The aetiology and cellular mechanism of chronic inflammatory processes are poorly understood. Macrophages act prominently in the inflammatory response and we report here that they express two calcium-binding proteins. The expression of these proteins, referred to as <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em>, is specific for cells of myeloid origin, namely granulocytes, monocytes and macrophages, and is observed in blood granulocytes and monocytes but not in normal tissue macrophages. In acutely inflamed tissues, macrophages can express <em>MRP</em>-<em>14</em> but not <em>MRP</em>-<em>8</em>, and in chronic inflammations, such as primary chronic polyarthritis, infiltrate macrophages express both <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em>. Characterization of <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em> could therefore be useful to the understanding of cellular processes induced in chronic inflammation.

BACKGROUND

Platelets participate in events that immediately precede acute myocardial infarction. Because platelets lack nuclear DNA but retain megakaryocyte-derived mRNAs, the platelet transcriptome provides a novel window on gene expression preceding acute coronary events.

RESULTS

We profiled platelet mRNA from patients with acute ST-segment-elevation myocardial infarction (STEMI, n=16) or stable coronary artery disease (n=44). The platelet transcriptomes were analyzed and single-gene models constructed to identify candidate genes with differential expression. We validated 1 candidate gene product by performing a prospective, nested case-control study (n=255 case-control pairs) among apparently healthy women to assess the risk of future cardiovascular events (nonfatal myocardial infarction, nonfatal stroke, and cardiovascular death) associated with baseline plasma levels of the candidate protein. Platelets isolated from STEMI and coronary artery disease patients contained 54 differentially expressed transcripts. The strongest discriminators of STEMI in the microarrays were CD69 (odds ratio 6.2, P<0.001) and myeloid-related protein-<em>14</em> (<em>MRP</em>-<em>14</em>; odds ratio 3.3, P=0.002). Plasma levels of <em>MRP</em>-<em>8</em>/<em>14</em> heterodimer were higher in STEMI patients (17.0 versus <em>8</em>.0 microg/mL, P<0.001). In the validation study, the risk of a first cardiovascular event increased with each increasing quartile of <em>MRP</em>-<em>8</em>/<em>14</em> (Ptrend<0.001) such that women with the highest levels had a 3.<em>8</em>-fold increase in risk of any vascular event (P<0.001). Risks were independent of standard risk factors and C-reactive protein.

CONCLUSIONS

The platelet transcriptome reveals quantitative differences between acute and stable coronary artery disease. <em>MRP</em>-<em>14</em> expression increases before STEMI, and increasing plasma concentrations of <em>MRP</em>-<em>8</em>/<em>14</em> among healthy individuals predict the risk of future cardiovascular events.

Analysis of the protein patterns of normal and psoriatic noncultured unfractionated keratinocytes has revealed several low-molecular-weight proteins that are highly up-regulated in psoriatic epidermis. Here, we have cloned and sequenced the cDNA (clone 10<em>8</em>5) for one of these proteins that we have termed psoriasin. The deduced sequence predicted a protein of molecular weight of 11,457 daltons and a pI of 6.77. The protein co-migrated with psoriasin as determined by two-dimensional (2D) gel analysis of [35S]-methionine-labeled proteins expressed by RK13 cells transfected with clone 10<em>8</em>5 using the vaccinia virus expression system. Analysis of the predicted sequence revealed a potential calcium-binding sequence of the EF-hand type, as well as the absence of a signal sequence at its amino terminal. Psoriasin is not related to other proteins that migrate closely in 2D gels (<em>MRP</em> <em>14</em>, also known as calgranulin B, L1 and calprotectin; <em>MRP</em> <em>8</em>, or calgranulin A and cystatin A or stefin A), and bears no significant sequence homology with any other protein of known primary structure. Increased expression of psoriasin mRNA in psoriatic keratinocytes was confirmed by Northern blotting and in situ hybridization. Psoriasin showed a restricted occurrence in fetal human tissues as determined by 2D gel electrophoresis. Of 21 tissues analyzed, only ear, skin, and tongue showed significant levels of this protein. Psoriasin was not detected in normal human fibroblasts, lymphocytes, endothelial cells and transformed epithelial cells of keratinocyte origin. Granulocyte extracts contained this protein suggesting that its overexpression by psoriatic keratinocytes may be linked to the inflammatory stimuli.

Two calcium-binding proteins, named migration inhibitory factor-related proteins-<em>8</em> (<em>MRP</em>-<em>8</em>) and <em>MRP</em>-<em>14</em>, are primarily expressed by circulating human neutrophils and monocytes. Evidence accumulating from the investigations of several independent groups is now leading to an improved understanding of the biology of these proteins. Both <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em> display features characteristic of members of the S100 family of calcium-binding proteins. Some of these features predict functions for <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em> but to date an exact and well-defined function remains elusive. Here we review the available information and highlight evidence that suggests the function of <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em> may be associated with both monocyte and neutrophil activation and the accumulation of these cells in inflammatory sites.

The <em>14</em>-kDa myeloid-related protein (<em>MRP</em>-<em>14</em>) and its heterodimeric partner, <em>MRP</em>-<em>8</em>, are members of the S100 family of calcium-binding proteins (S100A9 and S100A<em>8</em>, respectively). Their importance in neutrophil function is implied by their unusual abundance in neutrophil cytosol (approximately 40% of cytosolic protein). Previous work from our laboratory has demonstrated the extracellular association of these proteins with vascular endothelium adjacent to transmigrating leukocytes. We report here a function for <em>MRP</em>-<em>14</em> as a stimulator of neutrophil adhesion mediated by the beta 2 integrin, Mac-1. <em>MRP</em>-<em>14</em> is an affinity regulator of Mac-1 because it promotes binding of soluble ligand and expression of an "activation reporter" epitope of high affinity beta 2 integrins recognized by mAb24. The activity of <em>MRP</em>-<em>14</em> is confined to regulating integrin function because, unlike other inflammatory agonists, there was no release of L-selectin, up-regulation of cytosolic Mac-1, or induction of neutrophil respiratory burst or calcium flux. Furthermore, <em>MRP</em>-<em>14</em> does not act as a chemoattractant or cause alterations in cell shape or cytoskeleton. <em>MRP</em>-<em>8</em> has a regulatory role in <em>MRP</em>-<em>14</em> activity, inhibiting the adhesion induced by <em>MRP</em>-<em>14</em> through the formation of the heterodimer. In terms of mechanism of action, <em>MRP</em>-<em>14</em> does not increase Mac-1 function by direct binding to this integrin but recognizes a distinct receptor on neutrophils. This receptor interaction is pertussis toxin sensitive, indicating that <em>MRP</em>-<em>14</em>-generated signals leading to a Mac-1 affinity increase are heterotrimeric G protein dependent. We postulate that <em>MRP</em>-<em>14</em> and <em>MRP</em>-<em>8</em> are important in vivo candidates for the regulated adhesion of neutrophils through control of Mac-1 activity.

BACKGROUND

Myeloid-related protein (<em>MRP</em>)-<em>8</em> (S100A<em>8</em>) and <em>MRP</em>-<em>14</em> (S100A9) are members of the S100 family of calcium-modulated proteins that regulate myeloid cell function and control inflammation, in part, through activation of Toll-like receptor-4 and the receptor for advanced glycation end products. A transcriptional profiling approach in patients with acute coronary syndromes identified <em>MRP</em>-<em>14</em> as a novel predictor of myocardial infarction. Further studies demonstrated that elevated plasma levels of <em>MRP</em>-<em>8</em>/<em>14</em> heterodimer predict increased risk of first and recurrent cardiovascular events. Beyond its serving as a risk marker, whether <em>MRP</em>-<em>8</em>/<em>14</em> participates directly in vascular inflammation and disease remains unclear.

RESULTS

We evaluated vascular inflammation in wild-type and <em>MRP</em>-<em>14</em>-deficient (<em>MRP</em>-<em>14</em>(-/-)) mice that lack <em>MRP</em>-<em>8</em>/<em>14</em> complexes with experimental arterial injury, vasculitis, or atherosclerosis. After femoral artery wire injury, <em>MRP</em>-<em>14</em>(-/-) mice had significant reductions in leukocyte accumulation, cellular proliferation, and neointimal formation compared with wild-type mice. In a cytokine-induced local Shwartzman-like reaction that produces thrombohemorrhagic vasculitis, <em>MRP</em>-<em>14</em>(-/-) mice had significant reductions in neutrophil accumulation, lesion severity, and hemorrhagic area. In response to high-fat feeding, mice doubly deficient in apolipoprotein E and <em>MRP</em>-<em>8</em>/<em>14</em> complexes had attenuation in atherosclerotic lesion area and in macrophage accumulation in plaques compared with mice deficient in apolipoprotein E alone.

CONCLUSIONS

This study demonstrates that <em>MRP</em>-<em>8</em>/<em>14</em> broadly regulates vascular inflammation and contributes to the biological response to vascular injury by promoting leukocyte recruitment.

Myeloid-related protein <em>14</em> (<em>MRP</em>-<em>14</em>) and its heterodimeric partner, <em>MRP</em>-<em>8</em>, are cytosolic calcium-binding proteins, highly expressed in neutrophils and monocytes. To understand the function of <em>MRP</em>-<em>14</em>, we performed targeted disruption of the <em>MRP</em>-<em>14</em> gene in mice. <em>MRP</em>-<em>14</em>(-/-) mice showed no obvious phenotype and were fertile. <em>MRP</em>-<em>8</em> mRNA but not protein is present in the myeloid cells of these mice, suggesting that the stability of <em>MRP</em>-<em>8</em> protein is dependent on <em>MRP</em>-<em>14</em> expression. A compensatory increase in other proteins was not detected in cells lacking <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em>. Although the morphology of <em>MRP</em>-<em>14</em>(-/-) myeloid cells was not altered, they were significantly less dense. When Ca(2+) responses were investigated, there was no change in the maximal response to the chemokine MIP-2. At lower concentrations, however, there was reduced responsiveness in <em>MRP</em>-<em>14</em>(-/-) compared with <em>MRP</em>-<em>14</em>(+/+) neutrophils. This alteration in the ability to flux Ca(2+) did not impair the ability of the <em>MRP</em>-<em>14</em>(-/-) neutrophils to respond chemotactically to MIP-2. In addition, the myeloid cell functions of phagocytosis, superoxide burst, and apoptosis were unaffected in <em>MRP</em>-<em>14</em>(-/-) cells. In an in vivo model of peritonitis, <em>MRP</em>-<em>14</em>(-/-) mice showed no difference from wild-type mice in induced inflammatory response. The data indicate that <em>MRP</em>-<em>14</em> and <em>MRP</em>-<em>8</em> are dispensable for many myeloid cell functions.

The S100 family proteins <em>MRP</em>-<em>8</em> (S100A<em>8</em>) and <em>MRP</em>-<em>14</em> (S100A9) form a heterodimer that is abundantly expressed in neutrophils, monocytes, and some secretory epithelia. In inflamed tissues, the <em>MRP</em>-<em>8</em>/<em>14</em> complex is deposited onto the endothelium of venules associated with extravasating leukocytes. To explore the receptor interactions of <em>MRP</em>-<em>8</em>/<em>14</em>, we use a model system in which the purified <em>MRP</em>-<em>8</em>/<em>14</em> complex binds to the cell surface of an endothelial cell line, HMEC-1. This interaction is mediated by the <em>MRP</em>-<em>14</em> subunit and is mirrored by recombinant <em>MRP</em>-<em>14</em> alone. The cell surface binding of <em>MRP</em>-<em>14</em> was blocked by heparin, heparan sulfate, and chondroitin sulfate B, and the binding sites were sensitive to heparinase I and trypsin treatment but not to chondroitinase ABC. Furthermore <em>MRP</em>-<em>8</em>/<em>14</em> and <em>MRP</em>-<em>14</em> did not bind to a glycosaminoglycan-minus cell line. <em>MRP</em>-<em>14</em> has a high affinity for heparin (K(d) = 6.1 +/- 3.4 nm), and this interaction mimicked that with the endothelial cells. We therefore conclude that the <em>MRP</em>-<em>8</em>/<em>14</em> complex binds to endothelial cells via the <em>MRP</em>-<em>14</em> subunit interacting chiefly with heparan sulfate proteoglycans. CD36 and RAGE, two other putative receptors for <em>MRP</em>-<em>8</em>/<em>14</em>, were not expressed by HMEC-1 cells. This binding activity may explain the immobilization of the <em>MRP</em>-<em>8</em>/<em>14</em> complex on endothelium that is observed in vivo.

Using a monoclonal antibody to macrophage migration inhibition factor (MIF), two proteins were isolated from supernatants of Concanavalin A-stimulated human peripheral blood mononuclear cells which seem to have complexed to a third component carrying the MIF activity. They are therefore designated MIF-related proteins or <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em> according to their apparent molecular weights. Partial amino acid sequences have been determined and their cDNA have been cloned and expressed in Escherichia coli. Both are calcium-binding proteins and <em>MRP</em>-<em>8</em> seems to be largely homologous to the cystic fibrosis antigen (Dorin et al., 19<em>8</em>7). Antisera were raised in the rabbit against the recombinant proteins and their expression in cells and tissues studied using immunohistological techniques. The proteins are only found in blood granulocytes and monocytes. In culture the number of positive monocytes sharply increased and then declined with time, suggesting that their expression is associated with early stages of monocyte/macrophage differentiation and absent from resident macrophages in all tested tissues. In acute inflammatory reactions, e.g. gingivitis, <em>MRP</em>-<em>8</em> is never seen in the tissue, whereas <em>MRP</em>-<em>14</em> is expressed by intravascular monocytes and perivascular macrophages. In contrast, in chronic inflammation, e.g. rheumatoid arthritis, <em>MRP</em>-<em>8</em> is also expressed by macrophages in the tissue. From this it is concluded that <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em> are expressed sequentially at defined stages of monocyte/macrophage differentiation and that dysregulation of this process in chronic inflammation is mirrored by the presence of <em>MRP</em>-<em>8</em>-positive macrophages in the tissue.

In mycophenolate mofetil (MMF)-treated organ transplant recipients, lower mycophenolic acid (MPA) plasma concentrations have been found in cyclosporine (CsA) compared with tacrolimus (Tac)-based immunosuppressive regimens. We previously demonstrated that CsA decreases exposure to MPA and increases exposure to its metabolite MPA-glucuronide (MPAG), possibly by interfering with the biliary excretion of MPAG. To elucidate the role of the multidrug resistance-associated protein (<em>Mrp</em>)-2 in the interaction between MMF and CsA, we treated three groups of 10 <em>Mrp</em>2-deficient rats (TR- rat) for 6 days with either vehicle, CsA (<em>8</em> mg/kg) or Tac (4 mg/kg) by oral gavage. Hereafter, co-administration with MMF (20 mg/kg) was started in all groups and continued through day <em>14</em>. The 24-h MPA/MPAG area under the concentration-time curve (AUC) was determined after single (day 7) and multiple MMF doses (day <em>14</em>). On both study days, there were no significant differences in the mean MPA and MPAG AUC between CsA and Tac-treated animals. We conclude that the pharmacokinetics of MMF are comparable in <em>Mrp</em>2-deficient rats receiving either CsA or Tac as co-medication. This finding suggests that CsA-mediated inhibition of the biliary excretion of MPAG by the <em>Mrp</em>2 transporter is the mechanism responsible for the interaction between CsA and MMF.

The phenotypical and functional heterogeneity of different macrophage subpopulations are defined by discrete changes in the expression of two S100 calcium-binding proteins, migration inhibitory factor-related proteins (<em>MRPs</em>) <em>8</em> and <em>14</em>. To further our understanding of MRP<em>8</em> and MRP<em>14</em> in the developmental stages of inflammatory responses, overexpression of the <em>MRPs</em> was obtained through a combination of a T7-based expression vector and the Escherichia coli BL21 (DE3) cell line. An efficient, two-step chromatographic protocol was then developed for rapid, facile purification. Extensive biophysical characterization and chemical cross-linking experiments show that MRP<em>8</em> and MRP<em>14</em> form oligomers with a strong preference to associate as a heterodimer. Heteronuclear NMR experiments indicate that a specific well packed dimer is formed only in equimolar mixtures of the two proteins. Our results suggest that there is a unique complementarity in the interface of the MRP<em>8</em>/MRP<em>14</em> complex that cannot be fully reproduced in the MRP<em>8</em> and MRP<em>14</em> homodimers.

<em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em> are calcium-binding proteins belonging to the S-100 protein family which have been shown to be associated with specific stages of myeloic/monocytic cell differentiation. Members of this protein family are shown to form homo- and heterodimers. Complex formation has also been observed in preliminary experiments for <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em>. To evaluate the in vivo relevance of the <em>MRP</em> complex formation and the stoichiometric ratio of individual components complexes were isolated from granulocytes and monocytes by immunoaffinity chromatography using monospecific antibodies. The purified fraction of the <em>MRPs</em> was found to contain monomers and dimers as shown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by silver staining and immunoblotting. Similar results were obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of crude cell extracts. The existence of the <em>MRP</em> complexes in vivo was demonstrated by chemical cross-linking and subsequent isolation of complexes by immunoaffinity chromatography. Two new, highly abundant complexes were found in addition to the heterodimer, but neither monomers nor homodimers were detected. The two larger protein complexes (35.0 and 4<em>8</em>.5 kDa) were identified as [<em>MRP</em>-<em>8</em>)2.(<em>MRP</em>-<em>14</em>] trimer and [<em>MRP</em>-<em>8</em>)2.(<em>MRP</em>-<em>14</em>)2) tetramer, respectively. All complexes could be shown to be noncovalently associated in vivo. Furthermore, the association of <em>MRPs</em> was shown to be Ca2+ dependent.

Analysis by means of two-dimensional (2D) gel electrophoresis of the protein patterns of normal and psoriatic unfractionated non-cultured keratinocytes has revealed a few low-molecular-weight proteins that are highly up-regulated in psoriatic skin. These include psoriasin; calgranulin B, also known as <em>MRP</em> <em>14</em>, L1, or calprotectin; calgranulin A or <em>MRP</em> <em>8</em>; and cystatin A or stefin A. Here, we have cloned and sequenced the cDNA (clone 1592) encoding a new member of this group of low-molecular-weight proteins [isoelectric focusing (IEF) SSP 3007 in the keratinocyte 2D gel protein database] that we have termed PA-FABP (psoriasis-associated fatty acid-binding protein). The deduced sequence predicted a protein with molecular weight of 15,164 daltons and a calculated pI of 6.96, values that are close to those recorded in the keratinocyte 2D gel protein database. The protein comigrated with PA-FABP as determined by 2D gel analysis of [35S]-methionine-labeled proteins expressed by transformed human amnion (AMA) cells transfected with clone 1592 using the vaccinia virus expression system and reacted with a rabbit polyclonal antibody raised against 2D gel purified PA-FABP. Structural analysis of the amino acid sequence revealed 4<em>8</em>%, 52%, and 56% identity to known low-molecular-weight fatty acid-binding proteins belonging to the FABP family. Northern blot analysis showed that PA-FABP mRNA is indeed highly up-regulated in psoriatic keratinocytes. The transcript is present in human cell lines of epithelial and lymphoid (Molt 4) origin but cannot be detected in normal or SV40 transformed MRC-5 fibroblasts. 2D gel protein analysis of normal primary keratinocytes cultured for at least <em>8</em> d under conditions that promoted incomplete terminal differentiation [serum-free keratinocyte (SFK) medium supplemented with epidermal growth factor (EGF), pituitary extract, and 10% fetal calf serum] revealed a strong up-regulation of PA-FABP, psoriasin, calgranulins A and B, and a few other proteins that are highly expressed in psoriatic skin. The levels of these proteins exceeded by far those observed in non-cultured normal keratinocytes implying that the cultured cells have followed an altered pattern of differentiation that resembles--at least in part--that of non-cultured psoriatic keratinocytes. The implications of these results for the study of psoriasis are discussed.

OBJECTIVE

Fever of unknown origin is a diagnostic challenge in children, especially for differentiation of systemic-onset juvenile idiopathic arthritis (systemic-onset JIA) and infectious diseases. We undertook this study to analyze the relevance of myeloid-related proteins (<em>MRPs</em>) <em>8</em> and <em>14</em>, endogenous activators of Toll-like receptor 4, in diagnosis and pathogenesis of systemic-onset JIA.

METHODS

Serum concentrations of <em>MRP</em>-<em>8</em>/<em>MRP</em>-<em>14</em> were analyzed in 60 patients with systemic-onset JIA, <em>8</em>5 patients with systemic infections, 40 patients with acute lymphoblastic leukemia, 5 patients with acute myeloblastic leukemia, 1<em>8</em> patients with neonatal-onset multisystem inflammatory disease (NOMID), and 50 healthy controls. In addition, we investigated the link between interleukin-1beta (IL-1beta) and <em>MRP</em>-<em>8</em>/<em>MRP</em>-<em>14</em> in systemic-onset JIA.

RESULTS

Serum <em>MRP</em>-<em>8</em>/<em>MRP</em>-<em>14</em> concentrations were significantly (P < 0.001) elevated in patients with active systemic-onset JIA (mean +/- 95% confidence interval <em>14</em>,920 +/- 4,030 ng/ml) compared with those in healthy controls (340 +/- 70 ng/ml), patients with systemic infections (2,640 +/- 720 ng/ml), patients with acute lymphoblastic leukemia (650 +/- 2<em>8</em>0 ng/ml), patients with acute myeloblastic leukemia (<em>8</em>40 +/- 940 ng/ml), and patients with NOMID (2,<em>8</em>30 +/- 5<em>8</em>0 ng/ml). In contrast to C-reactive protein levels, <em>MRP</em>-<em>8</em>/<em>MRP</em>-<em>14</em> concentrations distinguished systemic-onset JIA from infections, with a specificity of 95%. <em>MRP</em>-<em>14</em> in serum of patients with systemic-onset JIA was a strong inducer of IL-1beta expression in phagocytes.

CONCLUSIONS

The analysis of <em>MRP</em>-<em>8</em>/<em>MRP</em>-<em>14</em> in serum is an excellent tool for the diagnosis of systemic-onset JIA, allowing early differentiation between patients with systemic-onset JIA and those with other inflammatory diseases. <em>MRP</em>-<em>8</em>/<em>MRP</em>-<em>14</em> and IL-1beta represent a novel positive feedback mechanism activating phagocytes via 2 major signaling pathways of innate immunity during the pathogenesis of systemic-onset JIA.

BACKGROUND

Using a transcriptional profiling approach, we recently identified myeloid-related protein <em>8</em>/<em>14</em> (<em>MRP</em>-<em>8</em>/<em>14</em>) to be expressed by platelets during acute myocardial infarction (MI). Elevated concentrations of <em>MRP</em>-<em>8</em>/<em>14</em> are associated with a higher risk for future cardiovascular events in apparently healthy individuals but have not been assessed with respect to prognosis in patients with acute coronary syndrome.

METHODS

We performed a nested case-control study (n = 237 case-control pairs) among patients enrolled in the Pravastatin or Atorvastatin Evaluation and Infection Therapy: Thrombolysis in Myocardial Infarction 22 (PROVE IT-TIMI 22) trial (mean follow-up 24 months) to investigate the risk of cardiovascular death or MI associated with <em>MRP</em>-<em>8</em>/<em>14</em> measured at 30 days after an acute coronary syndrome.

RESULTS

Patients with cardiovascular death or MI after 30 days (cases) had higher median [25th, 75th percentile] <em>MRP</em>-<em>8</em>/<em>14</em> levels than patients who remained free of recurrent events (5.6 [2.<em>8</em>, 13.5] mg/L vs 4.0 [1.9, 10.1] mg/L, P = .020). The risk of a recurrent cardiovascular event increased with each increasing quartile of <em>MRP</em>-<em>8</em>/<em>14</em> (P-trend = 0.007) such that patients with the highest levels had a 2.0-fold increased odds (95% CI 1.1-3.6, P = .029) of a recurrent event after adjusting for standard risk indicators, randomized treatment, and C-reactive protein. Patients with elevated levels of <em>MRP</em>-<em>8</em>/<em>14</em> and high-sensitivity C-reactive protein showed significantly increased risk of cardiovascular death or MI compared with patients with the lowest levels of both markers (adjusted odds ratio 2.1, 95% CI 1.2-3.<em>8</em>).

CONCLUSIONS

Myeloid-related protein <em>8</em>/<em>14</em> may be a useful biomarker of platelet and inflammatory disease activity in atherothrombosis and may serve as a novel target for therapeutic intervention.

Synovial macrophages play a pivotal role in the pathogenesis of chronic autoimmune arthritis by contributing to local inflammation and tissue damage and are therefore a primary target for therapeutic intervention. The aim of the present study was to investigate in more detail the relative contribution of different synovial macrophage subsets with potentially different inflammatory or anti-inflammatory functions by analysing the two most frequent forms of human autoimmune arthritis, spondyloarthropathy (SpA) and rheumatoid arthritis (RA). Both infiltrating macrophages from peripheral blood expressing myeloid-related proteins (<em>MRP</em>) <em>8</em> and <em>14</em>, and resident tissue macrophages expressing CD163 were abundant in inflamed synovium. Whereas the global number of synovial macrophages was similar in both diseases, infiltrating macrophages were increased in the RA lining layer in contrast with resident tissue macrophages, which were more frequently observed in SpA. Soluble <em>MRP</em><em>8</em>/<em>MRP</em><em>14</em> complexes, which were secreted locally in the joint during the infiltration process, were increased in the serum of arthritis patients and, in contrast with soluble CD163 shed from resident tissue macrophages, correlated well with global inflammatory parameters. Treatment in vivo with anti-TNFalpha had a rapid and pronounced effect on the infiltration of <em>MRP</em>-positive macrophages into tissues, as evidenced by histopathological analysis and serum <em>MRP</em><em>8</em>/<em>MRP</em><em>14</em> levels. Taken together, these data support an important role for infiltrating versus resident tissue macrophages in human autoimmune synovitis and indicate that macrophage products such as soluble <em>MRP</em><em>8</em>/<em>MRP</em><em>14</em> complexes are valuable biomarkers for the experimental and clinical monitoring of specific disease mechanisms in vivo.

BACKGROUND

Pediatric obstructive sleep apnea (OSA) leads to multiple end-organ morbidities that are mediated by the cumulative burden of oxidative stress and inflammation. Because not all children with OSA exhibit increased systemic inflammation, genetic and environmental factors may be affecting patterns of DNA methylation in genes subserving inflammatory functions.

METHODS

DNA from matched children with OSA with and without high levels of high-sensitivity C-reactive protein (hsCRP) were assessed for DNA methylation levels of 24 inflammatory-related genes. Primer-based polymerase chain reaction assays in a case-control setting involving 47 OSA cases and 31 control subjects were conducted to confirm the findings; hsCRP and myeloid-related protein (<em>MRP</em>) <em>8</em>/<em>14</em> levels were also assayed.

RESULTS

Forkhead box P3 (FOXP3) and interferon regulatory factor 1 (IRF1) showed higher methylation in six children with OSA and high hsCRP levels compared with matched children with OSA and low hsCRP levels (P < 0.05). In the case-control cohort, children with OSA and high CRP levels had higher log FOXP3 DNA methylation levels compared with children with OSA and low CRP levels and control subjects. IRF1 did not exhibit significant differences. FOXP3 DNA methylation levels correlated with hsCRP and <em>MRP</em> <em>8</em>/<em>14</em> levels and with apnea-hypopnea index (AHI), BMI z score, and apolipoprotein B levels. A stepwise multiple regression model showed that AHI was independently associated with FOXP3 DNA methylation levels (P < 0.03).

CONCLUSIONS

The FOXP3 gene, which regulates expression of T regulatory lymphocytes, is more likely to display increased methylation among children with OSA who exhibit increased systemic inflammatory responses. Thus, epigenetic modifications may constitute an important determinant of inflammatory phenotype in OSA, and FOXP3 DNA methylation levels may provide a potential biomarker for end-organ vulnerability.

The recruitment of monocytes from the bloodstream is crucial in the accumulation of macrophages and dendritic cells in type 1 diabetic pancreases. Adhesion via integrins to endothelium and extracellular matrix proteins, such as fibronectin (FN), and the production of myeloid-related protein (<em>MRP</em>)-<em>8</em>, -<em>14</em>, and -<em>8</em>/<em>14</em> by recently transmigrated monocytes are thought to be instrumental in such recruitment. We determined the FN-adhesive capacity and integrin expression of monocytes of type 1 and type 2 diabetic patients and related them to the subjects' serum levels of <em>MRP</em>-<em>8</em>, -<em>14</em> and -<em>8</em>/<em>14</em>. Monocytes of type 1 diabetic patients displayed an increased adhesion to fibronectin in comparison with type 2 patients and healthy control subjects but had a normal expression of the FN binding integrins CD29, CD49a, CD49d, and CD49e (although CD11b and CD1<em>8</em> expression was increased). <em>MRP</em>-<em>8</em>/<em>14</em>, which was increased in the sera of type 1 diabetic patients, induced healthy donor monocytes to adhere to FN and upregulate CD11b expression in a dosage-dependent manner. The observed <em>MRP</em>-induced increased adhesion of monocytes to FN and upregulation of CD11b most likely contributed to a facilitated accumulation of monocytes and monocyte-derived cells at the site of inflammation, in this case the pancreatic islets.

OBJECTIVE

Myeloid related proteins (<em>MRP</em>) <em>8</em> and <em>14</em> and the heterodimer <em>MRP</em><em>8</em>/<em>14</em> are myeloid differentiation markers present on infiltrating tissue macrophages in inflammation but not on resident tissue macrophages. We determined the pattern of expression of <em>MRP</em><em>8</em>, <em>MRP</em><em>14</em>, and the <em>MRP</em><em>8</em>/<em>14</em> heterodimer (27E10 antigen) in rheumatoid arthritis (RA) synovial membrane (SM).

METHODS

SM samples were obtained from patients with RA at joint replacement surgery or at arthroscopy and patients without joint disease (healthy subjects) and immunostained for <em>MRP</em><em>8</em>, <em>MRP</em><em>14</em>, 27E10 antigen (<em>MRP</em><em>8</em>/<em>14</em> heterodimer), and CD6<em>8</em>. Positive cell staining was measured by quantitative analysis.

RESULTS

SM from <em>8</em> patients with RA, including 7 who had paired samples from both adjacent to the cartilage-pannus junction (CPJ) and an area remote from the CPJ, and 2 healthy controls were analyzed. In RA, CD6<em>8</em>+ cells accumulated in greater numbers adjacent to the CPJ than remote from the CPJ [mean +/- standard error of the mean (SEM) 4<em>8</em><em>8</em>+/-103 and 2<em>8</em>6+/-76 cells/mm2, respectively; p = 0.01]. SM lining layer (LL) <em>MRP</em><em>8</em>, <em>MRP</em><em>14</em>, and 27E10 staining was observed predominantly adjacent to the CPJ and only in patients with active disease. Minimal or absent LL <em>MRP</em> staining was observed in non-CPJ sections. Synovial sublining layer (SL) <em>MRP</em><em>8</em>, <em>MRP</em><em>14</em>, and 27E10 staining was also observed only in patients with active disease, but in contrast to the LL, SL staining was observed predominantly in sections remote from the CPJ.

CONCLUSIONS

MRP antigens, representing activation markers on SM macrophages, were observed predominantly in the LL of sections adjacent to the CPJ samples. These original observations suggest altered activation and differentiation of lining layer macrophages at the site of maximal cartilage destruction in RA.

OBJECTIVE

To determine the long-term safety and efficacy of rilonacept, an anti-interleukin-1 fusion protein, in patients with active systemic juvenile idiopathic arthritis (JIA).

METHODS

In patients with systemic JIA, ages 4-20 years, the efficacy of rilonacept was evaluated using 30%, 50%, and 70% levels of improvement according to the adapted American College of Rheumatology (ACR) Pediatric 30, 50, and 70 response criteria, respectively. Efficacy and safety were evaluated during 23 months of open-label treatment (3 phases) after a 4-week, double-blind, placebo-controlled phase. Following double-blind treatment with 2.2 mg/kg or 4.4 mg/kg of rilonacept, patients were eligible to receive open-label treatment at their prior dose, with adjustments. Reductions in the median daily dose of oral prednisone and improvements in laboratory parameters of disease activity (i.e., decreased levels of D-dimer and myeloid-related proteins [MRPs]) were also evaluated.

RESULTS

Twenty-four patients entered the double-blind study and 23 entered the open-label period. Patients were predominantly white and female, and had a median age of <em>14</em>.0 years at baseline. No significant differences in efficacy were observed between the rilonacept- and placebo-treated patients during the double-blind phase, but fever and rash completely resolved by month 3 in all patients during the open-label treatment period and did not recur. Adapted ACR Pediatric 30, 50, and 70 response rates at 3 months from the start of the study were 7<em>8</em>.3%, 60.9%, and 34.<em>8</em>%, respectively; these responses were generally maintained over the study duration. Levels of D-dimer and <em>MRP</em>-<em>8</em>/<em>MRP</em>-<em>14</em> dramatically improved during the study, and in 22 of 23 patients, the prednisone dose was decreased or prednisone therapy was discontinued. No serious treatment-related adverse events were observed.

CONCLUSIONS

Sustained improvements in clinical and laboratory measures of the articular and systemic manifestations of systemic JIA were achieved in >50% of rilonacept-treated patients over 2 years. Treatment with rilonacept had a substantial steroid-sparing effect and was generally well-tolerated.

Myeloid-related proteins (<em>MRPs</em>) <em>8</em> and <em>14</em> are cytosolic proteins secreted from myeloid cells as proinflammatory mediators. Currently, the functional role of circulating extracellular MRP<em>8</em>/<em>14</em> is unclear. Our present study identifies extracellular MRP<em>8</em>/<em>14</em> as an autocrine player in the leukocyte adhesion cascade. We show that E-selectin-PSGL-1 interaction during neutrophil rolling triggers Mrp<em>8</em>/<em>14</em> secretion. Released MRP<em>8</em>/<em>14</em> in turn activates a TLR4-mediated, Rap1-GTPase-dependent pathway of rapid β2 integrin activation in neutrophils. This extracellular activation loop reduces leukocyte rolling velocity and stimulates adhesion. Thus, we identify Mrp<em>8</em>/<em>14</em> and TLR4 as important modulators of the leukocyte recruitment cascade during inflammation in vivo.

OBJECTIVE

To analyze which cellular compartments are involved in the initial phase of systemic-onset juvenile rheumatoid arthritis (JRA), and to investigate the role that myeloid-related protein <em>8</em> (<em>MRP</em>-<em>8</em>) and <em>MRP</em>-<em>14</em>, two S-100 proteins that are primarily expressed in phagocytes, play in the disease.

METHODS

Skin biopsy samples obtained during patients' acute episodes of systemic-onset JRA were analyzed by immunohistochemistry and in situ hybridization. Concentrations of <em>MRP</em>-<em>8</em>/<em>MRP</em>-<em>14</em> in serum were determined by enzyme-linked immunosorbent assay.

RESULTS

By analyzing biopsy samples from cutaneous rashes during the initial phase of systemic-onset JRA, we discovered infiltration of leukocytes expressing <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em>. Surprisingly, keratinocytes also showed de novo synthesis of these proinflammatory proteins, indicating activation of epithelial cells during systemic-onset JRA. Serum concentrations of <em>MRP</em>-<em>8</em>/<em>MRP</em>-<em>14</em> were 120-fold higher compared with healthy controls and approximately 12-fold higher compared with patients with other inflammatory diseases. Concentrations of <em>MRP</em>-<em>8</em>/<em>MRP</em>-<em>14</em> in patients with systemic-onset JRA fell dramatically after remission was induced.

CONCLUSIONS

The exceptionally high serum levels of <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em> in active systemic-onset JRA make them prime candidates as markers for monitoring disease activity and response to treatment. Since <em>MRP</em>-<em>8</em>/<em>MRP</em>-<em>14</em> exhibit direct effects on leukocyte adhesion to the vascular endothelium, their extensive expression in the epidermis indicates an active role for these S-100 proteins in the initial phase of this systemic autoimmune disease.

A rabbit polyclonal antibody raised against myeloid-related protein <em>8</em> (<em>MRP</em>-<em>8</em>), a protein of the S100 family, recognized another S100 protein (<em>MRP</em>-<em>14</em>) as well as a protein of 6.5 kDa (p6) in the cytosol of resting neutrophils. p6 was found to be a novel member of the S100 family. It consisted of two isoforms with pI values of 6.2 (the minor form, p6a) and 6.3 (the major form, p6b) and constituted 5% of the total cytosolic proteins. Both isoforms were also demonstrated in the cytosol of monocytes, but not in lymphocytes, as previously shown for <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em>. Only the major isoform bound radioactive Ca2+, as also observed for <em>MRP</em>-<em>8</em>, whereas the different variants of <em>MRP</em>-<em>14</em> were all labelled. On neutrophil activation with opsonized zymosan, a stimulant known to require extracellular Ca2+, 5<em>8</em>% of p6a and 42% of p6b was translocated to the membrane. With phorbol 12-myristate 13-acetate, a Ca(2+)-independent stimulant, no translocation was detected. This translocation pattern was similar to that observed with <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em>. In addition, p6, <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em> were specifically associated with the cytoskeletal fraction of the membrane. The Ca(2+)-dependent translocation of the novel S100 protein in parallel with <em>MRP</em>-<em>8</em> and <em>MRP</em>-<em>14</em> suggests a role for these proteins in regulating the Ca2+ signal to the membrane cytoskeleton and thus in regulating neutrophil activation.

Recent studies indicate that mucosal innate immune factors modulate HIV-1 infection in vitro. Our interest was to examine the levels of innate mucosal factors for their potential association with HIV-1 shedding in the female genital tract. Vaginal lavages were collected from HIV-1-infected women who had vaginal viral loads (VVL) that were below, within, or above the 90% confidence interval (CI) predicted by their matched plasma viral loads. Innate immune factors [cathepsin D, lactoferrin (Lf), myeloid related protein (<em>MRP</em>)-<em>8</em>, <em>MRP</em>-<em>8</em>/<em>14</em>, secretory leukocyte protease inhibitor, and gp340], cytokines (IL-1beta and TNF-alpha), and chemokines (MIP-1alpha, MIP-1beta, RANTES, and SDF-1alpha) were quantified by ELISA. Leukocyte levels were determined using a leukocyte reagent strip for urinalysis. Lf, <em>MRP</em>-<em>8</em>/<em>14</em>, gp340, and IL-1beta levels were significantly higher in vaginal lavages above the 90% CI and generally correlated with each other and with VVL. Leukocyte levels were significantly higher in the lavages that had virus shedding above the 90% CI and correlated strongly with Lf levels and VVL. In this group of women, these results suggest that the levels of certain innate immune factors are more closely associated with HIV-1 shedding in the genital mucosa than plasma virus concentrations.