Potential predictive biomarkers of adalimumab response in patients with hidradenitis suppurativa.

BACKGROUND: Adalimumab provides significant efficacy for patients with hidradenitis suppurativa (HS), which was demonstrated by at least 50% of patients achieving a clinical response by week 12 that was maintained through to week 168 in the PIONEER trials. OBJECTIVES: To identify whether there are biomarkers that could predict adalimumab response, as well as markers that differentially respond to adalimumab in patients with HS. METHODS: Baseline and week-12 plasma samples from the PIONEER studies were used to assess the levels of circulating proteins by multiplex and enzyme-linked immunosorbent assays. RESULTS: Analyses revealed significantly higher high-sensitivity C-reactive protein (hs-CRP) and chemokine (C-C motif) ligand (CCL) 16 (HCC-4) levels in nonresponders at baseline and identified a multivariate response signature of calprotectin, fractalkine and HCC-4, reaching an 86% predictive accuracy rate for adalimumab response. Additionally, post-treatment reduction of plasma C-X-C motif chemokine ligand (CXCL)9, CXCL8 (interleukin-8) and CCL19 (macrophage inflammatory protein 3ß) were greater in adalimumab super-responders than in nonresponders (P = 0·026, P = 0·044 and P = 0·026, respectively). These cytokines are involved in the recruitment of innate and adaptive inflammatory cells, and/or stimulation of certain inflammatory responses, suggesting that these pathways could be disease drivers in adalimumab nonresponders. CONCLUSIONS: These initial results suggest HCC-4, calprotectin and fractalkine could be potential predictive biomarkers of adalimumab response in HS and identified possible tumour necrosis factor-independent disease pathways.

In vitro and in vivo characterization of A-940894: a potent histamine H4 receptor antagonist with anti-inflammatory properties.

BACKGROUND AND PURPOSE: The histamine H4 receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In this report, we describe the in vitro and in vivo anti-inflammatory properties of a potent histamine H4 receptor antagonist, A-940894 (4-piperazin-1-yl-6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-d]pyrimidin-2-ylamine). EXPERIMENTAL APPROACH: We have analysed the pharmacological profile of A-940894 at mouse native, rat recombinant and human recombinant and native, histamine H4 receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan-induced peritonitis. KEY RESULTS: A-940894 potently binds to both human and rat histamine H4 receptors and exhibits considerably lower affinity for the human histamine H1, H2 or H3 receptors. It potently blocked histamine-evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine-induced shape change of mouse bone marrow-derived mast cells and chemotaxis of human eosinophils in vitro. In a mouse mast cell-dependent model of zymosan-induced peritonitis, A-940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D2 levels. Finally, A-940894 has good pharmacokinetic properties, including half-life and oral bioavailability in rats and mice. CONCLUSIONS AND IMPLICATIONS: These data suggest that A-940894 is a potent and selective histamine H4 receptor antagonist with pharmacokinetic properties suitable for long-term in vivo testing and could serve as a useful tool for the further characterization of histamine H4 receptor pharmacology.

The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation.

Atherosclerosis causes most acute coronary syndromes and strokes. The pathogenesis of atherosclerosis includes recruitment of inflammatory cells to the vessel wall and activation of vascular cells. CD44 is an adhesion protein expressed on inflammatory and vascular cells. CD44 supports the adhesion of activated lymphocytes to endothelium and smooth muscle cells. Furthermore, ligation of CD44 induces activation of both inflammatory and vascular cells. To assess the potential contribution of CD44 to atherosclerosis, we bred CD44-null mice to atherosclerosis-prone apoE-deficient mice. We found a 50-70% reduction in aortic lesions in CD44-null mice compared with CD44 heterozygote and wild-type littermates. We demonstrate that CD44 promotes the recruitment of macrophages to atherosclerotic lesions. Furthermore, we show that CD44 is required for phenotypic dedifferentiation of medial smooth muscle cells to the "synthetic" state as measured by expression of VCAM-1. Finally, we demonstrate that hyaluronan, the principal ligand for CD44, is upregulated in atherosclerotic lesions of apoE-deficient mice and that the low-molecular-weight proinflammatory forms of hyaluronan stimulate VCAM-1 expression and proliferation of cultured primary aortic smooth muscle cells, whereas high-molecular-weight forms of hyaluronan inhibit smooth muscle cell proliferation. We conclude that CD44 plays a critical role in the progression of atherosclerosis through multiple mechanisms.

A crucial role for CD44 in inflammation.

Current therapies for chronic inflammatory diseases typically act through the nonspecific downregulation of immune cell activation. However, it is becoming increasingly evident that parenchymal cells are also active participants in the inflammatory process. Future prospects for the treatment of inflammation should therefore include the targeting of specific inflammatory pathways in both immune cells and parenchymal cells. CD44, a cell-adhesion molecule that is ubiquitously expressed on leukocytes and parenchymal cells, has been implicated, together with its ligand hyaluronan (HA), in several inflammatory diseases. The mechanisms of action of CD44-HA interactions in inflammation might provide potential targets for therapy.

Interleukin-1beta-induced expression of monocyte chemotactic protein-1 in the rabbit retina: an in situ and immunohistochemical study.

In this study, the temporal and spatial expression of the chemotactic factor monocyte chemotactic protein-1 (MCP-1) was examined in the rabbit retina after challenge with the proinflammatory cytokine interleukin-1beta (IL-1). In these tissues, IL-1 induces an acute inflammatory response of the epiretinal vessels that peaks approximately 24 h postintraocular injection (pi) with the cytokine. At 2 h after challenge with IL-1, MCP-1 mRNA was expressed by perivascular microglial cells and astrocytes that form the glial limitans. Protein analysis at 3 h pi with IL-1 confirmed these sites of MCP-1 expression. The intensity of the mRNA and protein signals increased at 6 h and at 24 h. At these time points, MCP-1 message and protein also were detected in infiltrating macrophages and, at the latest time point, in endothelial cells as well. These data support the conclusion that IL-1 provides a strong stimulus for the rapid expression of MCP-1 mRNA and protein in retinal tissues, and they further support the role of endogenous glial cells as important sources of mediators involved in the regulation of inflammation occurring within the nervous system.

Lymphoid tissue homing chemokines are expressed in chronic inflammation.

Secondary lymphoid tissue chemokine (SLC) and B lymphocyte chemoattractant (BLC) are homing chemokines that have been implicated in the trafficking of lymphocytes and dendritic cells in lymphoid organs. Lymphotoxin-alpha (LTalpha), a cytokine crucial for development of lymphoid organs, is important for expression of SLC and BLC in secondary lymphoid organs during development. Here we report that transgenic expression of LTalpha induces inflammation and ectopic expression of SLC and BLC in the adult animal. LTbeta was not necessary for induction of BLC and SLC in inflamed tissues, whereas, in contrast, tumor necrosis factor receptor-1 was found to be important for the LTalpha-mediated induction of these chemokines. The ectopic expression of LTalpha is associated with a chronic inflammation that closely resembles organized lymphoid tissue and this lymphoid neogenesis can also be seen in several chronic inflammatory diseases, including in the pancreas of the prediabetic nonobese diabetic (NOD) mouse. Expression of SLC was also observed in the pancreas of prediabetic NOD mice. This study implicates BLC and SLC in chronic inflammation and presents further evidence that LTalpha orchestrates lymphoid organogenesis both during development and in inflammatory processes.

TGF-beta downmodulates cytokine-induced monocyte chemoattractant protein (MCP)-1 expression in human endothelial cells. A putative role for TGF-beta in the modulation of TNF receptor expression.

The expression of chemokines, including monocyte chemoattractant protein (MCP)-1, by many cell types contributes to the pathogenesis of inflammatory diseases. We examined MCP-1 expression in human umbilical vein endothelial cells (EC) following cytokine treatment. We specifically compared the effect of TGF-beta 1 on this cytokine-induced expression, as TGF-beta has been shown to have immunosuppressive effects on EC. EC expressed MCP-1 mRNA and protein in response to TNF alpha, IFN gamma or IL-1beta, but not TGF-beta1. TGF-beta1 in cotreatment with either TNF alpha or IL-1beta, but not IFN gamma, significantly decreased MCP-1 mRNA and protein expression, as compared to TNF alpha or IL-1beta treatment alone. Pretreatment with TGF-beta had no effect on any cytokine-induced MCP-1 expression. TGF-beta had no effect on MCP-mRNA stability. Examination of TNF receptor expression by flow cytometry revealed that TNF alpha treatment caused a decrease of p75 expression on the cell surface. The p55 receptor was not detected at the cell surface, but was localized intracellularly by confocal microscopy. Treatment of EC with TGF-beta alone decreased p75 surface expression and in cotreatment with TNF alpha, caused an additive decrease in p75 surface expression, as compared to TNF alpha treatment alone. Whereas mRNA expression for both receptors was increased with TNF alpha treatment, this was decreased with TGF-beta/TNF alpha cotreatment, as compared to TNF alpha treatment alone. Thus, the expression of TNF receptors was also down-modulated by TGF-beta. These findings indicate additional mechanisms by which TGF-beta exerts immunosuppressive properties on EC.

Differential induction of adhesion molecule and chemokine expression by LTalpha3 and LTalphabeta in inflammation elucidates potential mechanisms of mesenteric and peripheral lymph node development.

Lymphotoxin (LT) is a member of the proinflammatory TNF family of cytokines that plays a critical role in the development of lymphoid tissue. It has previously been reported that the presence of the LTalpha transgene under the control of the rat insulin promoter results in inflammation at the sites of transgene expression. LTalpha transgene expression results in expression of the adhesion molecules VCAM, ICAM, peripheral node addressin (a marker of peripheral lymph nodes), and mucosal addressin cellular adhesion molecule (a marker of mucosal lymphoid tissue, including mesenteric lymph nodes). In this study to determine the mechanisms by which LT promotes inflammation and lymphoid tissue organization, we analyzed the regulation of expression of adhesion molecules and chemokines in LT transgenic mice. The results demonstrate that LTalpha3 induces expression of the adhesion molecules VCAM, ICAM, and mucosal addressin cellular adhesion molecule as well as the chemokines RANTES, IFN-inducible protein-10, and monocyte chemotactic protein-1, while LTalphabeta is required for the induction of peripheral node addressin that may contribute to the recruitment of L-selectinhigh CD44low naive T cells. These data provide candidate mediators of LT-induced inflammation as well as potential mechanisms by which LTalpha and LTalphabeta may differentially promote the development of mesenteric and peripheral lymph nodes.

Lymphotoxin alpha3 induces chemokines and adhesion molecules: insight into the role of LT alpha in inflammation and lymphoid organ development.

Lymphotoxin (LT) plays an important role in inflammation and lymphoid organ development, though the mechanisms by which it promotes these processes are poorly understood. Toward this end, the biologic activities of a recently generated recombinant murine (m) LT alpha preparation were evaluated. This cytokine preparation was effective at inducing cytotoxicity of WEHI target cells with 50% maximal killing observed with 1.2 ng/ml. mLT alpha also induced the expression of inflammatory mediators in the murine endothelial cell line bEnd.3. rmLT alpha induced expression of the adhesion molecules VCAM, ICAM, E-selectin, and the mucosal addressin cellular adhesion molecule, MAdCAM-1. When mLT alpha, human (h) LT alpha, and mTNF-alpha were compared, mLT alpha was the most potent inducer of MAdCAM-1. None of these cytokines induced the peripheral node addressin, PNAd. mLT alpha also induced expression of the chemokines RANTES, IFN-inducible protein 10 (IP-10), and monocyte chemotactic protein 1 (MCP-1). mRNA levels peaked 4 h following treatment with mLT alpha and declined through the 24-h treatment period. LT alpha also induced chemokine protein within 8 h of treatment, which increased through the 24-h treatment period. These data demonstrate that the proinflammatory effects of LT alpha3 may be mediated in part through the induction of adhesion molecule and chemokine expression. Further, LT alpha3 may promote development of lymphoid tissue through induction of chemokines and the mucosal addressin MAdCAM-1. These data confirm previous observations in transgenic and knockout mice that LT alpha3 in the absence of LT beta carries out unique biologic activities.

Differential activities of secreted lymphotoxin-alpha3 and membrane lymphotoxin-alpha1beta2 in lymphotoxin-induced inflammation: critical role of TNF receptor 1 signaling.

Lymphotoxin (LT, LT alpha, TNF beta) is a member of the immediate TNF family that also includes TNF-alpha and lymphotoxin-beta (LT beta). LT is produced by activated lymphocytes and functions as either a secreted homotrimer or a membrane-associated heterotrimer that includes the transmembrane protein LT beta. Secreted LT alpha3 can bind to two cell surface receptors, TNFR1 and TNFR2, while the membrane-bound heterotrimer LT alpha1beta2 has been shown to interact with a distinct receptor, LT betaR. LT alpha induces inflammation at the sites of expression of a rat insulin promoter-driven lymphotoxin (RIPLT) transgene in the pancreas and kidney. To determine the role of the various ligands and their receptors in LT-induced inflammation, mice deficient in either TNFR1, TNFR2, or LT beta were crossed to RIPLT-transgenic mice. Our results indicate that LT alpha-induced inflammation is dependent on the interaction of LT alpha3 with TNFR1, and there is no obvious role for TNFR2, since in its absence, LT alpha-induced inflammation is quantitatively and qualitatively similar to that seen in the wild type. However, the absence of LT beta results in accentuated infiltration of the kidney with an increase in the proportion of memory cells in the infiltrate. These data show a crucial role for the secreted LT alpha3 signaling via TNFR1 in LT alpha-induced inflammation, and a separate and distinct role for the membrane LT alpha1beta2 form in this inflammatory process.

Mediators of inflammation.

Therapeutic studies and genetically engineered animals have elucidated the inflammatory roles of cytokines and chemokines in autoimmune disease. Most unexpected has been a continuum of recent evidence demonstrating that inflammatory mediators are crucial in lymphoid organ development, thus suggesting that these hitherto unrelated processes have common elements with implications for determinant spreading.

Differential effects of transforming growth factor-beta 1 on interleukin-1-induced cellular inflammation and vascular permeability in the rabbit retina.

Intra-vitreal injection of 300 U of interleukin (IL)-1 beta into the rabbit eye induces an inflammation of the retina characterized by hemorrhage, monocyte and neutrophil infiltration, and an increase in vascular permeability that peaks 24 h post-injection. Since the epiretinal vessels involved in this inflammation form part of the blood-retina barrier, we used this model to investigate the effects of the immunosuppressive cytokine TGF beta 1 on inflammation within the context of the central nervous system. We found that intra-vitreal injection of 1 microgram rh TGF beta administered concomitantly with rh IL-1 beta significantly reduced IL-1 beta-induced hemorrhage by 78%, and monocyte and neutrophil infiltration by 53% and 62%, respectively. In contrast, TGF beta did not reduce the IL-1 beta-induced increase in vascular permeability. However, TGF beta by itself caused a statistically significant increase in serum proteins in perfused tissues of the eye, to give a 3.1 +/- 0.4 fold increase in protein content over control values. No cellular inflammation accompanied this alteration in vascular permeability. These data indicate that whereas the local administration of TGF beta may be an effective inhibitor of cellular inflammation in the CNS, the effects on alterations in vascular permeability and accumulation of serum proteins may be more complex.

The ordered array of perivascular macrophages is disrupted by IL-1-induced inflammation in the rabbit retina.

In this study we have shown that an antibody to CD18 identified a population of cells in the rabbit retina that resembled the perivascular macrophage found in other regions of the central nervous system. In the normal retina these cells possessed a ramified morphology and presented in an ordered array on the vitreal surface in association with the epiretinal vessels. Approximately 50% of the perivascular macrophages constitutively expressed MHC class II. In response to interleukin-1 beta (IL-1 beta)-induced inflammation, these cells became activated, as evidenced by a change from a ramified to an ameboid morphology and increased expression of MHC class II, and migrated away from the vessels. These changes were first detected around 3 h post-intraocular challenge coincident with the onset of inflammation. At the peak of the inflammatory response (approximately 24 h post-challenge), many activated perivascular macrophages were no longer associated with the vessels and formed long "cord" of MHC class II+ cells associated with underlying deposits of fibrin. In eyes challenged with heat-inactivated IL-1, no change in the morphology or distribution of the perivascular macrophage was noted. At 3 weeks post-challenge with IL-1, the number and distribution of the perivascular macrophages were restored to baseline values, although with a reduced cell size. Since these changes closely resemble those that occur in non-lymphoid dendritic cells in the skin, heart, and/or kidney following activation with cytokines or bacterial products, the results suggest that the perivascular macrophage represents the dendritic cell of the retina and may thus play an important role in immune surveillance in the eye and maintenance of the blood-retina barrier.