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R S Mitra

Publications and source records attributed to R S Mitra.

At least 19 recordsLinked to original sources
PubMed1992

Epidermal growth factor and transforming growth factor-alpha decrease gamma interferon receptors and induction of intercellular adhesion molecule (ICAM-1) on cultured keratinocytes.

The link between the epidermal keratinocytes of the skin and the activated T lymphocytes of the immune system is mediated by a variety of cytokines, including gamma interferon (IFN-gamma). We studied the influence of keratinocyte mitogens such as transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and somatomedin-C (SM-C) on the ligand binding of 32P-labeled IFN-gamma to cultured keratinocytes derived from normal appearing adult human skin. Keratinocytes placed in a medium devoid of mitogens become growth arrested, and these quiescent cells expressed 2.4 times (28,900 versus 12,200 sites/cell) as many high affinity IFN-gamma receptors (Kd = 0.22 nM) compared to keratinocytes which were actively growing in medium containing TGF-alpha (25 ng/ml) or EGF (10 ng/ml). The reduction in IFN-gamma receptor sites by TGF-alpha/EGF was mitogen specific, as adding SM-C (500 ng/ml) did not have any effect on ligand binding, although it similarly stimulated keratinocyte growth. The reduction in IFN-gamma receptors was time dependent, occurring primarily after 24-48 hours of change in tissue culture conditions. The reduction in the number of high affinity IFN-gamma receptors by TGF-alpha/EGF had immunobiological consequences, because quiescent keratinocytes in basal medium had an increased expression of HLA-DR and intercellular adhesion molecule-1 (ICAM-1) induced by IFN-gamma, compared to actively growing TGF-alpha/EGF treated keratinocytes. These results suggest that rapidly proliferating keratinocytes exposed to TGF-alpha/EGF but not SM-C are capable of altering their response to IFN-gamma by decreasing their number of cell surface high affinity receptors for IFN-gamma.

Cell Adhesion Molecules↗
PubMed1992

Keratinocyte activation following T-lymphocyte binding.

T lymphocytes infiltrate the epidermis and follicular epithelium adhering to keratinocytes within hours following induction of cutaneous inflammation. To determine if the physical binding interaction between a T cell and keratinocyte induces transmission of activation pathways, CD3+ T cells (HUT 78) were allowed to directly bind to non-cytokine-treated cultured keratinocytes. When these T cells bound to keratinocytes, the keratinocytes were activated as evidenced by detection of tumor necrosis factor-alpha, interleukin-6, and intercellular adhesion molecule-1 mRNA. This induction was relatively mRNA specific, as several other mRNA were not found to be altered. This activation process appeared to be one-sided, as no change in HUT cell mRNA levels was detectable. The keratinocyte activation process was confined to cultures that had direct physical binding by HUT cells, because co-culturing the HUT cells immediately above the keratinocyte monolayer (but not in direct contact), resulted in no such mRNA alterations. This direct adhesion-mediated activation of keratinocytes by T lymphocytes may be important in the genesis of cutaneous inflammation by amplifying the original stimulus, as well as contributing to the trafficking pattern of inflammatory cells as they leave the general circulation and enter the skin.

Cell Adhesion Molecules↗
PubMed1992

HUT 78 T cells bind to noncytokine-stimulated keratinocytes using a non-CD18-dependent adhesion pathway.

The initial in vitro observation that cultured keratinocytes, when treated with cytokines such as gamma interferon, increased the binding of T lymphocytes, opened up a whole new avenue of research to understand epidermal trafficking patterns in inflammatory skin diseases. A growing body of data strongly supports the in vivo role of lymphocyte-function-associated antigen-1 (CD18) expression by T cells in the binding to intercellular adhesion molecule-1 (CD54) expressing keratinocytes. To further explore the molecular basis for other possible adhesive interactions involving T cells and skin-derived cellular constituents, the authors used 2 cell lines (HUT 78 cells and Jurkat cells) and added them to multipassaged human keratinocytes, fibroblasts, and melanocytes. The skin-derived cells were treated with cytokines alone, or in combination, with a phorbol ester. HUT cells were capable of binding to keratinocytes in the absence of pretreatment with cytokines at 25 degrees C, which was not inhibited by anti-CD18 antibodies, or sensitive to reducing the temperature of the adhesion assay to 7 degrees C. Fibroblasts and melanocytes also constitutively bound HUT cells, but the binding to fibroblasts was highly temperature-sensitive. When keratinocytes were pretreated for 48 hours with gamma interferon plus phorbol ester, a "superadhesive" state was induced, resulting in a synergistically increased binding ability of both HUT cells and Jurkat cells. This effect was related to quantitative increases in keratinocyte intercellular adhesion molecule-1 expression. Several other clear-cut qualitative and quantitative differences were detectable in the ability of HUT cells and JS cells to bind to nontreated and cytokine/phorbol ester-treated keratinocytes, fibroblasts, and melanocytes. These results emphasize the complexity of molecular associations underlying T-cell trafficking patterns, potentially operative in the dermal and epidermal compartments of the skin.

Antigens, CD↗
PubMed1991

Monocyte chemotaxis and activating factor production by keratinocytes in response to IFN-gamma.

Monocytes accumulate in the epidermis and along the dermo-epidermal junction in several different inflammatory skin diseases. To determine whether human epidermal keratinocytes elaborate a specific chemotaxin responsible for the accumulation of monocytes at these anatomic sites, monocyte chemotactic activity in conditioned 16-h cultured keratinocyte supernatants were assayed using human peripheral blood monocytes as the target cell. Dilutional analysis revealed directed monocyte migration in IFN-gamma-treated (100 U/ml) keratinocyte supernatants (80% maximal FMLP response) which was 10-fold more than IFN-gamma itself or untreated keratinocyte activity alone. Gel filtration chromatography revealed that this activity eluted just ahead of a 12.5-kDa molecular mass marker. Blocking studies demonstrated that a rabbit polyclonal antibody to monocyte chemotaxis and activating factor (MCAF) inhibited all monocyte chemotaxis by greater than 80%. Keratinocytes were metabolically labeled with 35S-cysteine/methionine, and after 16 h incubation the supernatants immunoprecipitated with the same anti-MCAF antibody. MCAF was detected as a protein doublet of 12 and 9 kDa only in IFN-gamma-treated (100 U/ml) keratinocyte supernatants. Incubation with IFN-gamma and TNF-alpha (250 U/ml) in combination resulted in increased production of MCAF protein. By Northern blot analysis, MCAF mRNA was constitutively expressed in keratinocytes and upregulated only in the presence of IFN-gamma. TNF-alpha, IL-1 beta, transforming growth factor-beta and phorbol esters had no positive or negative influence on MCAF mRNA. These studies demonstrate that biologically active MCAF is elaborated by human epidermal keratinocytes upon activation by IFN-gamma, a cytokine also required for the induction of adherence between monocytes and keratinocytes. Keratinocyte-derived MCAF is likely to be important in the regulation of cutaneous monocyte trafficking and may also be responsible for the recruitment of Langerhans cells and dermal dendrocytes, which share many phenotypic features with monocytes/macrophages, to their anatomic locations in skin.

Cells, Cultured↗
PubMed1991

Keratinocytes as initiators of inflammation.

Environmental stimuli responsible for inducing cutaneous inflammation include contact allergens and ultraviolet light. We postulate that these diverse stimuli trigger a cutaneous inflammatory response by directly inducing epidermal keratinocytes to elaborate specific pro-inflammatory cytokines and adhesion molecules. The consequences are activation of dermal microvascular endothelial cells and selective accumulation of specific mononuclear cells in the dermis and epidermis. Thus, keratinocytes may act as "signal transducers", capable of converting exogenous stimuli into the production of cytokines, adhesion molecules, and chemotactic factors (acting in an autocrine and paracrine fashion) responsible for initiation of "antigen-independent" cutaneous inflammation. The initiation phase may facilitate or promote an amplification phase with additional production of tumour-necrosis factor alpha and interferon gamma via an "antigen-dependent" pathway, and keratinocyte/T cell/antigen-presenting dendritic cellular associations. The direct activation of keratinocytes, with their ability to produce the complete repertoire of pro-inflammatory cytokines, can profoundly influence endogenous and recruited immunocompetent cells, thereby providing the critical trigger responsible for the swift and clinically dramatic alterations that occur following contact between the epidermis and a host of "noxious" agents.

Catechols↗
PubMed1991

Assessment of the role of DNA damage and repair in the survival of primary cultures of rat cutaneous keratinocytes exposed to bis(2-chloroethyl)sulfide.

Toxicity manifests itself as vesication in human skin exposed topically to bis(2-chloroethyl)sulfide (BCES). The destruction of the proliferating population of epidermal cells is a major component of the pathogenic process. Available data strongly suggest that damage to cellular DNA is a critical factor in the loss of these cells. However, the influence of DNA repair on this toxic response has not been adequately studied. Therefore, a study was undertaken to ascertain the influence of DNA repair on the survival of primary monolayer cultures of rat cutaneous keratinocytes exposed to BCES. The sensitive nucleoid sedimentation assay was employed for the determination of DNA damage in cultures exposed to very low levels of BCES. Initial experiments demonstrated that within 1 hr of exposure to as little as 0.1 microM BCES the structural integrity of cellular DNA was compromised, presumably resulting from the appearance of single-strand breaks in the nucleic acid. This same effect was demonstrated in basal cells derived from a stratified, cornified culture grown at the air-liquid interface and exposed topically to the vesicant. Further studies with the monolayer culture demonstrated that the gross structural integrity of the DNA in cells exposed to as much as 5 microM BCES was completely restored within the first 22 hr following the exposure. However, this repair process appeared to be inefficient since a depression of thymidine incorporation into DNA and a significant loss of DNA were exhibited in exposed cultures as long as 72 hr after the initial exposure.

Animals↗
PubMed1991

Modulation of keratinocyte-derived interleukin-8 which is chemotactic for neutrophils and T lymphocytes.

Interactions between T lymphocytes, neutrophils, and epidermal cells are believed to play a central role in the pathophysiology of psoriasis and other inflammatory cutaneous disorders. Although there is strong evidence that lymphocyte-function-associated antigen-1 (LFA-1) positive T cells are retained in the epidermis via intercellular adhesion molecule-1 (ICAM-1) expression induced on keratinocytes, the molecular basis for the directed migration of T cells or neutrophils towards the epidermis is not known. To investigate whether epidermal keratinocyte-derived products may be important in the migration of T cells and neutrophils into the epidermis, human keratinocytes were cultured in the presence of various cytokines and chemotactic activity of the supernatants were assessed. TNF-alpha stimulation produced directed migrational responses for both neutrophils and T-lymphocytes (both CD4 and CD8), but not B lymphocytes; 69% of T-cell movement and 80% of neutrophil migration induced by the TNF-alpha treated keratinocyte cell supernatants could be inhibited by anti-interleukin-8 (IL-8) serum. Using the same antibody, IL-8 was immunoprecipitated from the supernatants of TNF-stimulated 35S-labelled keratinocytes, and a single 7-kd band product detected by SDS-PAGE. In keeping with these biological activities and protein data, Northern blot analysis of total cellular RNA extracted from keratinocyte monolayers hybridized with a 32P-labelled 1-kb cDNA to IL-8 mRNA, revealed induction of the IL-8 gene in the presence of TNF-alpha and IL-1 beta, but not IFN-gamma. The protein kinase C agonist, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known stimulator of psoriasiform cutaneous inflammation when applied directly to murine epidermis, strongly induced keratinocyte elaboration of IL-8 mRNA. These studies demonstrate that activated human keratinocytes are capable of producing biologically active IL-8, and provide evidence that keratinocytes can play a key role in mediating the influx of T cells and neutrophils into the epidermis.

Blotting, Northern↗
PubMed1991

Cellular localization of interleukin-8 and its inducer, tumor necrosis factor-alpha in psoriasis.

The importance of immunologic mechanisms in psoriasis has been deduced from the ability of immunosuppressive therapies to ameliorate this common and chronic skin disease. Certainly the histology of psoriatic lesions suggests a dialogue between the hyperplastic keratinocytes and infiltrating T lymphocytes and macrophages. To begin dissecting the cytokine network involved in the pathophysiology of psoriasis, the location, in both epidermal and dermal compartments, of tumor necrosis factor-alpha, interleukin-8, intercellular adhesion molecule-1, and transforming growth factor-alpha at the protein and/or mRNA levels were identified. Tumor necrosis factor-alpha was selected as a potentially key regulatory cytokine, first because it induces cultured keratinocyte interleukin-8, intercellular adhesion molecule-1, and transforming growth factor-alpha production, and second because intercellular adhesion molecule-1 expression by keratinocytes in psoriatic epidermis had been identified previously. Using immunohistochemical localization, tumor necrosis factor-alpha was identified in 12 psoriatic lesions as intense and diffuse expression by dermal dendrocytes (macrophages) in the papillary dermis (without significant staining of endothelial cells, mast cells, or dermal Langerhans cells), and focally by keratinocytes and intraepidermal Langerhans cells. Functional interaction between the dermal dendrocytes and keratinocytes was suggested by the presence of interleukin-8 expression of suprabasal keratinocytes immediately above the tumor necrosis factor-alpha-positive dermal dendrocytes. Interleukin-8 mRNA and transforming growth factor-alpha mRNA were detectable in the epidermal roof of psoriatic lesions, but neither was detectable at the protein or mRNA levels in any normal skin specimens. Treatment of cultured human keratinocytes with phorbol ester (which experimentally produces psoriasiform changes on mouse skin) or tumor necrosis factor-alpha also increased interleukin-8 and transforming growth factor-alpha mRNAs. Further elucidation of the cellular and molecular basis for the genesis and evolution of psoriasis will provide the framework for a better evaluation of the cause and treatment of this skin disease.

Adult↗
PubMed1990

All-trans retinoic acid stimulates growth and extracellular matrix production in growth-inhibited cultured human skin fibroblasts.

All-trans retinoic acid was examined for effects on human dermal fibroblast proliferation and for effects on fibroblast production and expression of non-collagenous and collagenous components of the extracellular matrix in vitro. Fibroblast proliferation was blocked when the cells were cultured in the presence of a serum-free culture medium containing epidermal growth factor, hydrocortisone, insulin, ethanolamine, phosphoethanolamine, and bovine pituitary extract as growth supplements and 0.15 mM Ca++. This level of extracellular Ca++ is lower than that needed to support fibroblast growth. Under these conditions, growth was stimulated by all-trans retinoic acid. Proliferation was also stimulated in the same basal medium without the growth supplements. Growth-promoting concentrations of all-trans retinoic acid ranged from 0.5-2.0 micrograms/ml (1.7-6.6 X 10(-6) M). Stimulation of proliferation was not seen at higher or lower concentrations. Concentrations of all-trans retinoic acid that stimulated proliferation also induced increased production of fibronectin as indicated by biosynthetic labeling/immunoprecipitation and by enzyme-linked immunosorbent assay. Increased production was associated with increased staining for fibronectin in the extracellular matrix. Increased production of two other non-collagenous extracellular matrix component, i.e., thrombospondin and laminin, also occurred in all-trans retinoic acid-treated cells. At 0.5 micrograms/ml, all-trans retinoic acid also stimulated production of type I collagen by the dermal fibroblasts, but at higher concentrations (2.5 micrograms/ml) production of type I collagen was inhibited. These data indicate that all-trans retinoic acid can induce changes in dermal fibroblasts in vitro (i.e., increased proliferation and extracellular matrix production) that mimic the major changes seen in the dermis after topical treatment with this agent.

Cell Division↗
PubMed1990

Thrombospondin binding by keratinocytes: modulation under conditions which alter thrombospondin biosynthesis.

Our recent studies have shown that thrombospondin (TSP) is a potent adhesion factor for normal human keratinocytes. Stimulation of adhesion is presumed to result from the binding of TSP to high-affinity receptors on the surface of responsive cells. The present study indicates that keratinocytes bind TSP in a receptor-like manner. Binding is time- and concentration-dependent, saturable, reversible and specific. Approximately 180 ng of TSP can be bound per 1 x 10(5) cells at saturation and half-maximal binding occurs at 22 nM. A series of monoclonal antibodies to various regions of the TSP molecule were examined for effects on TSP binding and TSP-induced adhesion. An antibody directed against the heparian-binding domain of the TSP molecule significantly inhibited TSP binding but had no effect on adhesion. In contrast, three antibodies which recognize epitopes in the 140-kDa fragment of the molecule inhibited both binding and adhesion. In a previous study we showed that treatment of keratinocytes with interferon-gamma inhibited TSP production and inhibited adhesion under unstimulated conditions as well as in response to TSP. The present study shows that interferon-gamma also inhibits TSP binding by keratinocytes. When the data from the present study are taken together with our past data, they suggest that normal human keratinocytes have the capacity to bind TSP and use this capacity to bind endogenously synthesized TSP. This provides a mechanism for utilizing the endogenously produced TSP to stimulate adhesion.

Antibodies, Monoclonal↗
PubMed1990

Marked synergism between tumor necrosis factor-alpha and interferon-gamma in regulation of keratinocyte-derived adhesion molecules and chemotactic factors.

T lymphocytes and mononuclear cells preferentially accumulate in the epidermis in inflammatory skin disease. To determine the role of keratinocytes in both the chemotaxis and adhesion of these cells to the epidermis, cultured keratinocytes were incubated with IFN-gamma and tumor necrosis factor-alpha (TNF-alpha), and mRNA detected and quantitated for IL-8, monocyte chemotaxis and activating factor, and intercellular adhesion molecule-1. Whereas induction of these mRNAs was either absent, or relatively weak and transient, to either IFN-gamma or TNF-alpha alone, when administered in combination there was a dramatic increase and persistence in the induction of all three genes. Pretreatment of the keratinocytes with cycloheximide failed to eliminate transcription, implying that all three are primary response genes. Transforming growth factor-beta, which modulates other keratinocyte functions (not related to adhesion or chemotaxis of inflammatory cells) failed to induce any of the genes. These novel findings potentially explain the selective recruitment of T cells and monocytes observed in inflammatory skin disease, because IFN-gamma and TNF-alpha can co-ordinately regulate keratinocyte-derived chemoattractants and adhesion molecule production.

Cell Adhesion Molecules↗
PubMed1990

Retinoic acid stimulation of human dermal fibroblast proliferation is dependent on suboptimal extracellular Ca2+ concentration.

Human dermal fibroblasts failed to proliferate when cultured in medium containing 0.15 mmol/l (millimolar) Ca2+ (keratinocyte growth medium [KGM]) but did when the external Ca2+ concentration was raised to 1.4 mmol/l. All-trans retinoic acid (retinoic acid) stimulated proliferation in KGM but did not further stimulate growth in Ca2(+)-supplemented KGM. The ability of retinoic acid to stimulate proliferation was inhibited in KGM prepared without Ca2+ or prepared with 0.03 mmol/l Ca2+ and in KGM treated with 1 mmol/l ethylene-glycol-bis-(beta-aminoethyl ether)N,N'-tetra acetic acid. Using 45Ca2+ to measure Ca2+ influx and efflux, it was found that retinoic acid minimally increased Ca2+ uptake into fibroblasts. In contrast, retinoic acid treatment of fibroblasts that had been pre-equilibrated for 1 day with 45Ca2+ inhibited release of intracellular Ca2+ into the extracellular fluid. Retinoic acid also stimulated 35S-methionine incorporation into trichloroacetic acid-precipitable material but in contrast to its effect on proliferation, stimulation of 35S-methionine incorporation occurred in both high-Ca2+ and low-Ca2+ medium. These data indicate that retinoic acid stimulation of proliferation, but not protein synthesis, is dependent on the concentration of Ca2+ in the extracellular environment.

Calcium↗
PubMed1989

Production and utilization of extracellular matrix components by human melanocytes.

Normal human melanocytes were separated from keratinocytes and maintained in culture using KGM medium supplemented with 12-O-tetradecanoylphorbol acetate and cholera toxin. The melanocytes were examined for the production of extracellular matrix molecules including fibronectin, laminin, and thrombospondin and for the utilization of these molecules in adhesion and motility assays. Melanocytes produced significant amounts of fibronectin as indicated by biosynthetic labeling/immunoprecipitation and by enzyme-linked immunosorbent assay (ELISA). Fibronectin was expressed on the surface of these cells. Laminin was also produced by melanocytes and expressed on the cell surface. The amount of laminin produced was significantly less than the amount of fibronectin. In contrast, melanocytes did not produce measurable thrombospondin as indicated by biosynthetic labeling/immunoprecipitation. Only traces of thrombospondin were detected by ELISA and no surface fluorescence was observed. When examined in adhesion and motility assays, melanocytes were found to utilize fibronectin for both processes. Laminin also stimulated adhesion but it was much less effective than fibronectin. Thrombospondin did not stimulate either attachment and spreading or motility. The pattern of extracellular matrix molecule production and utilization by melanocytes is significantly different from that shown previously for human epidermal keratinocytes (J. Varani et al., 1988, J. Clin. Invest. 81, 1537). These differences may underlie the differences with which the two cell types interact with basement membranes in vivo.

Cells, Cultured↗
PubMed1989

All-trans retinoic acid stimulates growth of adult human keratinocytes cultured in growth factor-deficient medium, inhibits production of thrombospondin and fibronectin, and reduces adhesion.

Human epidermal keratinocytes were established in culture using a low-Ca2+ (0.15 mM), serum-free keratinocyte growth medium (KGM) as the culture medium. Early passage keratinocytes (i.e., between passages 3-8) were incubated for 1 or 2 d in KGM, in KGM supplemented with 1.4 mM Ca2+, or in growth factor-deprived keratinocyte basal medium (KBM). The cells were concomitantly treated with all-trans retinoic acid (0.1-2.5 micrograms/ml), and cell growth was quantitated at the end of the incubation period. The keratinocytes were simultaneously examined for adhesiveness and production of two extracellular matrix molecules, e.g., thrombospondin (TSP) and fibronectin (FN). Treatment with all-trans retinoic acid inhibited proliferation of keratinocytes that were rapidly growing in KGM. Proliferation was also inhibited in KGM supplemented with 1.4 mM Ca2+, but all-trans retinoic acid did not reverse the morphologic features associated with differentiation induced by high Ca2+. In contrast to these effects, all-trans retinoic acid treatment of keratinocytes in KBM, in which the cells were normally quiescent, stimulated growth. In the presence of optimal concentrations of all-trans retinoic acid (0.5 microgram/ml), the rate of keratinocyte proliferation in KBM was approximately 35% of the rate obtained in KGM (maximal proliferation rate). Keratinocyte adhesion (resistance to trypsin-mediated release from the substrate and attachment to the substrate) was inhibited by all-trans retinoic acid under all three conditions. In regard to extracellular matrix production, TSP production was inhibited by greater than 90% under all three conditions in the presence of all-trans retinoic acid. FN production was also inhibited but to a lesser degree. Concentrations of all-trans retinoic acid required to maximally inhibit keratinocyte adhesion and matrix production were higher (1.0-2.5 microgram/ml) than the concentration required to stimulate proliferation in KBM. These in vitro observations may have implications in the effects of retinoids on intact skin, including enhanced keratinocyte proliferation and thickening of the epidermis after topical application to photoaged skin and inhibition of proliferation and cell-cell cohesion after systemic administration in cases of psoriasis.

Calcium↗
PubMed1989

Inhibition of 125I-epidermal growth factor binding to cultured keratinocytes by antiproliferative molecules gamma interferon, cyclosporin A, and transforming growth factor-beta.

The growth of cultured human keratinocytes (KC) is inhibited by gamma interferon (IFN-gamma), cyclosporin A and transforming growth factor-beta, but not by tumor necrosis factor. When these antiproliferative molecules were added to KC they induced a concentration and time-dependent inhibition of 125I-epidermal growth factor (I-EGF) binding. These anti-proliferative molecules primarily reduced the number of binding sites by approximately 25%-50% without affecting the binding affinity. Tumor necrosis factor did not influence the ligand binding by I-EGF. In parallel with the ability of the antiproliferative molecules to inhibit I-EGF binding, there was an increase in transforming growth factor-alpha production. These results suggest that several different antiproliferative molecules may share a common mechanism to inhibit cell growth by reducing I-EGF binding to KC.

Cyclosporins↗