Showing papers on "Receptor expression published in 1993"

The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis

Abstract: The c-kit receptor tyrosine kinase belongs to the PDGF/CSF-1/c-kit receptor subfamily The kit-ligand, KL, also called steel factor, is synthesized from two alternatively spliced mRNAs as transmembrane proteins that can either be proteolytically cleaved to produce soluble forms of KL or can function as cell-associated molecules The c-kit receptor kinase and KL are encoded at the white spotting (W) and steel (Sl) loci of the mouse, respectively Mutations at both the W and the Sl locus cause deficiencies in gametogenesis, melanogenesis and hematopoiesis The c-kit receptor is expressed in the cellular targets of W and Sl mutations, while KL is expressed in their microenvironment In melanogenesis, c-kit is expressed in melanoblasts from the time they leave the neural crest and expression continues during embryonic development and in the melanocytes of postnatal animals In gametogenesis c-kit is expressed in primordial germ cells, in spermatogonia, and in primordial and growing oocytes, implying a role at three distinct stages of gametogenesis Many mutant alleles are known at W and Sl loci and their phenotypes vary in the degree of severity in the different cellular targets of the mutations While many W and Sl alleles severely affect primordial germ cells (PGC), several mild Sl alleles have weak effects on PGCs and exhibit differential male or female sterility Steel Panda (Sl(pan)) is a KL expression mutation in which KL RNA transcript levels are reduced in most tissues analyzed In female Sl(pan)/Sl(pan) mice, ovarian follicle development is arrested at the one layered cuboidal stage as a result of reduced KL expression in follicle cells, indicating a role for c-kit in oocyte growth Wsh is a c-kit expression mutation, which affects mast cells and melanogenesis While the mast cell defect results from lack of c-kit expression, the pigmentation deficiency appears to stem from ectopic c-kit receptor expression in the somitic dermatome at the time of migration of melanoblasts from the neural crest to the periphery It is proposed that the ectopic c-kit expression in Wsh mice affects early melanogenesis in a dominant fashion The "sash" or white belt of Wsh/+ animals and some other mutant mice is explained by the varying density of melanoblasts along the body axis of wild-type embryos

Nervous system distribution of the serotonin 5-HT3 receptor mRNA.

Abstract: The serotonin 5-HT3 receptor subtype has been implicated in many brain functions. Antagonists of this receptor have anxiolytic and antiemetic effects in humans and in animal models. To determine with cellular resolution the distribution of 5-HT3 receptor mRNA, in situ hybridization was performed in sections of mouse brain and dorsal root ganglia. Scattered labeled cells were observed throughout cortical regions, with highest densities in the piriform, cingulate, and entorhinal areas. Strong hybridization signals were seen in the hippocampal formation, where expression appeared primarily in interneurons. Labeled cells were most abundant in the posteroventral hippocampal region, particularly in the lacunosum moleculare layer of CA1. This distribution suggests that 5-HT3 receptors may mediate the known serotonergic inhibition of pyramidal cell populations via excitation of inhibitory interneurons. Labeled cells were also observed in the major subdivisions of the amygdaloid complex, the olfactory bulb, the trochlear nerve nucleus, the dorsal tegmental region, the facial nerve nucleus, the nucleus of the spinal tract of the trigeminal nerve, and the spinal cord dorsal horn. In the periphery, intense hybridization signals were seen in a subpopulation of cells in dorsal root ganglia. The data correlate generally with physiological, behavioral, and receptor autoradiographic studies, provide cellular resolution, and reveal regions of receptor expression not previously observed. The distribution of 5-HT3 receptor mRNA is consistent with roles for the receptor in cognition and affect and in the modulation of sensory input.

Molecular alterations in the neostriatum of human cocaine addicts

Abstract: Molecular changes in the neostriatum of human subjects who died with a history of cocaine abuse were revealed in discrete cell populations by means of the techniques of in situ hybridization histochemistry and in vitro receptor binding and autoradiography. Cocaine subjects had a history of repeated cocaine use and had cocaine and/or cocaine metabolites on board at the time of death. These subjects were compared to control subjects that had both a negative history and toxicology of cocaine use. Selective alterations in mRNA levels of striatal neuropeptides were detected in cocaine subjects compared to control subjects, especially for the opioid peptides. Marked reductions in the levels of enkephalin mRNA and mu opiate receptor binding were found in the caudate and putamen, concomitant with elevations in levels of dynorphin mRNA and kappa opiate receptor binding in the putamen and caudate, respectively. Dopamine uptake site binding was reduced in the caudate and putamen of cocaine subjects. The greater magnitude of changes in the dorsolateral striatum (caudate and putamen) as opposed to the ventromedial striatum (nucleus accumbens) suggests that cocaine abuse preferentially alters the biosynthetic activity of striatal systems associated with sensorimotor functioning. Additionally, an imbalance in the activity of the two major striatal output pathways in cocaine users is implicated because peptide mRNA levels were reduced in enkephalinergic striatopallidal neurons and increased in dynorphinergic striatonigral neurons. Another imbalance, that of reductions of transmitter mRNA and receptor expression associated with euphoria (enkephalin and mu opiate receptors), together with elevations in mRNAs of transmitter systems associated with dysphoria (dynorphin and kappa opiate receptors), suggests a model of dysphoria and craving in the human cocaine addict brain.

Parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acids are widely distributed in rat tissues.

Abstract: PTH/PTH-related peptide (PTHrP) receptor mRNAs are widely distributed in rat tissues. PTH and PTHrP, a peptide responsible for hypercalcemia associated with cancers, bind equivalently to common receptors that initially were cloned from rat bone and opossum renal cell cDNA libraries. In this study we used rat PTH/PTHrP receptor cDNA to probe for receptor expression in different rat tissues by Northern blot analysis. PTH/PTHrP receptor transcripts are highly expressed in PTH target tissues, kidney and bone. Receptor transcripts, however, also are expressed in many other tissues, including aorta, adrenal gland, bladder, brain, cerebellum, breast, heart, ileum, liver, lung, skeletal muscle, ovary, placenta, skin, spleen, stomach, uterus, and testes. The major transcript in most tissues is 2.3-2.5 kilobases in size. At least two larger mRNAs are observed in kidney and liver, and smaller transcripts are found in kidney, skin, and testes. The most abundant testicular transcript is 1.4-1.5 kilobases in size, and it hybridizes with two different cDNA probes that encode portions of the receptor sequence from the putative fourth transmembrane domain to its C-terminal end. It does not hybridize, however, with a probe encoding the first 107 residues of the receptor sequence. Although, PTH/PTHrP receptor mRNAs are highly expressed in kidney and bone, classic PTH targets that are associated with calcium homeostasis, their wide tissue distribution suggests that PTH and/or PTHrP have other physiological roles, particularly in these other tissues. The mechanisms leading to tissue-specific expression of PTH/PTHrP receptor transcripts of different sizes and the functions of these mRNAs remain to be determined.

Development of the embryonic neuromuscular synapse of Drosophila melanogaster

Abstract: We have examined the embryonic development of an identified neuromuscular junction (NMJ) of Drosophila melanogaster using whole-cell patch-clamp and a variety of physiological and morphological techniques. Synaptic current at the embryonic NMJ is carried through a large-conductance (200 pS) L-glutamate receptor. Early synaptic communication is characterized by frequent, brief (< 10 msec) currents carried through few (1-10) receptors and relatively rare, prolonged currents (up to seconds) of similar amplitude. The brief currents have a time course similar to the mature larval excitatory junction currents (EJCs), but the prolonged currents are restricted to early stages of synaptogenesis. The amplitude of EJCs rapidly increases, and the frequency of the prolonged currents decreases, after the initial stages of synaptogenesis. Early prolonged (seconds), nonspiking synaptic potentials are replaced with rapid (< 0.10 sec), spiking synaptic potentials later in development. The early synapse appears tenuous, easily fatiguable, and with inconsistent communication properties. Synaptogenesis can be divided into a sequence of progressive stages. (1) Motor axon filopodia begin neurotransmitter expression and concurrent exploration of the myotube surface. (2) Myotubes uncouple to form single-cell units soon after motor axon contact. (3) A small number of transmitter receptors are homogeneously displayed on the myotube surface immediately following myotube uncoupling. (4) Endogenous transmitter release from pioneering growth cones is detected; nerve stimulation elicits postsynaptic EJC response. (5) Motor axon filopodia and transmitter receptors are localized to the mature synaptic zone; filopodial localization is complete in advance of receptor localization. (6) A functional neuromuscular synapse is formed; endogenous muscular activity begins; nerve stimulation leads to muscle contraction. (7) Morphological presynaptic specializations develop; synapse develops mature morphology. (8) A second motor axon synapses on the myotube at the pre-established synaptic zone. (9) Vigorous neuromuscular activity, characteristic of larval locomotory movements, begins. (10) A second stage of receptor expression begins and continues through the end of embryogenesis. In general, Drosophila neuromuscular synaptogenesis appears similar to neuromuscular synaptogenesis in known vertebrate preparations. We suggest that this system provides a model for synaptogenesis in which investigation can be readily extended to a genetic and molecular level.

Infectious morbidity and defects of phagocytic function in end-stage renal disease: a review.

Abstract: Infectious diseases remain among the most morbid events and are an important cause of death in ESRD. These events are related to an acquired immunodeficiency that progresses during the development of uremic retention, as part of the broader spectrum, displayed by the "uremic syndrome". A central role in the hose defense against bacterial infection is played by the phagocytic polymorphonuclear white blood cells, which are characterized by the capacity to ingest bacteria (phagocytosis), which is followed by the destruction of those bacteria (killing capacity). This article reviews the mechanisms of development and the potential causes that have been held responsible for this aspect of the defective immune function. The observed changes are attributed to alterations in receptor expression, although more convincing evidence points in the direction of metabolic functional disturbances, especially in the NAD(P)H-oxidase-dependent production of oxygen free radicals. The most important causative factors are: uremic toxicity, iron overload, renal anemia, dialyzer bioincompatibility, and the type of renal replacement therapy. It is concluded that the phagocytic defect in ESRD is multifactorial and that each factor should be managed by specific therapeutic approaches.

Growth inhibition by transforming growth factor beta (TGF-beta) type I is restored in TGF-beta-resistant hepatoma cells after expression of TGF-beta receptor type II cDNA

Abstract: The growth of human hepatoma Hep 3B cells is potently inhibited by TGF-beta 1 (ID50 = 0.2 ng/ml, 8 pM). A mutant cell line was derived that was not inhibited in growth by TGF-beta 1 at 5 ng/ml (200 pM) and that lacked TGF-beta receptor type II (TGF-beta RII) gene. Transfection of the cloned cDNA for human TGF-beta RII to this mutant cell line restored receptor expression as well as the inhibition in growth by TGF-beta 1. In both wild-type and mutant cells stably transfected with TGF-beta RII cDNA, TGF-beta RII coimmunoprecipitated with TGF-beta receptor type I in the presence of ligand. These experiments provide direct evidence for the role of TGF-beta RII in the inhibitory effect of TGF-beta on growth and suggest that TGF-beta RII acts by means of a heteromeric surface complex with TGF-beta receptor type I.

Enhanced expression of the type II transforming growth factor β receptor in human pancreatic cancer cells without alteration of type III receptor expression

Abstract: We have recently found that human pancreatic adenocarcinomas exhibit strong immunostaining for the three mammalian transforming growth factor beta (TGF-beta) isoforms. These important growth-regulating polypeptides bind to a number of proteins, including the type I TGF-beta receptor (T beta R-I), type II TGF-beta receptor (T beta R-II), and the type III TGF-beta receptor (T beta R-III). In the present study we sought to determine whether T beta R-II and T beta R-III expression is altered in pancreatic cancer. Northern blot analysis indicated that, by comparison with the normal pancreas, pancreatic adenocarcinomas exhibited a 4.6-fold increase (P < 0.01) in mRNA levels encoding T beta R-II. In contrast, mRNA levels encoding T beta R-III were not increased. In situ hybridization showed that T beta R-II mRNA was expressed in the majority of cancer cells, whereas mRNA grains encoding T beta R-III were detectable in only a few cancer cells and were present mainly in the surrounding stroma. These findings suggest that enhanced levels of T beta R-II may have a role in regulating human pancreatic cancer cell growth, while T beta R-III may function in the extracellular matrix.

Interleukin involvement in anterior pituitary cell growth regulation: effects of IL-2 and IL-6.

Abstract: The pituitary gland plays a central role in the interactions between the immune and neuroendocrine systems. The expression of receptors for interleukin-1 (IL-1), IL-2, and IL-6 and the intrinsic production of these ILs by pituitary cells have been described. Previous studies have focused on the way cytokines influence hormone secretion. We have determined whether, in addition to these effects, ILs could affect pituitary cell proliferation. In GH3 cells, both IL-2 (1-100 U/ml) and IL-6 (10-500 U/ml) significantly stimulated [3H]thymidine incorporation and cell count. In contrast, inhibitory effects of both IL-2 and IL-6 at the same concentrations were observed on normal rat anterior pituitary cell growth. This finding was clearly evident when cells were cultured in Minimum Essential Medium-D-valine medium, a condition that results in cultures virtually free of fibroblasts. Autoradiographic studies confirmed that [3H]thymidine was only incorporated in the nucleus of nonfibroblastic pituitary cells. No direct correlation between the effects of IL-2 and IL-6 on cell growth and hormone secretion was apparent. By immunofluorescence, we observed IL-2 receptor expression on GH3 cells and, for the normal rat cultures, a high percentage of PRL-secreting and a lower percentage of GH-producing cells expressing IL-2 receptors, providing new evidence for a direct site of action of IL-2 on pituitary cells. Considering that uncontrolled division of cells may result from either excessive growth stimulation or deficient growth inhibition, the regulation of pituitary cell growth by IL-2 and IL-6 together with their intrinsic pituitary production could be of potential importance in pituitary adenoma pathogenesis.

Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor.

Abstract: Most G protein-coupled receptors contain a series of highly conserved proline and tryptophan residues within their hydrophobic transmembrane domains (TMD I-VII) To study their potential role in ligand binding and receptor function, the rat m3 muscarinic acetylcholine receptor was used as a model system A series of mutant receptors in which the conserved proline and tryptophan residues were individually replaced with alanine and phenylalanine, respectively, was created and transiently expressed in COS-7 cells [3H]N-methylscopolamine ([3H]NMS) saturation binding studies showed that three of the seven mutant receptors studied (Pro242-->Ala, TMD V; Pro505-->Ala, TMD VI; Pro540-->Ala, TMD VII) were expressed at 35-100 times lower levels than the wild-type receptor while displaying 'm3-like' antagonist binding affinities Pro201-->Ala (TMD IV) showed drastically reduced binding affinities (up to 450-fold) for both muscarinic agonists and antagonists Whereas most mutant receptors retained strong functional activity, Pro540-->Ala (TMD VII) was found to be severely impaired in its ability to stimulate carbachol-induced phosphatidyl inositol hydrolysis (Emax approximately 25% of wild type m3) Interestingly, this mutant receptor bound muscarinic agonists with 7- to 19-fold higher affinities than the wild type receptor The Trp-->Phe substitutions (Trp192-->Phe, TMD IV; Trp503-->Phe, TMD VI; Trp530-->Phe, TMD VII) resulted in less pronounced changes (compared with the Pro-->Ala mutant receptors) in both ligand binding and receptor function Our data indicate that the proline residues that are highly conserved across the entire superfamily of G protein-coupled receptors play key roles in receptor expression, ligand binding and receptor activation

Down-regulation of cytokine production and interleukin-2 receptor expression by pooled human IgG.

Abstract: The influence of pooled human IgG preparations for intravenous use (i.v.Ig) on in vitro-induced cytokine production was studied at the single-cell level using cytokine-specific monoclonal antibodies (mAb) and indirect immunofluorescent technique. Cultured mononuclear cells from peripheral blood from healthy adult donors were polyclonally stimulated for 96 hr by either direct ligation of T-cell receptors using immobilized anti-CD3 mAb or by a combination of a protein kinase C activator [phorbol 12-myristate 13-acetate (PMA)] and a calcium ionophore (ionomycin) in the absence or presence of i.v.Ig. A marked inhibition of proliferation and blast transformation was noted in all i.v.Ig exposed cultures, despite good cell survival. The production of the T-cell lymphokines interleukin-2 (IL-2), IL-10,interferon-gamma (IFN-gamma) and tumour necrosis factor-beta (TNF-beta) was significantly down-regulated during the whole studied period in the i.v.Ig containing anti-CD3 stimulated cultures. The synthesis of the monokine IL-8 was not suppressed and that of TNF-alpha, which was made by both lymphocytes and monocytes, was only moderately inhibited. Somewhat different and more transient effects were observed in the i.v.Ig-exposed PMA/ionomycin-activated cultures. The production of IL-2, IL-3, IL-4, IL-5, IL-10, TNF-beta and granulocyte-macrophage colony-stimulating factor (GM-CSF) was down-regulated during the initial phase of the cultures up to 48 hr, but not at 48-96 hr. The synthesis of IFN-gamma and TNF-alpha was unaffected of the influence of i.v.Ig during the entire culture period. The expression of IL-2 receptors (IL-2R) was significantly suppressed in the i.v.Ig-treated anti-CD3-activated cells, but not in the PMA/ionomycin-stimulated cultures. Taken together our results indicate that pooled IgG may mediate immunomodulation by direct effects on cytokine production and on T-cell proliferation.

Human serotonin1B receptor expression in Sf9 cells : phosphorylation, palmitoylation, and adenylyl cyclase inhibition

Abstract: Analysis of the primary protein structure of the human serotonin1B (5-HT1B) receptor reveals consensus sites for phosphorylation and a putative site for palmitoylation. To investigate these posttranslational modifications, we have expressed a c-myc epitope-tagged 5-HT1B (m5-HT1B) receptor in Sf9 cells. This strategy enabled receptors to be detected by immunoblot analysis and purified by immunoprecipitation using a monoclonal antibody, 9E10, specific for the c-myc epitope. Agonist radioligand [3H]5-HT binding studies showed that the expressed 5-HT1B and m5-HT1B receptors displayed the characteristic pharmacological profile of the neuronal 5-HT1B receptor. The expressed receptors displayed both high- and low-affinity states for [3H]5-HT, suggesting that the receptors were coupled to endogenous G-proteins. Indeed, agonist binding to the high-affinity receptor state was regulated in the presence of GTP gamma S, Gpp(NH)p, and pertussis toxin. [32P]ADP-ribosylation experiments identified a major approximately 41-kDa ADP-ribosylated protein present in Sf9 membranes that comigrated with partially purified bovine brain Gi alpha/G(o) alpha subunits. Measurements of adenylyl cyclase activity in membranes from cells expressing m5-HT1B receptors showed that serotonergic agonists mediated the inhibition of adenylyl cyclase activity with a rank order of potency comparable to their affinity constants. Immunoblot analysis of membranes prepared from cells expressing m5-HT1B receptors and photoaffinity labeling of the immunoprecipitated material revealed photolabeled species at approximately 95 and at approximately 42 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Inositol 1,4,5-trisphosphate receptor expression in cardiac myocytes.

Abstract: Calcium release from intracellular stores is the signal generated by numerous regulatory pathways including those mediated by hormones, neurotransmitters and electrical activation of muscle. Recently two forms of intracellular calcium release channels (CRCs) have been identified. One, the inositol 1,4,5-trisphosphate receptors (IP3Rs) mediate IP3-induced Ca2+ release and are believed to be present on the ER of most cell types. A second form, the ryanodine receptors (RYRs) of the sarcoplasmic reticulum, have evolved specialized functions relevant to muscle contraction and are the major CRCs found in striated muscles. Though structurally related, IP3Rs and RYRs have distinct physiologic and pharmacologic profiles. In the heart, where the dominant mechanism of intracellular calcium release during excitation-contraction coupling is Ca(2+)-induced Ca2+ release via the RYR, a role for IP3-mediated Ca2+ release has also been proposed. It has been assumed that IP3Rs are expressed in the heart as in most other tissues, however, it has not been possible to state whether cardiac IP3Rs were present in cardiac myocytes (which already express abundant amounts of RYR) or only in non-muscle cells within the heart. This lack of information regarding the expression and structure of an IP3R within cardiac myocytes has hampered the elucidation of the significance of IP3 signaling in the heart. In the present study we have used combined in situ hybridization to IP3R mRNA and immunocytochemistry to demonstrate that, in addition to the RYR, an IP3R is also expressed in rat cardiac myocytes. Immunoreactivity and RNAse protection have shown that the IP3R expressed in cardiac myocytes is structurally similar to the IP3R in brain and vascular smooth muscle. Within cardiac myocytes, IP3R mRNA levels were approximately 50-fold lower than that of the cardiac RYR mRNA. Identification of an IP3R in cardiac myocytes provides the basis for future studies designed to elucidate its functional role both as a mediator of pharmacologic and hormonal influences on the heart, and in terms of its possible interaction with the RYR during excitation-contraction coupling in the heart.

Tumor necrosis factor-alpha blockade prevents neutrophil CD18 receptor upregulation and attenuates acute lung injury in porcine sepsis without inhibition of neutrophil oxygen radical generation.

Abstract: Tumor necrosis factor (TNF alpha), both by direct action and by trafficking cells of the immune system, is implicated in cardiopulmonary derangements and PMN-mediated microvascular injury associated with gram-negative sepsis. We examined the effects of pretreatment with a monoclonal antibody to TNF alpha on PMN function, hemodynamic derangements, and alveolar capillary membrane damage in a septic porcine model. Anti-TNF alpha profoundly improved hemodynamic consequences in this model. Reduction in PMN CD11/18 receptor expression, lung myeloperoxidase activity, and attenuation of peripheral neutropenia (all P < 0.05) indicate that pretreatment significantly reduced lung sequestration of PMNs seen in septic controls. In contrast, PMN oxygen radical (O2-) generation was not significantly different from unprotected septic animals. Despite the presence of circulating PMNs primed for O2- burst, alveolar capillary membrane damage, assessed by bronchoalveolar lavage protein content and arterial PO2 was markedly attenuated in the treatment group (P < 0.05). We conclude that anti-TNF alpha suppresses systemic hemodynamic actions of TNF alpha. Further, it prevents upregulation of PMN adhesion receptors inhibiting PMN/endothelial cell interaction. This prevents formation of a "microenvironment," protected from circulating oxidant scavengers, into which sepsis-activated PMNs release their toxic products. Pretreatment with anti-TNF alpha monoclonal antibody thus affords global protection in porcine Gram-negative sepsis.

Transforming growth factor β exerts opposite effects from interleukin‐1β on cultured rabbit articular chondrocytes through reduction of interleukin‐1 receptor expression

Abstract: Objective. The aim of the study was to determine whether transforming growth factor β (TGFβ) modulates the effects and the receptor expression of interleukin-1 (IL-1) in rabbit articular chondrocytes (RAC). Methods. Collagen, glycosaminoglycan, and collagenase production, together with 125I-labeled IL-1 binding, were analyzed in RAC cultures. Results. TGFβ reduced both IL-1 effects on matrix metabolism and IL-1 receptor expression. Conclusion. TGF/3 acts as an antagonist of the effects of IL-1 through down-regulation of its receptor expression.

IL-1 and tumor necrosis factor-alpha each up-regulate both the expression of IFN-gamma receptors and enhance IFN-gamma-induced HLA-DR expression on human monocytes and a human monocytic cell line (THP-1).

Abstract: Stimulation of human blood monocytes (adherent mononuclear cells) and the monocytic cell line, THP-1, by IL-1 or TNF-alpha leads to the up-regulation of IFN-gamma receptors. Scatchard analysis using 125I-IFN-gamma revealed a twofold increase in the number of IFN-gamma receptors on THP-1 cells without an alteration in the affinity of the receptor. The potential functional significance of this induction of IFN-gamma receptors on monocytes and THP-1 cells was investigated by examining the effect of IFN-gamma on MHC class II Ag expression by these cells. Both IL-1 and TNF-alpha enhanced the IFN-gamma-induced HLA-DR expression (> twofold) and this effect was inhibited by antibody to IFN-gamma. In the case of human monocytes, IL-1 or TNF-alpha, each by themselves also increased HLA-DR expression, which was also abrogated by antibody to IFN-gamma. The data suggest that the immunopotentiating effects of IL-1 and TNF-alpha are mediated in part by enhancing IFN-gamma receptor expression on monocytes and macrophages. This presumably would increase the capacity of IFN-gamma to activate macrophages, enabling them to express HLA-DR and present Ag more effectively.

Fatty acids, inflammation and immune responses.

Abstract: Evidence obtained from experiments in vitro and in vivo suggests that certain unsaturated fatty acids (FA) may be safe and effective antiinflammatory and immunomodulatory agents. Generation of a unique eicosanoid profile with different biological effects by administration of FA precursors other than arachidonic acid is one approach under investigation. In addition to their role as eicosanoid precursors, FA are of major importance in maintaining cell membrane structure, are key determinants of membrane bound enzyme activity and receptor expression. FA can exert these functions directly and therefore may themselves be important regulators of immune responses. For example, certain FA influence cytokine production and proliferation of human T lymphocytes in a manner that is direct and not due to their conversion to eicosanoids. The observations indicate that FA can modulate immune responses by acting directly on T-cells and suggest that alteration of cellular FA may be a worthwhile approach to control of inflammation.

Decreased level of PDGF-stimulated receptor autophosphorylation by fibroblasts in mechanically relaxed collagen matrices

Abstract: The goal of our studies was to characterize the interrelationship between extracellular matrix organization and fibroblast proliferation in response to growth factors. We compared fibroblasts in monolayer culture with cells in contracted collagen matrices that were mechanically stressed or relaxed. In response to platelet-derived growth factor (PDGF), DNA synthesis by fibroblasts in mechanically relaxed collagen matrices was 80-90% lower than in monolayer culture and 50% lower than in mechanically stressed matrices. Fibroblasts in monolayer and contracted collagen matrix cultures contained similar levels of PDGF receptors, but differed in their autophosphorylation response. Cells in mechanically relaxed matrices showed lowest levels of autophosphorylation, 90% less than cells in monolayer culture. Experiments comparing receptor expression and capacity for PDGF-stimulated autophosphorylation showed that cells in mechanically relaxed collagen matrices never developed normal receptor autophosphorylation. Furthermore, when mechanically stressed collagen matrices were switched to mechanically relaxed conditions, capacity for receptor autophosphorylation decreased within 1-2 h and remained low. Based on immunomicroscopic observations and studies on down-regulation of receptors by PDGF binding, it appeared that most PDGF receptors in monolayer or contracted collagen matrix cultures were localized on the cell surface and accessible to PDGF binding. In related studies, we found that EGF receptors of fibroblasts in mechanically relaxed collagen matrices also showed low levels of autophosphorylation in response to EGF treatment. Based on these results, we suggest that mechanical interactions between cells and their surrounding matrix provide regulatory signals that modulate autophosphorylation of growth factor receptors and cell proliferation.

Selective up-regulation of the 75-kDa tumor necrosis factor (TNF) receptor and its mRNA by TNF and IL-1.

Abstract: In cells of the fibroblastoid line SV-80, rapid down-modulation of TNF binding in response to TNF itself, or to IL-1, was followed by a gradual recovery of binding, which occurred even in the continuous presence of the cytokines. Untreated cells carried mainly the 55-kDa receptor species. In cells treated with TNF or IL-1, the 55-kDa TNF-R, although increasing after initial down-modulation, remained lower than before treatment. Expression of the 75-kDa TNF-R species upon treatment with either cytokine was markedly increased. Both TNF and IL-1 also induced a strong increase of the mRNA for the 75-kDa receptors, whereas the amount of mRNA for the 55-kDa receptors decreased. The effects of TNF on cell surface expression of the TNF-R could not be blocked with antibodies to IL-1, nor could the effects of IL-1 be blocked with antibodies to TNF, indicating that each cytokine affects the cell surface expression of the receptors independently of the other. Applying both cytokines together resulted in much stronger increase in expression of the 75-kDa TNF-R than applying each alone. Similar changes in cell surface expression and mRNA levels of the two TNF-R as observed in SV-80 cells were also found in TNF and IL-1-treated human foreskin fibroblasts. It is suggested that these sustained changes in the pattern of receptor expression contribute to the adjustment of the cellular response to TNF when formation of TNF and IL-1 takes place over a prolonged period.

Regulation of protein kinase C isoform proteins in phorbol ester-stimulated Jurkat T lymphoma cells.

Abstract: Activation of protein kinase C (PKC) in T cells leads to a variety of responses including IL-2 production and IL-2 receptor expression. PKC consists of several isoforms that exhibit some different in vitro properties. We have set up a Western blotting system to explore the regulation of PKC isoforms during T cell activation. In Jurkat T lymphoma cells, PKC alpha, beta, delta, epsilon, and zeta were detected. PKC alpha and beta existed primarily in the cytosol, translocated to the membrane fraction after 10 minutes of treatment with PMA, and almost completely disappeared within 16 h. A larger fraction of PKC delta and epsilon existed in the membrane fraction compared to PKC alpha or beta, and PKC epsilon translocated to the membrane fraction rapidly. Translocation of PKC delta was not apparent after 1 h treatment with PMA, but total PKC delta protein was reduced within 4 to 6 h of treatment. Consistent with this, overnight treatment with PMA caused down-regulation of both PKC delta and epsilon, but to a lesser degree than was observed with PKC beta. Anti-PKC zeta antibody detected two bands at 82 and 75 kDa. The 75-kDa band existed mostly in the cytosol fraction and showed no translocation or down regulation after PMA. We present evidence that this 75-kDa band represents PKC zeta. Similar PMA-induced translocation responses were observed in murine thymocytes showing that the responses are not unique to PKC isoforms in Jurkat. These results demonstrate that it is possible for the PKC isoforms to be differentially regulated during T cell activation.

Interferon-alpha and interleukin 2 synergistically enhance basic fibroblast growth factor synthesis and induce release, promoting endothelial cell growth.

Abstract: To elucidate mechanisms underlying neovascularization that accompanies certain chronic immune/inflammatory disorders, the effects of interferon-alpha (IFN-alpha) and interleukin 2 (IL-2) on endothelial cell (EC) growth in vitro and angiogenesis in vivo were studied. Preincubation of cultured human ECs with IFN-alpha, followed by exposure to IL-2, resulted in effective stimulation of cell growth, whereas either cytokine alone had only a slight effect. The combination of IFN-alpha/IL-2 induced an angiogenic response in the rabbit cornea. IL-2 receptor expression was enhanced on IFN-alpha-treated ECs: p55 was increased and p70 was induced. 125I-IL-2 binding to ECs treated with IFN-alpha was enhanced (Kd from approximately 7 nM to approximately 260 pM with IFN-alpha), and anti-p55 IgG blocked 125I-IL-2/EC interaction as well as IL-2-mediated EC proliferation. Consistent with these findings in cell culture, immunohistologic studies demonstrated p55 and p70 antigen in the vasculature of rheumatoid joints, but not in normal joint tissue. Exposure of cultured ECs to IFN-alpha increased levels of intracellular EC basic fibroblast growth factor (bFGF), and subsequent addition of IL-2 led to bFGF release into the medium. The observation that anti-bFGF IgG largely blocked EC proliferation in response to IFN-alpha/IL-2 suggested that bFGF was a critical agent in this setting. These data suggest a mechanism rendering ECs responsive to IL-2 which may be relevant in immune/inflammatory disorders: IFN-alpha-mediated induction of functional EC receptors for IL-2, which drives cell proliferation by a mechanism dependent on increased synthesis and release of bFGF.

Effects of leflunomide on immune responses and models of inflammation

Abstract: Leflunomide is an antiphlogistic and immunomodulating agent that has been shown to be effective in preventing and healing autoimmune disorders and reactions leading to organ graft rejection. From our preliminary clinical data [4], we now have hopes that these effects, observed in experimental animals, can truly be transferred to humans. Although we are far from understanding the mode of action of leflunomide, we are slowly gathering some insight. A good many of the immunosuppressive effects of leflunomide can be attributed to the antagonistic effects it has on responses to many cytokines, most likely through receptor expression and signal transduction (tyrosine kinase inhibition). The inhibition of transplant rejection could be explained by interference with the activity of IL-2 and IL-4, i.e. the interference of cytotoxic T cell formation. Considering, further, that increased IL-3-like activity has been reported in autoimmune MRL/lpr mice [23], and that it is felt that this amplified activity may contribute to the pathology of their illness [23], then the interference of leflunomide with IL-3 may, together with the antagonistic activity of TRF and specifically IL-4, explain some of the disease modifying properties of this drug in animals with SLE-like and other autoimmune diseases. Also, interference with responses to IL-6 (Germann, personal communication) could be responsible for the suppression of acute-phase proteins observed in adjuvant-diseased rats [24]. Our data concerning tyrosine kinase inhibition as a hypothetical mechanism for the non-cytotoxic and reversible antiproliferative activity of A77 1726 are in many ways, intriguing. First of all, many known receptors for growth factors are associated with tyrosine kinase, i.e. EGF [35], IL-2 (the high binding, 75 kDa chain) [21], IL-3 [26], G-CSF, GM-CSF and TNF-α [9]. Leflunomide antagonizes all of these mediators. On the other hand, IL-1, which is not antagonized by leflunomide, is not associated with tyrosine kinase, but with threonine and serine kinase [11]. Much more work must be conducted before we can be sure that tyrosine kinase inhibition is important for the mode of action of leflunomide. Another important aspect of this drug is its inhibitory effect on the release of histamine from basophils and mast cells, because of its role in inflammatory reactions. Relating to our findings on the activity of leflunomide on murine SLE-like disorders, it has been reported recently that SLE patients often exhibit abnormal production of antibodies to IgE, and that these autoantibodies may, by activating mast cells and basophils, play a consequential part in the release of vasoactive amines, thus leading to generalized tissue injury [15]. We are confident that leflunomide will prove to be an effective drug in combating human autoimmune disorders. Indeed, we already have preliminary evidence for this, from studies of its effects on humans suffering from autoimmune diseases. Moreover, this drug provides a tool for gaining new insights into both the mechanisms leading to such ailments and their therapeutic control, and as such may facilitate the discovery of even more proficient drugs or other means to modulate these malfunctioning immune reactions.