hybridization signals for MIP-1 alpha mRNA. Most tissue eosinophils infiltrating nasal polyps were strongly positive for both TNF-alpha and MIP-1 alpha mRNA. By Northern blot analysis, highly enriched blood eosinophils from a patient with the idiopathic hypereosinophilic syndrome exhibited differential expression of TNF-alpha and MIP-1 alpha mRNA. These findings indicate that human eosinophils represent a potential source of TNF-alpha and MIP-1 alpha, that levels of expression of mRNA for both cytokines are high in the blood eosinophils of hypereosinophilic donors and in eosinophils infiltrating nasal polyps, that the eosinophils of normal subjects express higher levels of TNF-alpha than MIP-1 alpha mRNA, and that eosinophils purified from the blood of atopic donors can release TNF-alpha in vitro.     

Limited proteolysis by macrophage elastase inactivates human alpha 1- proteinase inhibitor

Inflammatory mouse peritoneal macrophages secrete a metalloproteinase that is not inhibited by alpha 1-proteinase inhibitor. This proteinase, macrophage elastase, recognizes alpha 1-proteinase inhibitor with macrophage elastase does not involve a stable proteinase-inhibitor complex and results in the proteolytic removal of a peptide of apparent molecular weight 4,000-5,000 from the inhibitor. After degradation by macrophage elastase, alpha 1-proteinase inhibitor is no longer able to inhibit human granulocyte elastase, a serine proteinase implicated in the pathogenesis of emphysema. Macrophage elastase apparently does not degrade human granulocyte elastase-alpha 1-proteinase inhibitor complexes or release active granulocyte elastase from these complexes. The ability of macrophage elastase to degrade alpha 1-proteinase inhibitor is inhibited by EDTA and alpha 2-macroglobulin.     

Serum amyloid A protein compared with C-reactive protein, alpha 1-antichymotrypsin and alpha 1-acid glycoprotein as a monitor of inflammatory bowel disease

The serum concentrations of serum amyloid A protein (SAA), C-reactive protein (CRP), alpha 1-antichymotrypsin (alpha 1-ACT) and alpha 1-acid glycoprotein (alpha 1-AGP) have been measured in eighty-six patients with Crohn's disease, twenty-five patients with ulcerative colitis and twenty-two patients with the irritable bowel syndrome. In the Crohn's and ulcerative colitis group significant increases in concentration were observed in all four proteins, which parallelled disease severity as defined by other conventional laboratory parameters formulated into a simple activity index. In the irritable bowel group no significant changes were seen. Serum amyloid A and CRP concentrations were significantly lower in ulcerative colitis than in Crohn's disease when mild, but did not differ significantly when severe. Serum amyloid A correlated well with CRP (r = 0.83) and alpha 1-ACT (r = 0.80), but less well with alpha 1-AGP (r = 0.65). Serum amyloid A was the most sensitive protein (77%) but had the lowest specificity (74%). C-reactive protein was less sensitive (58%) than SAA but had greater specificity (100%). Alpha 1-ACT had a sensitivity and specificity similar to CRP and, therefore, provided little or no additional information. Alpha 1-AGP, although also 100% specific, had the lowest sensitivity (34%) and, therefore, is probably the least useful acute phase monitor of inflammatory bowel disease. The role, and associated problems, of SAA measurements are discussed.     

Interaction of mouse macrophage elastase with native and oxidized human alpha 1-proteinase inhibitor.

Native and oxidized alpha 1-proteinase inhibitor (alpha 1-PI) were compared as substrates for the metalloproteinase macrophage elastase. At substrate concentrations at which native alpha 1-PI was readily degraded by macrophage elastase, oxidized alpha 1-PI was hardly degraded at all. Incubation of macrophage elastase with oxidized alpha 1-PI before the addition of native alpha 1-PI showed that oxidized alpha 1-PI was not an inhibitor of macrophage elastase. Competition experiments with up to twofold excess oxidized alpha 1-PI did not interfere with the degradation of native alpha 1-PI by macrophage elastase. Sequence analysis of amino acids in degraded native alpha 1-PI showed that macrophage elastase attacked a single peptide bond between Pro-357 and Met-358, the latter representing the P1 reactive-site residue of alpha 1-PI. In oxidized alpha 1-PI, Met-358 was converted to methionine sulfoxide and macrophage elastase hydrolyzed the bond between Phe-352 and Leu-353. These data suggest that methionine may be the primary cleavage site for macrophage elastase and not leucine, as previously thought.     

Human macrophage inflammatory protein 3alpha: protein and peptide nuclear magnetic resonance solution structures, dimerization, dynamics, and anti-infective properties

Human macrophage inflammatory protein 3alpha (MIP-3alpha), also known as CCL20, is a 70-amino-acid chemokine which exclusively binds to chemokine receptor 6. In addition, the protein also has direct antimicrobial, antifungal, and antiviral activities. The solution structure of MIP-3alpha was solved by the use of two-dimensional homonuclear proton nuclear magnetic resonance (NMR). The structure reveals the characteristic chemokine fold, with three antiparallel beta strands followed by a C-terminal alpha helix. In contrast to the crystal structures of MIP-3alpha, the solution structure was found to be monomeric. Another difference between the NMR and crystal structures lies in the angle of the alpha helix with respect to the beta strands, which measure 69 and approximately 56.5 degrees in the two structures, respectively. NMR diffusion and pH titration studies revealed a distinct tendency for MIP-3alpha to form dimers at neutral pH and monomers at lower pH, dependent on the protonation state of His40. Molecular dynamics simulations of both the monomeric and the dimeric forms of MIP-3alpha supported the notion that the chemokine undergoes a change in helix angle upon dimerization and also highlighted the important hydrophobic and hydrogen bonding contacts made by His40 in the dimer interface. Moreover, a constrained N terminus and a smaller binding groove were observed in dimeric MIP-3alpha simulations, which could explain why monomeric MIP-3alpha may be more adept at receptor binding and activation. The solution structure of a synthetic peptide consisting of the last 20 residues of MIP-3alpha displayed a highly amphipathic alpha helix, reminiscent of various antimicrobial peptides. Antimicrobial assays with this peptide revealed strong and moderate bactericidal activities against Escherichia coli and Staphylococcus aureus, respectively. This confirms that the C-terminal alpha-helical region of MIP-3alpha plays a significant part in its broad anti-infective activity.