Phillip L. Campbell

Epigallocatechin-3-gallate inhibits IL-6 synthesis and suppresses transsignaling by enhancing soluble gp130 production.

Regulation of IL-6 transsignaling by the administration of soluble gp130 (sgp130) receptor to capture the IL-6/soluble IL-6R complex has shown promise for the treatment of rheumatoid arthritis (RA). However, enhancing endogenous sgp130 via alternative splicing of the gp130 gene has not yet been tested. We found that epigallocatechin-3-gallate (EGCG), an anti-inflammatory compound found in green tea, inhibits IL-1β–induced IL-6 production and transsignaling in RA synovial fibroblasts by inducing alternative splicing of gp130 mRNA, resulting in enhanced sgp130 production. Results from in vivo studies using a rat adjuvant-induced arthritis model showed specific inhibition of IL-6 levels in the serum and joints of EGCG-treated rats by 28% and 40%, respectively, with concomitant amelioration of rat adjuvant-induced arthritis. We also observed a marked decrease in membrane-bound gp130 protein expression in the joint homogenates of the EGCG-treated group. In contrast, quantitative RT-PCR showed that the gp130/IL-6Rα mRNA ratio increased by ∼2-fold, suggesting a possible mechanism of sgp130 activation by EGCG. Gelatin zymography results showed EGCG inhibits IL-6/soluble IL-6R–induced matrix metalloproteinase-2 activity in RA synovial fibroblasts and in joint homogenates, possibly via up-regulation of sgp130 synthesis. The results of these studies provide previously undescribed evidence of IL-6 synthesis and transsignaling inhibition by EGCG with a unique mechanism of sgp130 up-regulation, and thus hold promise as a potential therapeutic agent for RA.

Monocyte Chemoattractant Protein-1 and Macrophage Inflammatory Protein-1α as Possible Biomarkers for the Chronic Pelvic Pain Syndrome

PURPOSE The chronic pelvic pain syndrome is characterized by pelvic pain, voiding symptoms and varying degrees of inflammation within expressed prostatic secretions. We evaluated the chemokines monocyte chemoattractant protein 1 (CCL2) and macrophage inflammatory protein-1alpha (CCL3) in expressed prostatic secretions to identify marker increases associated with inflammatory (IIIA) and noninflammatory (IIIB) chronic pelvic pain syndrome. In addition, chemokine levels were correlated with clinical pain as determined by the National Institutes of Health chronic prostatitis symptom index. MATERIALS AND METHODS Expressed prostatic secretions were collected by digital rectal examination, and evaluated by enzyme linked immunosorbent assays for monocyte chemoattractant protein 1 and macrophage inflammatory protein-1alpha in 154 patients including controls (13), those with benign prostatic hyperplasia (54), chronic pelvic pain syndrome IIIA (37) and IIIB (50). Monocyte chemoattractant protein 1 and macrophage inflammatory protein-1alpha levels were compared between IIIA, IIIB and the control subgroups, and correlated against the chronic prostatitis symptom index and pain subscore using a Spearman test. RESULTS Mean levels of monocyte chemoattractant protein 1 in the control, inflammatory benign prostatic hyperplasia, noninflammatory benign prostatic hyperplasia, inflammatory chronic pelvic pain syndrome and noninflammatory chronic pelvic pain syndrome were 599.4, 886.0, 1,636.5, 3,261.2 and 2,272.7 pg/ml, respectively. Mean levels of macrophage inflammatory protein-1alpha in the control, inflammatory benign prostatic hyperplasia, noninflammatory benign prostatic hyperplasia, IIIA chronic pelvic pain syndrome and IIIB chronic pelvic pain syndrome were 140.1, 299.4, 238.7, 1,057.8 and 978.4 pg/ml, respectively. For each cytokine both chronic pelvic pain syndrome subtypes had statistically higher levels than the control group and patients with benign prostatic hyperplasia (p = 0.0002). Receiver operating curves using monocyte chemoattractant protein 1 levels greater than 704 pg/ml and macrophage inflammatory protein-1alpha greater than 146 pg/ml identified patients with chronic pelvic pain syndrome with an accuracy of 90% from control patients. Macrophage inflammatory protein-1alpha levels (p = 0.0007) correlated with the pain subscore of the chronic prostatitis symptom index while monocyte chemoattractant protein 1 (p = 0.71) did not. CONCLUSIONS Monocyte chemoattractant protein 1 and macrophage inflammatory protein-1alpha within the prostatic fluid in both chronic pelvic pain syndrome subtypes provide candidate future biomarkers for chronic pelvic pain syndrome. In addition, macrophage inflammatory protein-1alpha increase in expressed prostatic secretions provides a new marker for clinical pain in chronic pelvic pain syndrome patients. Given these findings prostatic dysfunction likely has a role in the pathophysiology of this syndrome. These chemokines may serve as effective diagnostic markers and modulators against the chemokines could provide an attractive treatment strategy in individuals with chronic pelvic pain syndrome.

Targeting the Myofibroblast Genetic Switch: Inhibitors of Myocardin-Related Transcription Factor/Serum Response Factor–Regulated Gene Transcription Prevent Fibrosis in a Murine Model of Skin Injury

Systemic sclerosis (SSc), or scleroderma, similar to many fibrotic disorders, lacks effective therapies. Current trials focus on anti-inflammatory drugs or targeted approaches aimed at one of the many receptor mechanisms initiating fibrosis. In light of evidence that a myocardin-related transcription factor (MRTF)–and serum response factor (SRF)–regulated gene transcriptional program induced by Rho GTPases is essential for myofibroblast activation, we explored the hypothesis that inhibitors of this pathway may represent novel antifibrotics. MRTF/SRF-regulated genes show spontaneously increased expression in primary dermal fibroblasts from patients with diffuse cutaneous SSc. A novel small-molecule inhibitor of MRTF/SRF-regulated transcription (CCG-203971) inhibits expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and collagen 1 (COL1A2) in both SSc fibroblasts and in lysophosphatidic acid (LPA)–and transforming growth factor β (TGFβ)–stimulated fibroblasts. In vivo treatment with CCG-203971 also prevented bleomycin-induced skin thickening and collagen deposition. Thus, targeting the MRTF/SRF gene transcription pathway could provide an efficacious new approach to therapy for SSc and other fibrotic disorders.

Green tea extract inhibits chemokine production, but up-regulates chemokine receptor expression, in rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis

OBJECTIVE Evaluation of the efficacy of green tea extract (GTE) in regulating chemokine production and chemokine receptor expression in human RA synovial fibroblasts and rat adjuvant-induced arthritis (AIA). METHODS Fibroblasts isolated from human RA synovium were used in the study. Regulated upon activation normal T cell expressed and secreted (RANTES)/CCL5, monocyte chemoattractant protein (MCP)-1/CCL2, growth-regulated oncogene (GRO)alpha/CXCL1 and IL-8/CXCL8 production was measured by ELISA. Western blotting was used to study the phosphorylation of protein kinase C (PKC)delta and c-Jun N-terminal kinases (JNK). Chemokine and chemokine receptor expression was determined by quantitative RT-PCR. The benefit of GTE administration in rat AIA was determined. RESULTS GTE (2.5-40 microg/ml) inhibited IL-1beta-induced MCP-1/CCL2 (10 ng/ml), RANTES/CCL5, GROalpha/CXCL1 and IL-8/CXCL8 production in human RA synovial fibroblasts (P < 0.05). However, GTE inhibited MCP-1/CCL2 and GROalpha/CXCL1 mRNA synthesis in RA synovial fibroblasts. Furthermore, GTE also inhibited IL-1beta-induced phosphorylation of PKCdelta, the signalling pathway mediating IL-1beta-induced chemokine production. Interestingly, GTE preincubation enhanced constitutive and IL-1beta-induced CCR1, CCR2b, CCR5, CXCR1 and CXCR2 receptor expression. GTE administration (200 mg/kg/day p.o.) modestly ameliorated rat AIA, which was accompanied by a decrease in MCP-1/CCL2 and GROalpha/CXCL1 levels and enhanced CCR-1, -2, -5 and CXCR1 receptor expression in the joints of GTE administered rats. CONCLUSIONS Chemokine receptor overexpression with reduced chemokine production by GTE may be one potential mechanism to limit the overall inflammation and joint destruction in RA.

Association of Plasma Interleukin-18 Levels with Emotion Regulation and μ-Opioid Neurotransmitter Function in Major Depression and Healthy Volunteers

BACKGROUND Alterations in central neurotransmission and immune function have been documented in major depression (MDD). Central and peripheral endogenous opioids are linked to immune functioning in animal models, stress-activated, and dysregulated in MDD. We examined the relationship between μ-opioid receptor (OR)-mediated neurotransmission and a proinflammatory cytokine (interleukin [IL]-18). METHODS We studied 28 female subjects (14 MDDs, 14 control subjects) with positron emission tomography and [(11)C] carfentanil (μ-OR selective) during neutral and sadness states. With a simple regression model in SPM2 (Wellcome Trust, London, England) we identified brain regions where baseline μ-OR availability (nondisplaceable binding potential [BP(ND)]) and sadness-induced changes in μ-OR BP(ND) were associated with baseline IL-18. RESULTS Baseline IL-18 was greater in MDDs than control subjects [t(25) = 2.13, p = .04]. In control subjects IL-18 was correlated with negative emotional ratings at baseline and during sadness induction. In MDDs, IL-18 was positively correlated with baseline regional μ-OR BP(ND) and with sadness-induced μ-opioid system activation in the subgenual anterior cingulate, ventral basal ganglia, and amygdala. CONCLUSIONS This study links plasma IL-18 with sadness-induced emotional responses in healthy subjects, the diagnosis of MDD, and μ-opioid functioning, itself involved in stress adaptation, emotion regulation, and reward. This suggests that IL-18 represents a marker associated with emotion regulation/dysregulation at least in part through central opioid mechanisms.

Citrullination of epithelial neutrophil-activating peptide 78/CXCL5 results in conversion from a non-monocyte-recruiting chemokine to a monocyte-recruiting chemokine.

To examine whether the citrullinated chemokines epithelial neutrophil–activating peptide 78 (ENA‐78)/CXCL5, macrophage inflammatory protein 1α/CCL3, and monocyte chemotactic protein 1/CCL2 are detected in the biologic fluid of patients with rheumatoid arthritis (RA), and if so, to determine the biologic activities of these chemokines.

Fucosyltransferase 1 mediates angiogenesis, cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation

IntroductionWe previously reported that sialyl Lewisy, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewisy antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined.MethodsAssay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed.ResultsTotal α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation.ConclusionsThese data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA.

Acute experimental changes in mood state regulate immune function in relation to central opioid neurotransmission: A model of human CNS-peripheral inflammatory interaction

Although evidence shows depressed moods enhance risk for somatic diseases, molecular mechanisms underlying enhanced somatic susceptibility are ill-defined. Knowledge of these molecular mechanisms will inform development of treatment and prevention strategies across comorbid depressive and somatic illnesses. Existing evidence suggests that interleukin-18 (IL-18; an IL-1 family cytokine) is elevated in depression and implicated in pathophysiology underlying comorbid medical illnesses. We previously identified strong associations between baseline IL-18 and μ-opioid receptor availability in major depressive disorder (MDD) volunteers. Combined with the evidence in animal models, we hypothesized that experimental mood induction would change IL-18, the extent proportional to opioid neurotransmitter release. Using the Velten technique in a [11C]carfentanil positron emission tomography neuroimaging study, we examined the impact of experimentally induced mood (sad, neutral) on plasma IL-18 and relationships with concurrent changes in the central opioid neurotransmission in 28 volunteers (healthy, MDD). Results showed mood induction impacted IL-18 (F2,25=12.2, P<0.001), sadness increasing IL-18 (T27=2.6, P=0.01) and neutral mood reducing IL-18 (T27=−4.1, P<0.001). In depressed volunteers, changes in IL-18 were more pronounced (F2,25=3.6, P=0.03) and linearly proportional to sadness-induced μ-opioid activation (left ventral pallidum, bilateral anterior cingulate cortices, right hypothalamus and bilateral amygdala). These data demonstrate that dynamic changes of a pro-inflammatory IL-1 superfamily cytokine, IL-18, and its relationship to μ-opioid neurotransmission in response to experimentally induced sadness. Further testing is warranted to delineate the role of neuroimmune interactions involving IL-18 in enhancing susceptibility to medical illness (that is, diabetes, heart disease and persistent pain states) in depressed individuals.

Suppression of endothelial cell activity by inhibition of TNFα

IntroductionTNFα is a proinflammatory cytokine that plays a central role in the pathogenesis of rheumatoid arthritis (RA). We investigated the effects of certolizumab pegol, a TNFα blocker, on endothelial cell function and angiogenesis.MethodsHuman dermal microvascular endothelial cells (HMVECs) were stimulated with TNFα with or without certolizumab pegol. TNFα-induced adhesion molecule expression and angiogenic chemokine secretion were measured by cell surface ELISA and angiogenic chemokine ELISA, respectively. We also examined the effect of certolizumab pegol on TNFα-induced myeloid human promyelocytic leukemia (HL-60) cell adhesion to HMVECs, as well as blood vessels in RA synovial tissue using the Stamper-Woodruff assay. Lastly, we performed HMVEC chemotaxis, and tube formation.ResultsCertolizumab pegol significantly blocked TNFα-induced HMVEC cell surface angiogenic E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression and angiogenic chemokine secretion (P < 0.05). We found that certolizumab pegol significantly inhibited TNFα-induced HL-60 cell adhesion to HMVECs (P < 0.05), and blocked HL-60 cell adhesion to RA synovial tissue vasculature (P < 0.05). TNFα also enhanced HMVEC chemotaxis compared with the negative control group (P < 0.05) and this chemotactic response was significantly reduced by certolizumab pegol (P < 0.05). Certolizumab pegol inhibited TNFα-induced HMVEC tube formation on Matrigel (P < 0.05).ConclusionOur data support the hypothesis that certolizumab pegol inhibits TNFα-dependent leukocyte adhesion and angiogenesis, probably via inhibition of angiogenic adhesion molecule expression and angiogenic chemokine secretion.

Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines

OBJECTIVES Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. METHODS Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. RESULTS Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. CONCLUSION Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc.