Neutrophil Fc gamma and complement receptors involved in binding soluble IgG immune complexes and in specific granule release induced by soluble IgG immune complexes

We examined the effect of soluble IgG immune complex (IC) characteristics on the binding of IC to human neutrophils and IC-induced specific granule release of neutrophils via Fc gamma receptors (CD16 and CD32) and complement receptors (CR1 and CR3). A set of soluble IgG IC varying in size, IgG subclass, antigen epitope density and complement (C) incorporation were formed between 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) coupled to bovine serum albumin (BSA) and chimeric mouse-human anti-NIP monoclonal antibodies (mAb) of all four IgG subclasses. High and low epitope density IC of all four IgG subclasses induced specific granule release with C, but in the absence of C only IgG1 and IgG3 IC were functionally active. The Fc gamma and C receptors responsible for IgG IC-induced specific granule release and IC binding were determined using mAb specific for the ligand binding sites of CD16, CD32 and CR3, and recombinant soluble CR1. Each defined IC displayed a unique pattern of receptor preference, dependent upon subclass and antigenic epitope density. IC binding and IC-induced specific granule release was not mediated by the same receptor, or combination of receptors. High and low epitope density IgG3 IC binding and induction of specific granule release was mediated predominantly via CD16. Other IC subclasses bound differently, i.e. IgG1 IC used CD16 and CR3; IgG2 and IgG4 predominantly used complement receptors; but all three induced specific granule release via CD32. In vivo these results may translate into differential activation of neutrophils by soluble IC dependent upon their characteristics, leading to subtle nuances in the etiology, pathology and control of the immune response in IC-related diseases.     

Differential and time-dependent expression of monocyte chemoattractant protein-1 mRNA by astrocytes and macrophages in rat brain: effects of ischemia and peripheral lipopolysaccharide administration

Increasing evidence indicates a key role of chemoattractant cytokines in the accumulation of leukocytes in the central nervous system (CNS) during the course of inflammatory processes. Monocyte chemoattractant protein (MCP-1/JE), a member of the beta-chemokine (C-C chemokine) family, functions as a potent chemoattractant and activator for monocytes. We have investigated the induction of MCP-1 mRNA using in situ hybridization histochemistry (ISH) and characterized its cellular source by combination of ISH and immunocytochemistry in ischemic rat brains as well as in brains of endotoxin-treated rats. Our results show that 6 h-2 d after middle cerebral artery occlusion (MCAO), MCP-1 mRNA is present in astrocytes surrounding the ischemic tissue (penumbra). At later time points (after 4 d), MCP-1 mRNA is found in macrophages and reactive microglia in the infarcted tissue. Peripheral administration of the bacterial lipopolysaccharide (LPS) induced MCP-1 mRNA throughout the brain in a time-dependent manner (1 h-1 d, peak of expression 6-8 h) and was found in astrocytes. In summary, we have found expression of MCP-1 in (a) astrocytes and to a lesser extent in macrophages/reactive microglia after MCA-occlusion and in (b) astrocytes after peripheral administration of LPS. These findings support that MCP-1 is involved in the CNS response to acute trauma or infection and thus may play a key role in inflammatory processes of the brain.     

MaMi, a human endometrial stromal sarcoma cell line that constitutively produces interleukin-6, interleukin-8, and monocyte chemoattractant protein-1

BACKGROUND: Uterine endometrial stromal sarcoma is a rare neoplasm with a morphology that closely resembles that of the proliferative endometrial stroma. To understand its pathologic characteristics, we established a novel cell line, MaMi, from a primary culture of an endometrial stromal sarcoma obtained from a 65-year-old Japanese woman. METHODS: We observed the morphology of MaMi cells and performed immunohistochemical analysis on the primary tumor and transplants in nude mice. Prolactin, insulin-like growth factor-binding protein-1, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1, and fibronectin production in the culture medium of MaMi cells were also examined. RESULTS: MaMi cells were shown to exhibit a fibroblast-like morphology in vitro, and they adopted a more elongated appearance in response to 12-O-tetradecanoyl phorbol-13-acetate (TPA). On injection into nude mice, the cells gave rise to subcutaneous tumors. Immunohistologically, both the primary tumor and MaMi cell-induced tumors stained positively with antibodies to neuron-specific enolase or vimentin. MaMi cells constitutively produced IL-6, IL-8, and monocyte chemoattractant protein-1 in vitro. Interleukin-1beta, (100 pmol/L), tumor necrosis factor-alpha (1 nmol/L), and lipopolysaccharide (1 microg/mL) each increased the release of IL-6, IL-8, and monocyte chemoattractant protein-1 by MaMi cells. TPA (10 nmol/L) also stimulated the production of IL-6 and IL-8 by these cells, but inhibited that of monocyte chemoattractant protein-1. CONCLUSIONS: We demonstrated that MaMi cells closely resemble proliferative endometrial stromal cells not only morphologically, but also functionally. This cell line may prove valuable in understanding the role of cytokines produced by tumor cells in the pathogenesis of endometrial stromal sarcoma and may also be useful as an in vitro model of functioning endometrial stromal cells.     

Chemokine receptor CCR2 expression and monocyte chemoattractant protein-1-mediated chemotaxis in human monocytes. A regulatory role for plasma LDL

The subendothelial accumulation of macrophage-derived foam cells is one of the hallmarks of atherosclerosis. The recruitment of monocytes to the intima requires the interaction of locally produced chemokines with specific cell surface receptors, including the receptor (CCR2) for monocyte chemoattractant protein-1 (MCP-1). We have previously reported that monocyte CCR2 gene expression and function are effectively downregulated by proinflammatory cytokines. In this study we identified low density lipoprotein (LDL) as a positive regulator of CCR2 expression. Monocyte CCR2 expression was dramatically increased in hypercholesterolemic patients compared with normocholesterolemic controls. Similarly, incubation of human THP-1 monocytes with LDL induced a rapid increase in CCR2 mRNA and protein. By 24 hours the number of cell surface receptors was doubled, causing a 3-fold increase in the chemotactic response to MCP-1. The increase in CCR2 expression and chemotaxis was promoted by native LDL but not by oxidized LDL. Oxidized LDL rapidly downregulated CCR2 expression, whereas reductively methylated LDL, which does not bind to the LDL receptor, had only modest effects on CCR2 expression. A neutralizing anti-LDL receptor antibody prevented the effect of LDL, suggesting that binding and internalization of LDL were essential for CCR2 upregulation. The induction of CCR2 expression appeared to be mediated by LDL-derived cholesterol, because cells treated with free cholesterol also showed increased CCR2 expression. These data suggest that elevated plasma LDL levels in conditions such as hypercholesterolemia enhance monocyte CCR2 expression and chemotactic response and potentially contribute to increased monocyte recruitment to the vessel wall in chronic inflammation and atherogenesis.     

Plasma levels of monocyte chemoattractant protein-1 but not those of macrophage inhibitory protein-1alpha and RANTES correlate with virus load in human immunodeficiency virus infection

Plasma levels of proinflammatory cytokines, cytokine inhibitors, and the beta chemokines RANTES, macrophage inhibitory protein (MIP)-1alpha, and monocyte chemoattractant protein (MCP)-1 were studied in relationship with virus load in 40 patients exhibiting plasma levels of HIV RNA ranging between undetectable and levels >10(6) copies/mL. Mean plasma levels of MCP-1 were increased in patients with high virus load compared with HIV-seropositive subjects with undetectable plasma viral RNA and healthy controls. MCP-1 levels were directly correlated with plasma levels of HIV RNA. No correlation was observed between virus load and plasma concentrations of MIP-1alpha and RANTES. The results suggest that low rates of viral replication in vivo are not dependent on increased production of the suppressive chemokines RANTES and MIP-1alpha. Since MCP-1 upregulates viral replication in vitro, the results may suggest a role for MCP-1 in triggering viral replication in HIV disease.     

Anti-fucosyl-GM1 ganglioside IgG and IgM autoantibodies in human serum: no link to pathology

Anti-fucosyl-GM1 ganglioside antibodies were detected in sera of five persons: four patients with autoimmune neuropathies and more recently, IgG antibodies in one with Graves' disease (Adler et al., Autoimmunity 18, 149-152, 1994) [1]. In the latter case, we were unable to find any relation between the occurrence of antibodies and thyroid disease. Now we report a detailed study on the anti-glycolipid antibodies in this patient. We found that her serum contained not only IgG but also a high level of anti-FucGM1 IgM antibodies, with a titer stable over a period of 5 years of treatment and follow-up. The carbohydrate structure of the epitope recognized by IgG and some of IgM antibodies seems to consist of Fuc-Gal-GalNAc-Gal- or a part of this sequence. Moreover, this patient's serum contained other IgM antibodies active against FucGM1 and also asialo GM1 glycolipids. Our results indicate that anti-FucGM1 ganglioside antibodies of G and M classes occur in serum of this patient with no apparent adverse health effects.     

Expression of monocyte chemoattractant protein-1 in monocytes and effects of native and oxidized very low density lipoproteins

Rheumatoid neutrophilic dermatitis (RND) is a rare cutaneous finding in patients with severe rheumatoid arthritis or with high-titer rheumatoid factors. Most commonly, these lesions are erythematous papules or plaques distributed symmetrically on extensor surfaces. On histologic examination a dense dermal neutrophilic infiltrate without vasculitis is apparent. The pathogenesis of RND is unclear, and few treatments are known. With careful clinical and histologic examination, RND may be differentiated from the wide array of other cutaneous findings in rheumatoid arthritis.     

Expression of monocyte chemoattractant protein-1 and other beta-chemokines by resident glia and inflammatory cells in multiple sclerosis lesions

Beta-chemokines induce the directional migration of monocytes and T lymphocytes and are thus associated with chronic inflammation. Using immunocytochemistry and in situ hybridisation (ISH) techniques, we have examined the expression of the beta-chemokines monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated upon activation, normal T cell expressed and secreted) in post-mortem human brain from multiple sclerosis (MS) cases, at different stages of lesion development. In actively demyelinating MS plaques RANTES expression was restricted to the blood vessel endothelium, perivascular cells and surrounding astrocytes, suggesting a role in the recruitment of inflammatory cells from the circulation. MCP-1 was expressed by astrocytes and macrophages within acute MS lesions, but was restricted to reactive astrocytes in the parenchyma surrounding the lesion. MIP-1alpha was expressed by astrocytes and macrophages within the plaque, while MIP-1beta was expressed by macrophages and microglia within the lesion, and by microglia in surrounding white matter. Glial cells may be stimulated to produce chemokines and continue the local inflammatory response by forming chemotactic gradients to attract T cells and mononuclear phagocytes from the circulation and surrounding tissue.     

Interleukin 8 and monocyte chemoattractant protein-1 in patients with juvenile rheumatoid arthritis. Relation to onset types, disease activity, and synovial fluid leukocytes

OBJECTIVE: To measure serum and synovial fluid (SF) levels of interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in patients with juvenile rheumatoid arthritis (JRA) and to compare them with adult rheumatoid factor-positive rheumatoid arthritis (RA). METHODS: IL-8 and MCP-1 were measured by immunoassay (1) in sera obtained from 55 children with JRA and from 16 adults with RA, and (2) in SF obtained from 30 children with JRA and 11 adults with RA. RESULTS: Patients with active systemic JRA had serum levels of IL-8 and MCP-1 higher than in controls (p<0.01) and in patients with active polyarticular or pauciarticular JRA (p<0.05). In patients with RA serum MCP-1 levels were higher than in patients with the 3 JRA onset types, while no difference was found for IL-8 levels. Patients with systemic JRA and with current systemic features had serum levels of IL-8 and MCP-1 higher (p = 0.03 and p = 0.04, respectively) than patients in which systemic features had subsided. No significant differences in SF IL-8 or MCP-1 levels were found among the 3 JRA onset types or adults with RA. In patients with JRA SF leukocyte counts were correlated with SF IL-8 levels (p = 0.002), but not with MCP-1 levels. Moreover, SF levels of both IL-8 and MCP-1 were correlated with those of IL-1beta (p<0.001) and IL-6 (p<0.01), but not with those of TNF-alpha. CONCLUSION: Elevated serum levels of IL-8 and MCP-1 in patients with systemic JRA with current systemic features at sampling suggest systemic production of the 2 chemokines during systemic phases of the disease. Similar SF levels of IL-8 and MCP-1 among the 3 JRA onset-types and RA suggest comparable local production of the 2 chemokines.     

Characterization of elevated neutrophil-associated IgG in various autoimmune disorders: not anti-neutrophil autoantibodies, but possibly immune complexes, bind to neutrophils

Resistance to toxicants is a convenient model for investigating whether adaptive changes are associated with pleiotropic fitness costs. Despite the voluminous literature devoted to this subject, intraspecific comparisons among toxicant resistance genes are rare. We report here results on the pleiotropic effect on adult survival of Culex pipiens mutants involved in the same adaptation: the resistance to organophosphorus insecticides. This field study was performed in southern France where four resistance genes sequentially appeared and increased in frequency in response to intense insecticide control. By repeated sampling of overwintering females through winter, we analysed the impact of each of three resistance genes on adult survival. We showed that (i) the most recent gene seems to be of no disadvantage during winter, (ii) the oldest affects survival in some environmental conditions, and (iii) the third induces a constant, severe and dominant survival cost. Such variability is discussed in relation to the physiological changes involved in resistance.     

Production and regulation of monocyte chemoattractant protein-1 in lipopolysaccharide- or monosodium urate crystal-induced arthritis in rabbits: roles of tumor necrosis factor alpha, interleukin-1, and interleukin-8

The production of monocyte chemoattractant protein-1 (MCP-1) and its regulation by TNFalpha, IL-1, and IL-8 were investigated in two rabbit models of arthritis induced by intra-articular injection of lipopolysaccharide (LPS) or monosodium urate (MSU) crystals. We first prepared recombinant rabbit MCP-1 and antibodies and then developed an immunoassay. The immunoassay detected 3 pg/ml rabbit MCP-1 and did not cross-react with other rabbit chemokines such as IL-8 or GRO. MCP-1 was first detected in synovial fluid (SF) at 1 hour, and peaked at 4 or 2 hours after the injection of LPS or MSU crystals, respectively. Immunohistochemically, MCP-1 was detected in synovial lining cells and infiltrating neutrophils. The amounts of MCP-1 detected in SF from neutrophil-depleted rabbits were similar to those in normal rabbits, suggesting that synovial lining cells were the main source of MCP-1 detected in SF. The peak level of MCP-1 in SF after LPS-injection was inhibited by 57% with anti-TNFalpha mAb and by 41% with IL-1 receptor antagonist (IL-1Ra). Coadministration of anti-TNFalpha mAb and IL-1Ra inhibited 90% of MCP-1 production. In contrast, the peak level of MCP-1 in SF after MSU crystal-injection was not affected by any cytokine inhibitor, but was reduced by 52% with coadministration of anti-TNFalpha mAb and IL-1Ra. Anti-IL-8 IgG had no effect on the production of MCP-1 in either model. Thus, the production of MCP-1 in LPS-induced arthritis was mostly regulated by TNFalpha and IL-1, whereas half the extent of MCP-1 production in MSU crystal-induced arthritis was independent of TNFalpha or IL-1. IL-8 does not seem to regulate the production of MCP-1 in SF either directly or indirectly. Finally, administration of neutralizing anti-MCP-1 antibody inhibited LPS- and MSU crystal-induced monocyte infiltration by 58.4% and 44.9%, respectively, suggesting that synovial production of MCP-1 plays an important role in the recruitment of monocytes in these arthritis models.     

Regulation of monocyte chemoattractant protein-1 expression in adult human non-neoplastic astrocytes is sensitive to tumor necrosis factor (TNF) or antibody to the 55-kDa TNF receptor

A trifluoroacetamide analogue of siastatin B, (3S,4S,5R,6R)-6-(trifluoroacetamido)-4,5-dihydroxy-3-piperidine carboxylic acid has been chemically synthesized. This compound, as well as the previously synthesized analogue, (3R,4R,5R,6R)-6-(trifluoroacetamido)-3,4,5-trihydroxy-3-piperid inecarboxylic acid, showed marked inhibitory activity against beta-glucuronidase and significant inhibition of experimental pulmonary metastasis of the highly metastatic melanoma B16.     

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.     

The first subcomponent of complement, C1q, triggers the production of IL-8, IL-6, and monocyte chemoattractant peptide-1 by human umbilical vein endothelial cells

We and others have demonstrated previously the occurrence of cC1qR/CaR, a receptor for the collagen-like stalks of complement component C1q, on endothelial cells. In the present study we investigated whether binding of C1q to endothelial cells resulted in enhancement of cytokine or chemokine production. HUVEC produced 82 +/- 91 pg/ml of IL-8, 79 +/- 113 pg/ml of IL-6, and 503 +/- 221 pg/ml of monocyte chemoattractant peptide-1 (MCP-1) under basal conditions. Incubation with C1q resulted in a time- and dose-dependent up-regulation of IL-8 (1012 +/- 43 pg/ml), IL-6 (392 +/- 20 pg/ml), and MCP-1 (2450 +/- 101 pg/ml). This production is dependent on de novo protein synthesis, as demonstrated by the detection of specific mRNA after C1q stimulation, and inhibition of peptide production in the presence of cycloheximide. The production of all factors was inhibited (69 +/- 7%) by the collagenous fragments of C1q, while the C1q globular heads only induced 13 +/- 11% inhibition. When HUVEC were incubated with C1q in the presence of aggregated IgM, enhanced production of IL-8 (2500 +/- 422 pg/ml), IL-6 (997 +/- 21 pg/ml), and MCP-1 (5343 +/- 302 pg/ml) was found. Furthermore, F(ab')2 anti-calreticulin partially inhibited the production of IL-8, confirming at least the involvement of cC1qR/CaR. These experiments suggest that in an inflammatory response C1q not only is able to activate the complement pathway, but when presented in a proper fashion also might induce the production of factors that contribute to acute phase responses and recruitment of inflammatory cells.     

Effect of interleukin-8 and monocyte chemotactic protein-1 on adhesion of circulating granulocytes and monocytes from asthma patients to human venous endothelial cells

Four mouse vomeronasal receptors (mV1Rs) have been isolated by similarity to rat vomeronasal receptor (V1R) motifs. The four mV1Rs identified in this study are members of two distinct subfamilies. Specific in situ hybridization probes (ISH) derived from the 3' non-coding regions of the mV1R genes, were used to detect expression of a single receptor and probes from the homologous coding regions were used to detect expression of subfamily members. The ISH results showed that the mV1Rs expressing neurons were scattered in the middle/upper layer of the vomeronasal organ (VNO) sensory epithelium in serial VNO sections but were excluded from the deeper layers of the VNO sensory epithelium and these neurons were found to co-express the mRNA for the G-protein Galphai2, and were distinct from the deeper layers of the VNO sensory epithelium where the mRNA for Galphao positive neurons was located.     

Chemotactic activity on mononuclear cells in the cerebrospinal fluid of patients with viral meningitis is mediated by interferon-gamma inducible protein-10 and monocyte chemotactic protein-1

In viral meningitis the inflammatory response involves activated T cells and monocytes which are recruited into the subarachnoid space. To identify the chemotactic signals attracting the cells to the site of infection in the meninges, we measured the levels of two CXC chemokines, interferon-gamma (IFN-gamma) inducible protein (IP)-10 and monokine induced by IFN-gamma, four CC chemokines, monocyte chemotactic protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, as well as the cytokines interleukin (IL)-15 and IL-16 in the cerebrospinal fluid (CSF) of patients suffering from viral meningitis. The results point to an involvement of two chemokines, MCP-1 and IP-10, since (1) unlike the other cytokines, MCP-1 and IP-10 were present in 97% and 79% of the CSF, respectively, at concentrations sufficient to induce chemotaxis of mononuclear cells; (2) more than 90% of the CSF of viral meningitis induced chemotaxis of peripheral blood mononuclear cells (PBMC) and all of them induced chemotaxis of activated T cells, and (3) the CSF-mediated chemotaxis of PBMC was inhibited by anti-MCP-1 antibodies and chemotaxis of activated T cells was abolished by the combination of anti-MCP-1 and anti-IP-10 antibodies. Our data provide evidence that MCP-1 and IP-10 lead to accumulation of activated T cells and monocytes in the CSF compartment in viral meningitis.