RDP58, a novel immunomodulatory peptide with anti-inflammatory effects. A pharmacological study in trinitrobenzene sulphonic acid colitis and Crohn disease.

BACKGROUND: Tumour necrosis factor (TNF) plays a key role in the pathogenesis of Crohn disease (CD). RDP58 is a novel anti-inflammatory decapeptide which was developed using a novel rational design strategy. Recently, RDP58 has proved to be a potent inhibitor of TNF production at a post-transcriptional step. The aims of this study were to investigate the anti-inflammatory properties of RDP58 ex vivo in human CD and in vivo in an experimental model colitis. METHODS: Biopsies and lamina propria mononuclear cells from inflamed colonic mucosa of 18 CD patients were cultured for 24 h in the presence or absence of RDP58. TNF was quantified in a bioassay: interferon (IFN)-gamma and interleukin (IL)-1beta levels were measured by enzyme-linked immunosorbent assays. Colitis was induced by intra-rectal administration of 2, 4, 6 trinitrobenzene sulphonic acid (TNBS) in rats. Inflammation was assessed following 7 days of oral therapy with RDP58 or vehicle alone. RESULTS: RDP58 led to decreased TNF and IFN-gamma (but not IL-1beta) production by biopsies and lamina propria mononuclear cells from CD patients. In rats with TNBS-induced colitis, oral RDP58 therapy reduced weight loss and diarrhoea and improved macroscopic and histological inflammation scores. CONCLUSIONS: Our results suggest that RDP58 may be an effective therapy for CD with the clinical advantage of an oral administration.

RDP58, a Novel Immunomodulatory Peptide with Anti-Inflammatory Effects. A Pharmacological Study in Trinitrobenzene Sulphonic Acid Colitis and Crohn Disease.

BACKGROUND: Tumour necrosis factor (TNF) plays a key role in the pathogenesis of Crohn disease (CD). RDP58 is a novel anti-inflammatory decapeptide which was developed using a novel rational design strategy. Recently, RDP58 has proved to be a potent inhibitor of TNF production at a post-transcriptional step. The aims of this study were to investigate the anti-inflammatory properties of RDP58 ex vivo in human CD and in vivo in an experimental model colitis. METHODS: Biopsies and lamina propria mononuclear cells from inflamed colonic mucosa of 18 CD patients were cultured for 24 h in the presence or absence of RDP58. TNF was quantified in a bioassay; interferon (IFN)-γ and interleukin (IL)-1ß levels were measured by enzyme-linked immunosorbent assays. Colitis was induced by intra-rectal administration of 2, 4, 6 trinitrobenzene sulphonic acid (TNBS) in rats. Inflammation was assessed following 7 days of oral therapy with RDP58 or vehicle alone. RESULTS: RDP58 led to decreased TNF and IFN-γ (but not IL-1ß) production by biopsies and lamina propria mononuclear cells from CD patients. In rats with TNBS-induced colitis, oral RDP58 therapy reduced weight loss and diarrhoea and improved macroscopic and histological inflammation scores. CONCLUSIONS: Our results suggest that RDP58 may be an effective therapy for CD with the clinical advantage of an oral administration.

Butyrate inhibits inflammatory responses through NFkappaB inhibition: implications for Crohn's disease.

BACKGROUND/AIM: Proinflammatory cytokines are key factors in the pathogenesis of Crohn's disease (CD). Activation of nuclear factor kappa B (NFkappaB), which is involved in their gene transcription, is increased in the intestinal mucosa of CD patients. As butyrate enemas may be beneficial in treating colonic inflammation, we investigated if butyrate promotes this effect by acting on proinflammatory cytokine expression. METHODS: Intestinal biopsy specimens, isolated lamina propria cells (LPMC), and peripheral blood mononuclear cells (PBMC) were cultured with or without butyrate for assessment of secretion of tumour necrosis factor (TNF) and mRNA levels. NFkappaB p65 activation was determined by immunofluorescence and gene reporter experiments. Levels of NFkappaB inhibitory protein (IkappaBalpha) were analysed by western blotting. The in vivo efficacy of butyrate was assessed in rats with trinitrobenzene sulphonic acid (TNBS) induced colitis. RESULTS: Butyrate decreased TNF production and proinflammatory cytokine mRNA expression by intestinal biopsies and LPMC from CD patients. Butyrate abolished lipopolysaccharide (LPS) induced expression of cytokines by PBMC and transmigration of NFkappaB from the cytoplasm to the nucleus. LPS induced NFkappaB transcriptional activity was decreased by butyrate while IkappaBalpha levels were stable. Butyrate treatment also improved TNBS induced colitis. CONCLUSIONS: Butyrate decreases proinflammatory cytokine expression via inhibition of NFkappaB activation and IkappaBalpha degradation. These anti-inflammatory properties provide a rationale for assessing butyrate in the treatment of CD.

Butyrate and trichostatin A effects on the proliferation/differentiation of human intestinal epithelial cells: induction of cyclin D3 and p21 expression.

BACKGROUND: Sodium butyrate, a product of colonic bacterial fermentation, is able to inhibit cell proliferation and to stimulate cell differentiation of colonic epithelial cell lines. It has been proposed that these cellular effects could be linked to its ability to cause hyperacetylation of histone through the inhibition of histone deacetylase. AIM: To analyse the molecular mechanisms of butyrate action on cell proliferation/differentiation and to compare them with those of trichostatin A, a well known inhibitor of histone deacetylase. METHODS: HT-29 cells were grown in the absence or presence of butyrate or trichostatin A. Cell proliferation and cell cycle distribution were studied after DNA staining by crystal violet and propidium iodide respectively. Cell cycle regulatory proteins were studied by western blot and reverse transcription-polymerase chain reaction. Cell differentiation was followed by measuring brush border enzyme activities. Histone acetylation was studied by acid/urea/Triton acrylamide gel electrophoresis. RESULTS: Butyrate blocked cells mainly in the G(1) phase of the cell cycle, whereas trichostatin A was inhibitory in both G(1) and G(2) phases. Butyrate inhibited the mRNA expression of cyclin D1 without affecting its protein expression and stimulated the protein expression of cyclin D3 without affecting its mRNA expression. Trichostatin A showed similar effects on cyclin D1 and D3. Butyrate and trichostatin A stimulated p21 expression both at the mRNA and protein levels, whereas their effects on the expression of cyclin dependent kinases were slightly different. Moreover, butyrate strongly stimulated the activity of alkaline phosphatase and dipeptidyl peptidase IV, whereas trichostatin A had no effect. Finally, a six hour exposure to butyrate or trichostatin A induced histone H4 hyperacetylation. At 15 and 24 hours, histone H4 remained hyperacetylated in the presence of butyrate, whereas it returned to control levels in the presence of trichostatin A. CONCLUSIONS: The data may explain how butyrate acts on cell proliferation/differentiation, and they show that trichostatin A does not reproduce every effect of butyrate, mainly because of its shorter half life.

Pathways and receptors involved in peptide YY induced contraction of rat proximal colonic muscle in vitro.

BACKGROUND: Peptide YY (PYY) is involved in the regulation of several gut functions, including secretion and motility. It exerts its effects through a family of six receptors, commonly named the Y receptor family. AIMS: To characterise the effects of PYY on strips of rat proximal colon in vitro, and to determine the pathways and receptors involved. METHODS: Contractions of strips removed from the muscle layer of rat proximal colon were recorded under isometric conditions, using PYY, Y receptor agonists and antagonists, and nerve blockers. Reverse transcription-polymerase chain reaction was also performed to detect the presence of mRNA coding for Y receptors. Finally, smooth muscle cells were isolated to estimate the cell length and intracellular Ca(2+) concentration in the presence and absence of PYY. RESULTS: PYY, neuropeptide Y (NPY), pancreatic polypeptide (PP) and [Leu31,Pro34]NPY induced a dose dependent contraction of strips from proximal colon. Tetrodotoxin partially inhibited the PYY and NPY induced contractions, and strongly inhibited the PP induced contraction. Specific antagonists showed the involvement of cholinergic nicotinic receptors and NK1 receptor. BIBP 3226, a specific Y1 antagonist, did not modify the colonic smooth muscle response to PYY, whereas blocking L-type Ca(2+) channels with D-600 abolished its effects. Moreover, PYY induced an increase in intracellular Ca(2+) concentration, associated with a reduction in cell length. mRNA encoding Y1 and Y4 receptors were detected in the muscle strips. CONCLUSIONS: These findings suggest that PYY stimulates colonic contractile activity in vitro through (a) a nervous Y4 dependent pathway and (b) a pathway involving a potential new receptor on myocytes.

Lack of interleukin 10 regulation of antigen presentation-associated molecules expressed on colonic epithelial cells.

BACKGROUND: Colonic epithelial cells may behave as antigen-presenting cells. Interleukin 10 (IL-10) is known to play a major role in the intestinal immune system; however, it remains to be determined whether human intestinal epithelial cells express IL-10 receptor, and whether this cytokine modulates their expression of antigen presentation-associated molecules. METHODS: The binding of biotinylated IL-10 was studied in SW 1116, HT-29 and T84 human colonic epithelial cell lines and freshly isolated normal colonic epithelial cells. Reverse transcription-polymerase chain reaction was also performed to detect IL-10 receptor mRNA. The effect of IL-10 on antigen presentation associated molecules was assessed by flow cytometry. RESULTS: Biotinylated IL-10 bound to SW 1116, HT-29, T84, and normal colonic epithelial cells. IL-10 receptor mRNA was detected in SW 1116 and normal epithelial cells. SW 1116 and HT-29 cells expressed MHC class I and ICAM-1, but not CD80, and SW 1116 constitutively expressed HLA-DR. Interferon-gamma up-regulated HLA-DR and ICAM-1 expression on both cells, whereas lipopolysaccharide increased ICAM-1 expression only on SW 1116. IL-10 failed to modulate these antigens, even after stimulation by lipopolysaccharide or interferon-gamma. Moreover, these molecules decreased IL-10 binding in both lines. CONCLUSION: The presence of IL-10 receptor on intestinal epithelial cells suggest that IL-10 may play a role in mucosal physiology, however its effect on the immune response remains to be determined.

Proteins spontaneously released by rat insulinoma (RIN) cells are anchored on cell membrane by a glycosyl-phosphatidyl-inositol link and inhibit increased RIN cell adhesion of lymphocytes from type 1 diabetic patients and non-obese diabetic mice in vitro.

Our group previously reported an assay for the study of lymphocyte adhesion to insulin-producing cells in which xenogeneic rat insulinoma (RIN) cells were used as targets. The present study found an increased number of RIN-cytoadherent lymphocytes in 63 patients with Type 1 diabetes compared with 150 control subjects and in 211 NOD mice compared with 104 BALB/c mice (p < 0.001). Proteins concentrated from spontaneous RIN cell culture supernatants inhibited increased RIN-adhesion of NOD splenocytes or lymphocytes from diabetic patients (p < 0.001). In addition, increased RIN binding was dose-dependently abolished by RIN membrane extracts. The fact that RIN binding was inhibited by proteins from both membrane and the culture supernatant from RIN cells suggests that soluble inhibitory proteins were spontaneously released into the supernatant from a hydrophobic membrane-bound form. This tended to be confirmed since inhibition obtained with both preparations involved a 55-75 kDa HPLC protein fraction. The possibility that the membrane form of the inhibitory protein was anchored by a glycosylphosphatidylinositol (GPI) tail was evaluated. When RIN cells were treated with PI-PLC, their ability to bind lymphocytes from diabetic patients or NOD splenocytes decreased (p < 0.001) to control levels. Co-incubation with the 55-75 kDa fraction of proteins cleaved from RIN cells by PI-PLC also lowered the number of RIN-adherent NOD splenocytes to control levels. SDS-PAGE and IEF analyses of the 55-75 kDa inhibitory fraction from RIN cell supernatant revealed a major band with Mr 66 kDa and PI5.4, which may correspond to a protein with similar characteristics noted on 2-D electrophoresis of proteins cleaved from RIN cells by PI-PLC. Specific labelling of GPI moieties with 3H-ethanolamine, 3H-glucosamine, or 14C-glucosamine, as well as conversion of the hydrophobic Triton-X114 solubilised form into a hydrophilic form after PI-PLC treatment, confirmed the presence of a GPI anchor in this approximately 66 kDa RIN protein, which could thus be the molecule inhibiting adhesion in the system. Our data suggest that GPI-proteins from insulin-producing cells may influence the immune system both in their membrane-anchored and soluble forms. When considering the binding model, in which beta cells were tumoral and xenogeneic to diabetic lymphocytes, this potential influence of GPI-proteins suggests possible implications in situations of lymphocyte-beta cell interaction, i.e. anti-beta cell autoimmunity, immune reaction against insulinomas, and reaction against islet xenografts.

Induction of fibroblast gelatinase B expression by direct contact with cell lines derived from primary tumor but not from metastases.

During cancer progression, tumor cells interact with stromal cells. As a consequence, matrix metalloproteinases are produced that contribute to the degradation of the extracellular matrix. This study used coculture systems to investigate fibroblast interaction with three colon cancer cell lines isolated from a single patient. Cells from primary colorectal carcinoma, but not from corresponding liver or lymph node metastases, induced gelatinase B expression by fibroblasts of different tissue origin. Remarkably, direct cell-cell contact was required for this induction, which occurred at the pretranslational level (as revealed by Northern blot analysis) and was completely blocked by anti-beta1 integrin monoclonal antibody, but only partially blocked by anti-alpha5 or anti-alpha(v). Induction was also inhibited by cytochalasin D, staurosporine, or dexamethasone, suggesting the need, respectively, for an organized actin cytoskeleton, protein kinase C, and AP-1-driven gene transcription. Our data suggest that direct tumor-stromal cell contact is one inductive event involved in matrix metalloproteinase expression by stromal cells.

Characterization of structural determinants and molecular mechanisms involved in pro-stromelysin-3 activation by 4-aminophenylmercuric acetate and furin-type convertases.

Stromelysin-3 (ST3) is a matrix metalloproteinase (MMP) which has been implicated in cancer progression and in a number of conditions involving tissue remodelling. In contrast to other MMPs which are secreted as zymogens requiring extracellular activation, ST3 is found in the extracellular space as a potentially active mature form, suggesting that the activation of the ST3 proform differs from that of other MMPs. We show in the present study that the ST3 proform is not autocatalytically processed in the presence of 4-aminophenylmercuric acetate (APMA). By using ST3/ST2 chimeras, we demonstrate that resistance to APMA is due to properties associated with both the ST3 pro- and catalytic domains. In agreement with the observation made by Pei and Weiss [Pei and Weiss (1995) Nature (London) 375, 244-247], we find that the requirement for activation of the ST3 proform by the furin convertase is entirely contained within a stretch of 10 amino acids located at the junction between the ST3 pro- and catalytic domains. Furin cleaves human and mouse ST3 equally well. However, PACE-4, a furin-like convertase, is much more efficient on the mouse enzyme, suggesting that ST3 protein determinants other than the conserved Ala-Arg-Asn-Arg-Gln-Lys-Arg sequence preceding the furin cleavage site are implicated in PACE-4 action. Finally, we show that processing of the ST3 proform is inhibited by a furin inhibitor in human MCF7 breast cancer cells stably transfected to constitutively express a full-length human ST3 cDNA. Using brefeldin A, we demonstrate that, in these MCF7 cells, the 56 kDa precursor form of ST3 is post-translationally modified in the cis- or media-Golgi into a 62 kDa proform. Thereafter, its processing into the 47 kDa mature form occurs in the trans-Golgi network and is followed by secretion into the extracellular space.

Characterization of monoclonal antibodies against stromelysin-3 and their use to evaluate stromelysin-3 levels in breast carcinoma by semi-quantitative immunohistochemistry.

Stromelysin-3 (ST3) is a matrix metalloproteinase which is expressed in fibroblastic cells of most human invasive carcinomas and represents a potential new prognostic indicator. Expression of recombinant ST3 forms in Escherichia coli from cDNA constructs indicated that high levels of expression were achieved when the ST3 pro-domain was deleted. The putative mature form of ST3 thus produced and recovered from bacterial inclusion bodies was used to prepare monoclonal antibodies (MAbs) against ST3 by immunization of BALB/C mice. Ten hybridomas producing MAbs against ST3 were obtained and analyzed for their ability to detect endogenous ST3 in breast cancer and in conditioned media from human fibroblasts. One of these MAbs (5ST-4A9) was found to be suitable for the routine detection of ST3 on breast cancer tissue sections, thus opening the possibility to evaluate ST3 prognostic value in breast cancer using semi-quantitative immunohistochemistry.

The 28-kDa N-terminal domain of mouse stromelysin-3 has the general properties of a weak metalloproteinase.

The putative matrix metalloproteinase mouse stromelysin-3 was expressed from Escherichia coli and from a mouse myeloma cell line. In the former case a single major protein of 58-kDa was detectable by immunoblotting, but no proteolytic activity could be elicited by zymography or trypsin or organomercurial treatment as would be expected for a typical matrix metalloproteinase. In the latter case immunodetectable proteins of 55-58 and 27-28-kDa were produced. The effect of trypsin or organomercurial treatment of the 55-58-kDa forms was to generate a 51-kDa form and lower molecular mass fragments. Upon zymographic analysis only the 27-28-kDa forms showed caseinolytic activity. N-terminal sequencing and immunoblotting analysis with antibodies specific to distinct domains of stromelysin-3 indicated that the 27-28-Da stromelysin-3 forms had lost the predicted propeptide and the majority of the C-terminal domain. The purified 28-kDa form of stromelysin-3 could weakly degrade a number of extracellular matrix proteins and was inhibited by TIMP. However, the evidence that mature full-length stromelysin-3 is a metalloproteinase could not be substantiated and the precise role of this protein in vivo remains to be elucidated. By partial analogy with interstitial collagenase, one hypothesis is that stromelysin-3 with an intact C-terminal domain has specific properties for an as yet undefined substrate.

B cell-adherent splenocytes precede the onset of diabetes in low-dose streptozotocin-treated mice.

Splenocytes from low-dose (40 mg.kg-1.day-1) streptozotocin-treated mice were tested for their binding ability to rat insulinoma (RINm-5F) cells in a rosette-forming cell assay, before and during the onset of diabetes. They displayed a higher (p less than 0.0001) RIN-adherence than control splenocytes. Such an enhanced binding of splenocytes from diabetic mice was observed on another B-cell (HIT cell) line, but not on non-B cells (particularly on exocrine pancreatic cells, endocrine cells or natural killer-target cells), suggesting that the increased RIN-binding is B-cell specific. This B-cell specificity was also suggested by the use of increasing splenocytes/RIN ratios showing a saturation of RIN-binding in streptozotocin-treated mice. Depletion of lymphocyte subsets revealed that supernumerary RIN-adherent splenocytes from diabetic mice were mainly T lymphocytes, involving both L3T4+ and Lyt2+ cells. Overall, the increased splenocyte-RIN binding was concomitant with the occurrence of islet destruction, but preceded the onset of hyperglycaemia by five days and even the islet immune infiltration. An increased number of RIN-binding splenocytes was also found in mice treated with 33 mg.kg-1.day-1 of streptozotocin, displaying insulitis but not hyperglycaemia. This phenomenon was not found with splenocytes from mice displaying a "toxic" diabetes induced by a single high dose of streptozotocin. No correlation could thus be found between numbers of RIN-binding splenocytes and blood glucose levels, indicating that this phenomenon was not due to metabolic disturbances. These data describe a new marker of cellular immunity in this animal model.(ABSTRACT TRUNCATED AT 250 WORDS)

Class II MHC antigen induction on rat insulinoma (RINm5F) and colon carcinoma (TS) cells by co-culture with diabetic and normal xenogenic lymphocytes.

Two MHC Class II-negative rat epithelial cell lines (RINm5F beta-cells and TS colic cells) were co-cultured with xenogenic lymphocytes from Type I diabetic patients or from low-dose streptozotocin (SZ) diabetic mice. MHC Class II antigens (Ag) were easily induced on both cell lines in such co-culture conditions, representing an experimental approach to insulitis. Our data indicate that: (1) lymphocytes from diabetic patients or from SZ mice were more efficient than lymphocytes from healthy controls in inducing Class II Ag on RIN cells. Lymphocytes from patients with autoimmune thyroid diseases were also more efficient than control lymphocytes, indicating that the ability to induce Class II may be related to the activation of lymphocytes rather than being diabetes-specific. (2) Rat colon carcinoma cells (TS) were also induced to express high levels of Class II Ag upon co-culture with SZ or control mouse lymphocytes. (3) Class II+ RIN cells were observed after 24 h of co-culture; their number increased after 48 and 72 h. The number of class II+ RIN increased proportionally to the number of lymphocytes in the culture. (4) Induction of Class II Ag was obtained by cell-free supernatants of mouse lymphocytes/RIN co-cultures and was inhibited by cyclosporine A, suggesting that Class II induction in this model is mediated by lymphokines. (5) Depletion experiments indicate that both monocytes and lymphocytes play a role in this Class II induction.

In vitro relationship of CD4 cells from type I diabetic patients and xenogeneic beta-cell membranes.

In a rosette assay, 63 patients with recent-onset type I (insulin-dependent) diabetes mellitus had a higher (P less than .001) number of lymphocytes adhering to rat insulinoma RINm5F cells (diabetic rosettes) than 153 healthy control (background rosettes) or 20 nondiabetic subjects with other organ-specific autoimmune diseases. Furthermore, lymphocytes from diabetic patients displayed a highly correlated (r = .97, P less than .001) binding on two different xenogeneic beta-cell lines (RIN and hamster insulinoma HIT cells). This phenomenon was not found on a panel of seven non-beta-cell lines (e.g., exocrine pancreatic cells, endocrine cells). By increasing lymphocyte-to-RIN ratios (0.25:1 to 30:1), the supernumerary RIN-adherent lymphocytes from diabetic patients, expressed as the percentage of lymphocytes involved conjugates, were only detectable at lower ratios (0.25:1 to 4:1), and their binding efficiency was two times higher than that of control lymphocytes. This efficiency fell at higher ratios (greater than 4:1) to the level of background rosettes that remained constant through the ratio scale. This specific RIN-rosette formation was abrogated when lymphocytes from diabetic patients were preabsorbed on beta-cells (either HIT or RIN) but not on non-beta-cells, whereas preabsorption of control lymphocytes did not modify the number of background rosettes. In addition, diabetic rosettes, but not background rosettes, were inhibited by competition with RIN membrane extracts but not by non-beta-cell extracts. Moreover, diabetic rosettes were inhibited during blocking experiments with anti-CD3 monoclonal antibody (MoAb) but not with unrelated MoAbs.(ABSTRACT TRUNCATED AT 250 WORDS)