Peripheral Inflammatory Sites Research Articles

Immunoreactive corticotropin-releasing hormone and its binding sites in the rat ovary.

Corticotropin-releasing hormone (CRH), the principal neuropeptide regulator of pituitary ACTH secretion, is also produced at peripheral inflammatory sites, where it acts as a proinflammatory cytokine, and by the Leydig cell of the testis, where it exerts autocrine inhibition of testosterone biosynthesis. Because key ovarian functions, such as ovulation and luteolysis, represent aseptic inflammatory responses, and because the theca cell is the functional equivalent of the Leydig cell, we explored the CRH presence in the ovary, first, by specific CRH immunohistochemistry of adult cycling female Sprague-Dawley rat ovaries. We detected cytoplasmic immunoreactive CRH (IrCRH) in theca and stromal cells and in cells within the corpora lutea, at all phases of the estrous cycle. Using a specific radioimmunoassay, we measured IrCRH in extracts of rat ovaries (0.042-0.126 pmol/g wet tissue). The mobility of the ovarian IrCRH molecule was similar to that of rat/human CRH by reverse phase HPLC. To investigate the CRH action in the ovary, we identified, characterized, and localized CRH receptors in the rat ovary. Binding was linear with increasing tissue concentration, saturable, and of high affinity. Scatchard analysis of 125I-Tyr-ovine CRH competitive displacement curves indicated a high affinity binding site with a Kd of approximately 6 nM and a Bmax value of approximately 61 fM/mg protein. Autoradiographic studies revealed CRH receptors primarily in ovarian theca and stroma. We conclude that IrCRH and CRH receptors are present in rat ovaries, suggesting that this neuropeptide may play a regulatory role in this gonad, perhaps through its proinflammatory properties and/or by participating in the auto/paracrine regulation of steroid biosynthesis. Functional studies are necessary to define the role(s) of CRH in the ovary.

Regulated activity of the distal promoter-like element of the human corticotropin-releasing hormone gene and secondary structural features of its corresponding transcripts

Corticotropin-releasing hormone (CRH) plays a major role in the coordination of the stress response. Its gene is expressed in multiple brain regions, the peripheral sympathetic system and the placenta, as well as in peripheral inflammatory sites where CRH acts as a pro-inflammatory cytokine. The human (h) CRH gene, in addition to its primary promoter (TATA box I), has a second distal promoter-like structure (TATA box II) and a functional cyclic adenosine monophosphate-responsive element, all of which are preserved in the rat and ovine genes. To examine the functionality of TATA II, we positioned a 881-bp-long segment of the 5′ flanking region of the hCRH gene containing TATA II, but lacking TATA I, upstream from a chloramphenicol acetyltransferase (CAT) reporter gene cloned in a pUC vector. We transfected COS-7 cells with this construct and examined responsiveness of CAT activity to potential stimulants and inhibitors. Phorbol ester (TPA) and forskolin had mild but clear stimulatory effects on CAT expression (~ 1.5- and ~ 1.3-fold, respectively), with a combined effect of ~ 1.9-fold. Dexamethasone (DEX) inhibited TPA-stimulated CAT activity by ~ 2.6-fold. In contrast, in the presence of a co-transfected glucocorticoid receptor cDNA expression plasmid, DEX augmented TPA-stimulated CAT expression by ~ 3.1-fold. The predicted secondary structures of the primary transcripts employing the distal and proximal promoters had significant differences, which could affect their stability and translatability. These observations suggest that TATA II functions weakly in the heterologous system examined, independently of TATA I, and directs the synthesis of the second largest pool of CRH transcripts with a longer 5′ non-coding region than the transcripts driven by TATA I. The former possess structural features that may differentiate them from the latter in terms of stability and translatability.

Local secretion of corticotropin-releasing hormone in the joints of Lewis rats with inflammatory arthritis.

Corticotropin-releasing hormone (CRH), the principal regulator of the hypothalamic-pituitary-adrenal axis, is also secreted in peripheral inflammatory sites, where it acts as a local proinflammatory agent. Arthritis-susceptible LEW/N rats have profoundly deficient hypothalamic CRH responses to inflammatory stimuli and other stressors. Arthritis-resistant F344/N rats, on the other hand, have a robust increase in hypothalamic CRH in response to the same stimuli. Contrasting with these hypothalamic CRH responses, we now show that CRH expression is markedly increased in the joints and surrounding tissues of LEW/N rats with streptococcal cell wall- and adjuvant-induced arthritis, whereas it is not increased in similarly treated F344/N rats and is only transiently increased in congenitally athymic nude LEW.rnu/rnu rats. Glucocorticoid treatment suppressed, but did not eliminate, CRH immunoreactivity in the joints of LEW/N rats. CRH mRNA was present in inflamed synovia, as well as in spinal cord, and inflamed synovia also expressed specific CRH-binding sites. We compared CRH expression in inflamed joints with another well-characterized proinflammatory neuropeptide, substance P (SP), and found that SP immunoreactivity paralleled that of CRH. In summary, although LEW/N rats have deficient hypothalamic CRH responses to inflammatory stimuli compared with F344/N rats, they express relatively high levels of CRH at the site of inflammation. Analogous to SP, CRH may be delivered to the inflammatory site by peripheral nerves and/or synthesized at the inflammatory site. These data provide further support for the concept that CRH not only triggers the pituitary-adrenal antiinflammatory cascade, but also functions as an antithetically active local mediator of acute and chronic inflammatory arthritis. These data also illustrate the complex interrelationships of the nervous, endocrine, immune, and inflammatory systems.

Interleukin‐6 in peripheral blood and inflammatory sites in Behçet′s disease

Interleukin-6, a potent pro-inflammatory cytokine, might be involved in Behçet's disease (BD) pathological pathways. We investigated IL-6 levels in sera and synovial fluids collected from BD patients. The IL-6 production was also studied in vivo, by measuring its activity in culture supernatants of PBMC and alveolar macrophages, stimulated or not with LPS. The patients with BD were compared to RA patients and healthy controls. High IL-6 levels were observed in sera, synovial fluid and LPS stimulated PBMC supernatants, from active BD patients, similar to those of RA patients. Alveolar macrophages production of IL-6 was significantly elevated in two active BD patients with an interstitial pneumonia, when compared to controls. These elevated levels of IL-6 suggest its involvement in the inflammatory sites of BD, which may be related to the progression of the acute lesions, at least in the joints and in the lungs.

Autocrine or Paracrine Inflammatory Actions of Corticotropin-Releasing Hormone in Vivo

Corticotropin-releasing hormone (CRH) functions as a regulator of the hypothalamic-pituitary-adrenal axis and coordinator of the stress response. CRH receptors exist in peripheral sites of the immune system, and CRH promotes several immune functions in vitro. The effect of systemic immunoneutralization of CRH was tested in an experimental model of chemically induced aseptic inflammation in rats. Intraperitoneal administration of rabbit antiserum to CRH caused suppression of both inflammatory exudate volume and cell concentration by approximately 50 to 60 percent. CRH was detected in the inflamed area but not in the systemic circulation. Immunoreactive CRH is therefore produced in peripheral inflammatory sites where, in contrast to its systemic indirect immunosuppressive effects, it acts as an autocrine or paracrine inflammatory cytokine.

Control of granulocyte-macrophage colony-stimulating factor production in normal endothelial cells by positive and negative regulatory elements.

Granulocyte-macrophage (GM)-CSF is an acidic glycoprotein involved in the hemopoietic response to inflammation and in the functional activation of mature blood cells. The protein is produced in response to a number of inflammatory mediators by mesenchymal cells present in the hemopoietic microenvironment and at peripheral sites of inflammation. To understand the molecular basis for the regulation of GM-CSF gene expression, nuclear run-on assays and a functional analysis of potential regulatory sequences were performed in normal human endothelial cells. These studies suggest that GM-CSF is regulated by both transcriptional and post-transcriptional mechanisms. By using hybrid constructs containing a reporter gene and varying lengths of the regions flanking the human GM-CSF gene, a 14-bp sequence was identified in the region upstream of the GM-CSF cap site which increased reporter expression in response to a number of inflammatory mediators. In addition, despite a failure to detect GM-CSF-specific RNA or protein in unstimulated cells, basal transcription from the GM-CSF promoter was readily detectable. To account for this apparent discrepancy, sequences present in the 3' untranslated region of the GM-CSF gene were found to substantially reduce the level of reporter gene expression in a number of cell types. Homologous sequences are found in other genes which share a similar pattern of expression, and may provide the molecular basis for the coordinate regulation of multiple inflammatory response genes.