Biomedical subjects

A E Postlethwaite

Publications and source records attributed to A E Postlethwaite.

At least 19 recordsLinked to original sources

PubMed1995

Identification of a chemotactic epitope in human transforming growth factor-beta 1 spanning amino acid residues 368-374.

TGF-beta 1 plays a critical role in inflammatory and repair processes due in part to its ability to provide a potent chemotactic stimulus for inflammatory cells such as neutrophils and monocytes and for fibroblasts which initiate the fibrogenic response. In the present study, we have used synthetic oligopeptides representing the amino acid sequence of the 12.1 kDa monomer of human TGF-beta 1 in an effort to identify a chemotactic epitope on the molecule. A seven residue peptide containing residues 368-374, Val Tyr Tyr Val Gly Arg Lys, was demonstrated to be capable of inducing chemotactic migration of human peripheral blood neutrophils, monocytes, monocyte leukemia cell line THP-1, and infant foreskin fibroblasts. Furthermore, larger peptides from the carboxy-terminal portion of TGF-beta 1 that contained residues 368-374 also induced migration of these cell types. None of the peptides representing the complete amino acid of TGF-beta 1 monomer were able to compete with [125I]hrTGF-beta 1 for binding to TGF-beta cell surface receptors or fibroblasts or THP-1 cells. Implications of these observations are discussed.

Amino Acid Sequence↗

PubMed1995

Role of T cells and cytokines in effecting fibrosis.

A number of humoral and cellular immune abnormalities are present in patients with early scleroderma (systemic sclerosis). Most of these abnormalities reflect ongoing autoimmune reactions of the cellular and humoral types, resulting in a variety of autoantibodies to cellular and tissue constituents. Evidence exists for a defect(s) in immunoregulation favoring excessive helper T cell activity. The presence of circulating cytokines and shed interleukin-2 receptors suggest ongoing cellular immune reactions are occurring, generating cytokines and lymphokines that are capable of effecting the vascular and fibrotic lesions that are hallmarks of the disease. Future directions for research are suggested that would focus on determining if, and at what point, fibroblasts might function autonomously to generate excessive matrix components and on determining the nature of the original antigenic stimulus that starts the scleroderma process.

Cytokines↗

PubMed1994

Interleukin 1 beta propeptide is detected intracellularly and extracellularly when human monocytes are stimulated with LPS in vitro.

Human interleukin 1 beta (IL-1 beta) is synthesized as an inactive precursor that is cleaved by IL-1 converting enzyme (ICE) between Asp116 and Ala117 to form COOH-terminal mature IL-1 beta and NH2-terminal IL-1 beta propeptide. Little is known about the fate of the NH2-terminal cleavage product. In this study, human recombinant (hr)IL-1 beta propeptide (amino acids 2-116) was produced and used to prepare specific antibodies which do not recognize mature human IL-1 beta. These anti-propeptide antibodies were used for immunoprecipitation of biosynthetically labeled proteins from lipopolysaccharide-stimulated human monocytes. Analysis of immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed that these antibodies recognize precursor IL-1 beta and two unique proteins: one migrating at 17.5 kD and one at 14 kD. The larger of these two proteins has a migration nearly identical to that of the recombinant IL-1 beta propeptide, and most likely represents naturally derived propeptide. The protein migrating at 14 kD may result from a second cleavage by ICE, between Asp27 and Gly28. These proteins accumulate intracellularly and extracellularly during pulse-chase experiments, and therefore represent stable products of precursor IL-1 beta cleavage.

Antibody Specificity↗

PubMed1994

Osteogenic protein-1, a bone morphogenic protein member of the TGF-beta superfamily, shares chemotactic but not fibrogenic properties with TGF-beta.

We have previously shown that recombinant human osteogenic protein-1 (rhOP-1), a bone morphogenetic protein member of the TGF-beta superfamily, can induce new bone formation when implanted with an appropriate carrier at subcutaneous sites in rats and can restore completely large diaphyseal segmental defects in laboratory animals. The role of OP-1 in the early events of bone induction viz, chemotaxis of phagocytic leukocytes, and fibroblastic mesenchymal cells is currently unknown. In the present study, we examined the effect of rhOP-1 on chemotaxis of phagocytic leukocytes (human neutrophils and monocytes) and fibroblastic mesenchymal cells (infant foreskin fibroblasts). Since OP-1 is structurally related to TGF-beta 1, we assessed the effects of OP-1 on several other fibroblast functions (in addition to chemotaxis) known to be modulated by TGF-beta 1. Our results demonstrated that rhOP-1, like TGF-beta 1, is a potent chemoattractant for human neutrophils, monocytes, and fibroblasts. However, in contrast to TGF-beta 1, OP-1 does not to stimulate fibroblast mitogenesis, matrix synthesis [collagen and hyaluronic acid (hyaluronan)], or production of tissue inhibitor of metalloproteinase (TIMP), i.e., fibroblast functions associated with fibrogenesis. These results clearly demonstrate a dichotomy between these two members of the TGF-beta superfamily with a regard to fibrogenic effects on fibroblasts but a similarity in their chemotactic properties.

Bone Morphogenetic Protein 7↗

PubMed1994

Connective tissue metabolism including cytokines in scleroderma.

The currently published literature on systemic sclerosis underscores the importance of vascular-leukocyte adhesion and perturbations of cytokines and growth factors that reflect T-cell activation and abnormal stimulation of fibroblast growth and matrix synthesis. Articles dealing with these topics are reviewed.

Cell Adhesion↗

PubMed1993

Intercellular adhesion molecule-1 expression induced by interleukin (IL)-1 beta or an IL-1 beta fragment is blocked by an IL-1 receptor antagonist and a soluble IL-1 receptor.

The effects of a recombinant human interleukin-1 (IL-1) receptor antagonist (IL-1ra) and a recombinant human soluble IL-1 receptor (sIL-1R) on cytokine-induced intercellular adhesion molecule-1 (ICAM-1) expression in a human glioblastoma cell line and a neuroblastoma cell line were determined. Cells were incubated with IL-1 beta, tumor necrosis factor (TNF) alpha and interferon (IFN) gamma. Cells were also tested under identical conditions with an IL-1 beta synthetic peptide fragment (IL-1 beta 208-240) previously shown to possess biological activity. IL-1 beta, TNF alpha and IFN gamma potentiated ICAM-1 expression in both cell lines in a dose-related manner. The IL-1 beta 208-240 fragments, corresponding to the rabbit, rat and human sequences, enhanced ICAM-1 expression in glioblastoma cells at high doses. ICAM-1 expression induced by IL-1 beta, rabbit IL-1 beta 208-240 and human IL-1 beta 208-240 was blocked by the IL-1ra, while TNF alpha- and IFN gamma-induced ICAM-1 expression were not. ICAM-1 expression induced by IL-1 beta and human IL-1 beta 208-240 was also blocked by the sIL-1R. Our findings suggest that IL1 beta 208-240 acts as an IL-1 beta agonist in enhancing ICAM-1 expression in vitro and that this effect is receptor-mediated.

Amino Acid Sequence↗

PubMed1993

Connective tissue metabolism including cytokines in scleroderma.

A growing body of evidence is shedding light on how vascular injury, tissue mononuclear cell infiltrates, and fibrogenesis are related in the pathogenesis of systemic sclerosis. Recent evidence points to a role for adhesion molecules in the binding of mononuclear leukocytes to endothelium and transvascular migration of these cells to connective tissue sites. Further interaction of mononuclear leukocyte integrins with connective tissue components at these sites leads to cytokine and growth factor release, resulting in upregulation of matrix production by fibroblasts and alterations of the fibroblast phenotype, leading to fibrogenesis.

Cell Adhesion↗

PubMed1992

Interleukin 1 receptor antagonist blocks somnogenic and pyrogenic responses to an interleukin 1 fragment.

Previously it was shown that human interleukin 1 (huIL-1) and a huIL-1 fragment, huIL-1 beta 208-240, are somnogenic and pyrogenic in rabbits. However, the amino acid sequences for IL-1 are species-specific and it was unknown whether rabbit (rb) IL-1 beta 208-240 and rat (rt) IL-1 beta 208-240 were active in their respective species. Furthermore, it was unknown whether these fragments elicited their effects via the IL-1 receptors. Two doses of rbIL-1 beta 208-240 (6.0 and 12.0 nmol) were intracerebroventricularly administered to rabbits. The 6.0-nmol dose had little effect, whereas the 12.0-nmol dose greatly increased non-rapid-eye-movement sleep across a 6-hr recording period and induced a febrile response. Rats injected intracerebroventricularly with rtIL-1 beta 208-240 at dark onset responded to three doses of the peptide (1.2, 2.4, and 4.8 nmol). The 1.2-nmol dose did not greatly affect sleep but did induce a moderate febrile response. The 2.4- and 4.8-nmol doses increased non-rapid-eye-movement sleep across the 12-hr recording period. Maximal brain temperature elevations relative to controls after the 2.4- and 4.8-nmol doses of the peptide were 0.9 +/- 0.2 degrees C and 0.7 +/- 0.2 degrees C, respectively. These responses in both rabbits and rats were completely blocked or significantly attenuated when the animals were pretreated with an IL-1 receptor antagonist. These results suggest that the biological activities of IL-1 beta 208-240 are mediated via the IL-1 beta receptors.

Amino Acid Sequence↗

PubMed1992

Somnogenic, pyrogenic, and anorectic activities of tumor necrosis factor-alpha and TNF-alpha fragments.

Exogenously administered tumor necrosis factor-alpha (TNF-alpha) elicits several symptoms of generalized infections such as fever, increased sleep, and anorexia. The aim of the present work was to localize these effects of TNF-alpha to specific amino acid sequences of the parent molecule by characterizing the in vivo and in vitro activities of several synthetic TNF-alpha fragments. Intracerebroventricular injection of TNF-alpha elicited dose-dependent fevers and increases in non-rapid-eye-movement sleep (NREMS) in rabbits. Four fragments also promoted NREMS and five elicited monophasic fevers. All of the somnogenic fragments share the amino acid sequence 31-36. In rats, TNF-alpha and one of the fragments [TNF-alpha-(69-100)] suppressed 12-h food intake. Furthermore, TNF-alpha increased the expression of the intercellular adhesion molecule-1 and enhanced interferon-gamma-induced HLA-DR expression in human glioblastoma cell line. In contrast, none of the fragments possessed these in vitro activities. Our in vivo results support the concept that there are biologically active regions in the TNF-alpha molecule.

Animals↗

PubMed1992

Human fibroblasts synthesize elevated levels of extracellular matrix proteins in response to interleukin 4.

Interleukin 4 (also known as "B cell stimulatory factor-1"), a cytokine product of T lymphocytes and mast cells, stimulates synthesis of the extracellular matrix proteins, types I and III collagen and fibronectin, by human dermal fibroblasts in vitro. Stimulation of collagen by human recombinant (hr)IL-4 was also demonstrated in several fibroblastic synovial cell lines obtained from patients with rheumatoid arthritis and osteoarthritis. The stimulatory effect of hrIL-4 on fibroblast collagen synthesis was specifically neutralized by rabbit anti-hrIL-4 Ig. IL-4 specifically increased the steady-state levels of types I and III procollagen and fibronectin mRNAs, with no effect on cytoplasmic beta-actin mRNA. Quantitative analysis of the levels of Pro alpha 1(I) collagen transcripts in IL-4-treated fibroblast cultures was also corroborated by antisense RNA-mRNA hybridization and RNAse resistant hybrids which showed that IL-4-treated fibroblasts expressed higher levels of Pro alpha 1(I) collagen transcripts. Nuclear run-off transcription experiments indicated that IL-4 stimulated the rates of mRNA biogenesis. Based on these observations we conclude that IL-4 exerts its effect on collagen and fibronectin synthesis at the pretranslational level, resulting in synthesis of these extracellular matrix proteins. These and other data suggest that IL-4 may be a "fibrogenic cytokine" that could be important in promoting biogenesis of extracellular matrix proteins in normal wound healing and in pathological fibrosis in which mast cells and T lymphocytes play a central role.

Cells, Cultured↗

PubMed1991

Transforming growth factor beta 1 (TGF-beta 1) induced neutrophil recruitment to synovial tissues: implications for TGF-beta-driven synovial inflammation and hyperplasia.

We have studied the consequences of introducing human recombinant transforming growth factor beta 1 (hrTGF-beta 1) into synovial tissue of the rat, to begin to better understand the significance of the fact that biologically active TGF-beta is found in human arthritic synovial effusions. Within 4-6 h after the intra-articular injection of 1 microgram of hrTGF-beta 1 into rat knee joints, extensive recruitment of polymorphonuclear leukocytes (PMNs) was observed. Cytochemistry and high resolution histological techniques were used to quantitate the influx of PMNs, which peaked 6 h post-injection. In a Boyden chamber assay, hrTGF-beta 1 at 1-10 fg/ml elicited a chemotactic response from PMNs greater in magnitude than that evoked by FMLP, establishing that TGF-beta 1 is an effective chemotactic agent for PMNs in vitro as well as in vivo. That PMNs may represent an important source of TGF-beta in inflammatory infiltrates was strongly suggested by a demonstration that stored TGF-beta 1 was secreted during phorbol myristate acetate-stimulated degranulation in vitro. Acid/ethanol extracts of human PMNs assayed by ELISA contained an average of 355 ng of TGF/beta 1 per 10(9) cells potentially available for secretion during degranulation of PMNs. [3H]Thymidine incorporation in vivo and autoradiography of tissue sections revealed that widespread cell proliferation was triggered by TGF-beta 1 injection. Synovial lining cells and cells located deep within the subsynovial connective tissue were identified as sources of at least some of the new cells that contribute to TGF-beta 1-induced hyperplasia. Our results demonstrate that TGF-beta is capable of exerting pathogenic effects on synovial tissue and that PMNs may represent a significant source of the TGF-beta present in synovial effusions.

Animals↗

PubMed1991

Kupffer cells from carbon tetrachloride-injured rat livers produce chemotactic factors for fibroblasts and monocytes: the role of tumor necrosis factor-alpha.

Conditioned media from cultured Kupffer and mononuclear macrophagic cells obtained 48 hr after CCl4 administration to rats contains chemotactic factors for human skin fibroblasts and human monocytes. The chemotactic mediator for fibroblasts was approximately 17 kD and was more prominent at early stages of culture. It induced a dose-dependent chemotactic response in fibroblasts. Although the conditioned medium from cultured Kupffer cells of normal rats also contained detectable biological activity, it was significantly less than that in conditioned medium from cultured Kupffer cells from CCl4-treated rats. The activity obtained after purification by high-performance liquid chromatography was completely ablated by incubation with tumor necrosis factor-alpha antibody. Transforming growth factor-beta antibody diminished biological activity by 20%. Human recombinant tumor necrosis factor-alpha and transforming growth factor-beta used in the assay as control showed significant chemotactic activity. The chemotactic activity present in whole normal conditioned medium was only present after 24 and 48 hr of culture. Furthermore, this activity was not neutralized by human recombinant tumor necrosis factor-alpha or transforming growth factor-beta antibodies. Incubation of whole 6-hr conditioned medium with human recombinant tumor necrosis factor-alpha and transforming growth factor-beta antibodies demonstrated and confirmed that tumor necrosis factor-alpha plays a major role in inducing the chemotactic response. On acidification of this supernatant, we found a notable increase in the biological response that could be neutralized by transforming growth factor-beta antibody. Thus tumor necrosis factor-alpha and transforming growth factor-beta may sequentially provide important signals for fibroblast and monocyte recruitment in vivo at initial stages of liver injury.

Animals↗

PubMed1991

The specialized centers of research in rheumatoid arthritis. Recent progress and prospects for future advances.

Specialized Centers of Research (SCOR) in arthritis are interdisciplinary research programs to investigate disease pathogenesis as well as advance diagnosis and treatment. A recent meeting of investigators from the three SCOR programs in rheumatoid arthritis demonstrated progress in several important research areas. Because of the multiplier effects of SCOR programs, new investigators have been enlisted into arthritis research as issues related to this disease become a focus of investigation throughout universities and medical centers. Continued progress by the SCOR programs should provide new targets for therapeutic intervention as well as strategies for monitoring disease activity.

Academic Medical Centers↗

PubMed1991

Interleukin 1 stimulates phosphatidylinositol kinase activity in human fibroblasts.

IL-1 mediates multiple cellular immune and inflammatory responses, but little is known of the intracellular biochemical mechanisms involved in IL-1 actions. We studied the effects of IL-1 on phosphatidylinositol (PtdIns) metabolism and confirmed reports indicating that IL-1 does not stimulate increased PtdIns turnover; however, we observed the accumulation of PtdIns-4-phosphate (PtdInsP) in response to IL-1. Using a fibroblast membrane preparation, we were able to detect stimulated PtdInsP accumulation within 10 s of IL-1 addition. Increased PtdInsP accumulation was due to stimulated PtdIns kinase activity, not the inhibition of PtdInsP hydrolysis by phospholipase(s). PtdIns kinase activity was magnesium dependent, increased as a function of IL-1 concentration, and specifically phosphorylated the D4 position of inositol. Stimulated PtdIns kinase activity could be detected at 10(-12) M IL-1 in fibroblast membranes, a concentration within the physiological range for IL-1 action; half-maximal activity was reached at approximately 10(-10) M IL-1. Heat denaturation of IL-1 or treatment of IL-1 with anti-IL-1 antibody abrogated the IL-1 effect. These findings demonstrate the direct, IL-1-mediated, stimulation of PtdIns kinase. IL-1-stimulated PtdIns kinase activity represents an important physiological regulatory effect by IL-1 as it could control the synthesis and/or maintenance of phosphorylated derivatives of PtdIns which comprise only a very small pool of substrates for the generation of the second messengers inositol 1,4,5-triphosphate and diacylglycerol.

1-Phosphatidylinositol 4-Kinase↗

PubMed1991

Fibroblast chemotaxis induction by human recombinant interleukin-4. Identification by synthetic peptide analysis of two chemotactic domains residing in amino acid sequences 70-88 and 89-122.

Interleukin-4 is a T lymphocyte- and mast cell-derived cytokine with pleiotropic properties with biological effects on a variety of target cells including B and T lymphocytes, macrophages, hematopoietic cells, mast cells, and fibroblasts. In addition to the proliferation effect of IL-4 on fibroblasts, which has been previously described, in this report the chemotactic properties of IL-4 for fibroblasts is described. Human recombinant IL-4 induced the chemotactic migration of dermal fibroblasts in vitro in modified Boyden-type chambers at concentrations between 10(-12) and 10(-11) M. The chemotactic activity of IL-4 was neutralized by anti-human recombinant IL-4 IgG antibodies. Oligopeptides representing the complete deduced amino acid sequence of human IL-4 were synthesized by the Merrifield technique and tested for their ability to induce fibroblast chemotaxis. Two peptides representing residues 70-88 and 89-122 induced fibroblast migration. Peptide 70-88 was the more potent of the two causing chemotaxis of fibroblasts at 10(-8)-10(-6) M while peptide 89-129 induced migration at 10(-7)-10(-5) M. Although the mechanism by which IL-4 and these two peptides induce fibroblast chemotaxis is unknown, each of these three compounds were able to chemotactically desensitize fibroblasts to the chemotactic effects of the other two but not to a structurally unrelated chemotactic cytokine, transforming growth factor beta-1. These studies suggest that IL-4 might function in vivo to induce the accumulation of fibroblasts at sites of tissue injury, inflammatory and immune reactions in which T lymphocytes and mast cells participate.

Amino Acid Sequence↗

PubMed1991

Alterations in influence of granuloma-derived cytokines on fibrogenesis in the course of murine Schistosoma mansoni infection.

Schistosomiasis is the main cause of hepatic fibrosis worldwide, yet its pathogenesis remains unknown. We previously reported that conditioned medium from cultures of hepatic egg granulomas (isolated from mice acutely infected with Schistosoma mansoni) can stimulate fibroblast proliferation and matrix production in vitro. We have proposed that initiation of hepatic fibrosis in this infection might be under the control of granuloma-derived cytokines. We now report that conditioned medium from cultures of schistosomal egg granulomas isolated from liver of chronically infected mice has reduced fibrogenic activity compared with medium from cultures of granulomas obtained from more acutely infected mice. In related studies, we adoptively transferred splenocytes from infected mice and examined the fibrogenic activity in culture supernatants of hepatic egg granulomas isolated from the recipients. Those prepared from recipients of splenocytes from chronically infected mice contained substantially less fibrogenic activity than did those from recipients of splenocytes of acutely infected mice. These findings suggest that the fibrogenic influence of schistosomal egg granuloma products decreases during the course of chronic murine S. mansoni infection. Furthermore, our preliminary findings suggest that immunoregulatory cells may be responsible for the down-regulation of this influence. Previous observations indicating that in murine schistosomiasis hepatic collagen and hyaluronate synthesis and deposition are elevated in acute but not chronic infection might be explained on the basis of our observations.

Animals↗

PubMed1990

Sphingosine potentiates IL-1-mediated prostaglandin E2 production in human fibroblasts.

IL-1 stimulates PGE2 production in human fibroblasts by stimulating arachidonic acid (AA) mobilization and cyclooxygenase synthesis. Cyclooxygenase is the first enzyme in the pathway that converts AA to PGE2. To examine the role of protein kinase C (PKC) in IL-1-mediated PGE2 production, we treated cells with PMA, which stimulated PGE2 production suggesting a positive role for PKC activation in the regulation of PGE2 synthesis. Therefore, we tested the effect of sphingosine, a PKC inhibitor, on IL-1-induced PGE2 production. Alone, sphingosine had little effect on PGE2 production. However, when sphingosine was added with IL-1, or IL-1 was added to sphingosine-pretreated cells, PGE2 production increased severalfold, suggesting that the inhibition of PKC results in enhanced IL-1-mediated PGE2 production; structural analogs of sphingosine did not potentiate the IL-1 effect. In cells made deficient in PKC by prolonged exposure to PMA, IL-1-mediated PGE2 production was enhanced compared with normal cells, further suggesting that functional PKC is not required for, and may down-modulate, IL-1-mediated PGE2 production. These findings also suggest that PMA and IL-1 stimulate PGE2 synthesis via fundamentally different pathways. In separate studies on the effect of IL-1 on AA mobilization, we found that IL-1 induced an increase in phospholipase A2 (PLA2) activity and that cycloheximide blocked the increase, suggesting the requirement for new protein synthesis. We also found that the PLA2 activity increased as a result of IL-1 exposure was further stimulated by sphingosine. Thus, in addition to its primary effects on the cell, which are likely mediated via PKC, we present evidence suggesting that sphingosine may also play a role in potentiating an IL-1-induced PLA2 activity, resulting in increased availability of AA for conversion to PGE2.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1990

Stimulation of fibroblast chemotaxis by human recombinant tumor necrosis factor alpha (TNF-alpha) and a synthetic TNF-alpha 31-68 peptide.

Macrophages are a major source of fibrogenic factors that promote healing of injured tissue. The recruitment of fibroblasts to sites of tissue injury is a prerequisite for optimal repair of tissue damage. In the present study, human recombinant tumor necrosis factor alpha (hrTNF-alpha), a major macrophage-derived cytokine, was demonstrated to be a potent fibroblast chemoattractant, inducing migration at picomolar concentrations. Anti-hrTNF-alpha monoclonal antibody neutralized most of the fibroblast chemotactic activity generated during short-term culture of human peripheral blood monocytes stimulated with bacterial lipopolysaccharide, suggesting that TNF-alpha is a major monocyte-derived fibroblast chemoattractant. The portion of the human TNF-alpha molecule responsible for its chemotactic stimulation of fibroblasts appears to reside in residues 31-68. This region is highly conserved between TNF-alpha and lymphotoxin. This peptide is not only itself chemotactic but is also able to block the chemotactic response of fibroblasts to hrTNF-alpha and vice versa, suggesting that they each mediate fibroblast migration through similar mechanisms. These data further underscore the potential importance of TNF-alpha in modulating a variety of fibroblast functions, including chemotaxis and synthesis of collagen, glycosaminoglycans, interleukin 1 alpha (IL-1 alpha) and -beta, human histocompatibility leukocyte antigen A and B antigens, collagenase, prostaglandin E2, and IL-6.

Amino Acid Sequence↗