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V B Morhenn

Publications and source records attributed to V B Morhenn.

At least 37 records · Page 2Linked to original sources
PubMed1989

Effects of recombinant interleukin 1 and interleukin 2 on human keratinocytes.

The effects of recombinant interleukin 1 alpha and beta, as well as recombinant interleukin 2, on human keratinocyte proliferation were studied in serum-containing as well as defined media. Both interleukin 1 preparations did not stimulate keratinocyte growth; interleukin 2 also did not stimulate keratinocyte growth. To determine whether interleukin 1 beta binds to keratinocytes, a cell membrane assay was developed for these cells. Iodinated interleukin 1 beta binds to keratinocytes with a kD of 6.2 nm and 2500 receptors per cell. To determine the effects of interleukin 1 beta on protein synthesis, the molecular patterns of radiolabeled cell extracts of interleukin 1 beta-treated and nontreated keratinocytes were compared using two-dimensional polyacrylamide gel electrophoresis. No significant changes in the molecular pattern of newly synthesized proteins were detected. Finally, none of these lymphokines induced HLA-DR expression by keratinocytes.

Cell Division↗
PubMed1988

Gamma interferon-induced expression of class II major histocompatibility complex antigens by human keratinocytes. Effects of conditions of culture.

Normal human keratinocytes grown in MCDB 153 plus bovine pituitary extract and treated with recombinant gamma interferon (rIFN-gamma) express HLA-DR, DP and DQ antigens. The expression of these class II MHC antigens is time dependent: DR and DP appear before DQ. The delay in HLA-DQ expression is not due to the effects of trypsinization of cultures prior to analysis. Increasing the calcium ion concentration from 0.1 to 1.8 mM does not alter the expression of these antigens. Keratinocytes grown without serum proteins or bovine pituitary extract exhibited markedly delayed expression of DR. By contrast, keratinocytes grown in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal calf serum express DR and DP but only very small amounts of DQ after treatment with rIFN-gamma. Expression of HLA-DR occurs at doses of rIFN-gamma that are too low to cause growth inhibition. The cells of the squamous cell carcinoma cell line SCL-1, whether grown in MCDB 153 plus bovine pituitary extract or DMEM plus 10% fetal calf serum, express HLA-DQ and DP on only small numbers of cells after treatment with the lymphokine. Thus, the conditions of culture, possibly the presence of a serum factor(s), influence the expression of class II antigens in normal keratinocytes. Furthermore, rIFN-gamma does not induce DP and DQ antigens readily in transformed squamous cells cultured in either serum-containing or serum-free medium.

Antigens, Surface↗
PubMed1988

Further characterization of the keratinocyte somatomedin-C/insulin-like growth factor I (SM-C/IGF-I) receptor and the biological responsiveness of cultured keratinocytes to SM-C/IGF-I.

Somatomedin-C/insulin-like growth factor I (SM-C/IGF-I) binds to both cultured keratinocytes trypsinized to single-cell suspensions and fresh epidermal sheets derived from 1 M NaBr-treated 6-mm punch biopsies. A gradual increase of 1-4% in specific binding of SM-C/IGF-I to fresh epidermal sheets was observed during a 5-hour incubation period at 15 degrees C. The human keratinocyte SM-C/IGF-I receptor was isolated by polyacrylamide gel electrophoresis after cross-linking with iodinated SM-C/IGF-I and migrated at 135,000 daltons under reducing conditions. Both a partially purified preparation of SM-C/IGF-I, as well as recombinant DNA-derived thr-59-SM-C/IGF-I (25-100 ng/ml), stimulated keratinocyte replication in a serum-free system, with an approximately equivalent effect as insulin (10 micrograms/ml). Since fibroblasts produce SM-C/IGF-I, our data suggest that some dermal-epidermal interactions in diseases such as psoriasis may be mediated, in part, by SM-C/IGF-I.

Cell Division↗
PubMed1987

Inhibition of attachment and growth of tumor cells on collagen by a monoclonal antibody.

A murine monoclonal antibody, VM-1, which binds to basal cells of normal human epidermis, reduces the ability of human squamous cell carcinoma cells (SCL-1) derived from the skin to attach and spread on collagen by about 50% and causes cell rounding. Similar effects have been previously shown using normal human keratinocytes. The attachment of cell lines derived from human lung squamous cell carcinomas (SW1271 and SW900), melanoma A375, glioblastoma 126, and fibrosarcoma HT1080 is also inhibited by this antibody. VM-1 antibody does not bind to normal human fibroblasts, benign nevus cells, or the human B-cell-derived line 8866. VM-1 antibody inhibits the growth of SCL-1 cells in vitro as measured by cell numbers and [3H]thymidine ([3H]TdR) incorporation. It is not cytolytic in the presence of complement as measured by 51Cr release. Repeated treatment of SCL-1 cells with VM-1 antibody significantly reduces the proportion of SCL-1 cells that attach to collagen. In addition, after treatment of SCL-1 cells with VM-1 antibody, several proteins can no longer be demonstrated by gel electrophoresis of the cell-free supernatant. The VM-1 antibody effect on attachment and spreading is partially reversed by pretreatment of the collagen surface with laminin and fibronectin, but not with the carbohydrates chondroitin-6-sulfate or hyaluronic acid or with the protein lysozyme. By fluorescence staining, the antigen recognized by VM-1 antibody is membrane-bound and Triton X-100 extractable. The VM-1 antigen is excluded from Bio-Sil TSK-400 and sediments at about 10.5 S. It has a covalent molecular weight on the order of 10(6). Proteinase K digestion produces VM-1 antibody reactive fragments, assumed to be polysaccharides, with a polydisperse molecular weight distribution in the range 5000 to 30,000. The VM-1 antigen is partially lost from solution on boiling and is no longer detectable in the aqueous or organic phase after chloroform-methanol extraction. The properties of the VM-1 antigen are consistent with those of a proteoglycan involved in attachment and spreading of keratinocytes and certain tumor cells on collagen.

Antibodies, Monoclonal↗
PubMed1987

Use of a monoclonal antibody (VM-2) plus the immunogold-silver technique to stain basal cell carcinoma cells.

A newly described murine monoclonal antibody, VM-2, stains both basal cell carcinoma cells and squamous cell carcinoma cells of the skin. We have combined this monoclonal antibody with a newly described technique, the immunogold-silver stain, to produce a method for rapid characterization of basal cell carcinoma cells. This method does not require the use of a fluorescence microscope and gives a very intense stain of the tumor cells. Tumor-containing tissue from 12 patients was stained and included 2 cases of morphealike basal cell carcinoma. All the tumors stained with VM-2 gold. Thus, staining with this technique may be helpful in diagnosing tumors of epithelial origin and may also find application in delineating residual tumor cells in microscopically controlled excision.

Antibodies, Monoclonal↗
PubMed1987

Hyperthermia for treatment of human split-thickness skin. Differential effects on HLA-DR-expressing cells and keratinocytes.

Human split-thickness skin was hyperthermia-treated and then the tissue was trypsinized to obtain dispersed cell suspensions. Cells consisting mainly of keratinocytes, obtained from the treated epidermis, retained their capacity to grow in vitro. By contrast, HLA-DR-expressing cells, dispersed from the epidermis as well as the dermis, lost their capacity to stimulate allogeneic peripheral blood mononuclear leukocytes after heat treatment (1.5 hr at 45 degrees C). The heat-treated epidermal cells did not release a factor that inhibited allogeneic lymphocyte proliferation. Even though the cells expressing HLA-DR antigen were no longer capable of stimulating allogeneic lymphocytes after hyperthermia, they still expressed HLA-DR antigen on their surface up to 72 hr after exposure to heat. The inhibition of stimulation of allogeneic lymphocytes by heat treatment was at least partially reversed by the addition of exogenous interleukin-2, but not interleukin-1. Thus, functions of the various cell types present in split-thickness skin were differentially sensitive to heat, a characteristic of skin cells that may be useful in skin transplantation.

Cell Division↗
PubMed1987

Interleukin-2 stimulates resting human T lymphocytes' response to allogeneic, gamma interferon-treated keratinocytes.

In order to investigate the biologic significance of HLA-DR expression by human keratinocytes, we have examined the possibility that DR-positive keratinocytes become alloantigen presenting cells for resting T cells in the presence of interleukin-2. Using this system, gamma interferon-treated, DR-positive keratinocytes stimulate the proliferation of allogeneic, resting T cells approximately 3-fold whereas non-gamma interferon-treated, DR-negative keratinocytes do not. Because a monoclonal antibody against recombinant gamma interferon inhibits this proliferation, the stimulation is dependent on pre-incubation with gamma interferon. By contrast, since the stimulation is not inhibited by a monoclonal antibody against HLA-DR, it is not clear that the stimulation is due to class II antigen expression by keratinocytes. To rule out that gamma interferon increases the expression of class I antigens, leading to stimulation of resting T cells on that basis, we determined whether gamma interferon treatment enhances class I antigen expression by keratinocytes. The lymphokine treated cells did not demonstrate more class I antigen expression than untreated keratinocytes. Thus, the observed stimulation of allogeneic, resting T cells by gamma interferon-treated keratinocytes in the presence of IL-2 is not due to increased class I antigen expression but is due to other cell surface antigen(s) induced by recombinant gamma interferon treatment. These results suggest that gamma interferon-exposed keratinocytes in the presence of interleukin-2 may augment the activation of resting T lymphocytes and, in this manner, may contribute to cutaneous inflammation.

Antibodies, Monoclonal↗
PubMed1987

Induction of new proteins by gamma interferon in cultured human keratinocytes.

Human epidermal keratinocytes incubated with recombinant gamma interferon (r-IFN-gamma) show, on two-dimensional gel electrophoresis, both the appearance of new proteins and the loss of others. Among [35S]methionine-labeled proteins, which are induced in an actinomycin- or alpha-amanitin-sensitive manner, is a prominent group with an apparent relative molecular mass of 53,000 and pI of 5.3-5.8. The synthesis of these proteins continues for at least 4 days in the presence of gamma interferon (IFN-gamma). Over the concentration range tested, up to 670 pM, there is no inhibition of protein synthesis, so the appearance of these proteins cannot be explained by overall inhibition of protein synthesis. Furthermore, at 4 pM we found only minor inhibition of DNA (21%) and RNA (29%) synthesis. Half-maximal induction of the prominent 53 kD proteins occurs at an interferon concentration of 0.8-3.5 pM which may be compared with a range of 1.5-30 pM for HLA-DR induction. The same prominent proteins are also induced by type I interferons. The 53 kD protein complex appears to consist of at least 4 different proteins, one of which is phosphorylated and another one of which is not induced in fibroblasts treated with IFN-gamma. We could obtain no evidence that the proteins were related by glycosylation. The presence of these proteins provides a sensitive means of identifying keratinocytes responding to interferons. Lack of these proteins in normal epidermis indicates that interferon does not play a major role in the control of keratinocyte behavior in sound skin.

Cells, Cultured↗
PubMed1987

Plasma somatomedin-C levels in psoriasis.

We have measured plasma levels of somatomedin-C, one of the principal mediators of growth hormone action, in 25 patients with plaque-type psoriasis of varying severity and compared these levels with those found in healthy controls. Plasma somatomedin-C levels were not elevated in the psoriatic patients regardless of the severity of their disease, suggesting that in these patients growth hormone secretion is within normal limits.

Adult↗
PubMed1987

Use of recombinant interferon gamma administered intramuscularly for the treatment of psoriasis.

Twenty-three patients with chronic plaque-type psoriasis were treated with intramuscular administration of human recombinant interferon gamma. Patients were treated with doses of 0.01 to 0.25 mg/m2 daily (five out of seven days) for four weeks, or 0.25 mg/m2 three times weekly for one week with escalation to 0.5 mg/m2 for the subsequent seven weeks. Some patients treated with the 0.25-mg/m2 dose showed improvement coincident with their therapy. Although recombinant interferon gamma may have some therapeutic activity in certain patients' psoriasis, the magnitude of this effect is at best small. This result is in contrast to interferon alfa, which has been reported to cause an exacerbation of this disease. Staining of posttreatment biopsy specimens with a monoclonal antibody against HLA-DR antigen using an immunoperoxidase technique demonstrated HLA-DR expression by keratinocytes in some of the patients treated at the higher doses. No obvious correlation was seen between clinical improvement of the psoriasis and intensity or extent of HLA-DR antigen expression by keratinocytes in the skin biopsy specimens.

Biopsy, Needle↗
PubMed1986

Human keratinocyte-lymphocyte reactions in vitro.

To extend our observation that recombinant gamma interferon (r-IFN-gamma) induces the synthesis and expression of HLA-DR antigen we have investigated 2 major areas including the modulation of r-IFN-gamma-induced HLA-DR expression and the possible immunologic consequences of keratinocyte HLA-DR expression in vitro. The induction of keratinocyte HLA-DR expression was greater for continuous compared with pulse dosage (0.5-24 h) of r-IFN-gamma and was markedly decreased after trypsinization of attached monolayers into single cell suspensions. The r-IFN-gamma caused induction of HLA-DR and this was not influenced by either pretreatment with irradiation, PGE2, or indomethacin. Both HLA-DR+ and HLA-DR- cultured keratinocytes induced RNA synthesis and gamma interferon production by allogeneic peripheral blood mononuclear leukocytes (PBMLs) indicating mononuclear cell activation. However, this activation was not followed by significant mitogenesis and only slightly increased levels of [3H]thymidine incorporation (maximal = 5800 cpm) by the PBMLs was observed. Cultured keratinocytes apparently inhibit both lectin-driven and mixed-lymphocyte reactions by producing a soluble mediator which is not dialyzable, or inhibited by pretreatment with indomethacin or anti-alpha, -beta, -gamma interferon antibodies. These results suggest that lymphocyte-keratinocyte reactions in vitro are complex and may be mediated by a variety of cytokines, lymphokines, and prostaglandins.

Cell Division↗
PubMed1986

Characterization of insulin-like growth factor-I/somatomedin-C receptors on human keratinocyte monolayers.

A membrane receptor for insulin on cultured human keratinocyte monolayers has recently been reported. It has also been established by previous investigators that the receptors for insulin and the somatomedin peptide, insulin-like growth factor-I/somatomedin-C (IGF-I), have similar oligomeric composition. However, these 2 receptors can be distinguished by their high affinity for their respective peptides. The present study was undertaken to identify and characterize IGF-I receptors on human keratinocytes, using time courses at different temperatures, competitive displacement of [125I]IGF-I by unlabeled IGF-I and insulin, and comparative binding of IGF-I, IGF-II, and insulin. The binding of [125I]IGF-I was time and temperature dependent, achieving steady state after 6 h of incubation at 15 degrees C. Specific binding at 15 degrees C averaged 4.33% for 0.8-1.0 million cells. Competition for binding was observed at IGF-I concentrations as low as 1 ng/ml, with half maximal displacement of [125I]IGF-I at IGF-I concentration of 17 ng/ml. Insulin, on the other hand, was 100-fold less potent than IGF-I in displacing [125I]IGF-I. Scatchard analysis of the competitive binding data revealed a curvilinear plot, with a calculated Kd = 1.4 X 10(-9) mol/liter. When the binding of [125I]IGF-I, -IGF-II, and -insulin was compared in the same experiment, the specific binding of [125I]IGF-I was 3.43% as compared with 5.60% for [125I]insulin and 0.90% for [125I]IGF-II. The finding of specific IGF-I receptors on cultured human keratinocytes suggests a possible role for this mitogenic peptide in epidermal cell proliferation.

Binding, Competitive↗
PubMed1985

Recombinant gamma interferon differentially regulates class II antigen expression and biosynthesis on cultured normal human keratinocytes.

Recombinant gamma interferon induces class II antigen (HLA-DR) biosynthesis and expression on normal cultured human keratinocytes. HLA-DR expression was not induced on keratinocytes by recombinant alpha or beta interferons in a similar dose range nor by Con A or PHA. HLA-DR (L243) expression, as determined by FACS analysis, was detected as early as 1-2 days after addition of r-IFN-gamma to the cultures and was maximal after 4-8 days. Keratinocytes were analyzed for expression of another class II antigen, HLA-DC (Leu-10). Little or no expression of Leu-10 (DC) was detectable on these cells although Fc receptors for the IgG1 isotype were increased. These data indicate a unique role for gamma interferon in the differential regulation of keratinocyte class II antigen biosynthesis and expression. Induction of HLA-DR on keratinocytes may be functionally important in expanding the number of antigen presenting cells in the skin for the induction of an immune response and/or targeting these keratinocytes for cytolysis.

Cells, Cultured↗
PubMed1985

A murine monoclonal antibody (VM-1) against human basal cells inhibits the growth of human keratinocytes in culture.

Using epidermal cells from psoriatic plaques as the immunogen, an IgG1 murine monoclonal antibody, VM-1, has been produced which stains basal keratinocytes on frozen sections of skin obtained from normal individuals and from psoriatic plaques. In some areas of both normal and psoriatic epidermis, the cell layer immediately above the basal cells is also stained. Cells in the external root sheath of the hair follicles also bind VM-1. The antibody binding site is trypsin-resistant, and is not blocked by bullous pemphigoid serum. If dispersed epidermal cells are preincubated with VM-1 for 1 h or more before plating, the majority of the cells do not attach and spread out on a collagen-coated Petri dish surface or on a fibroblast feeder layer. When added to attached, preconfluent cultures of keratinocytes, VM-1 inhibits growth and alters cell morphology. The growth inhibition is specific for keratinocytes, and viability studies show that it is not due to an immediate toxic effect of the antibody. The VM-1-induced inhibition of keratinocyte growth is not reversed by soy bean or lima bean trypsin inhibitors added at the time of cell plating or at the time of addition of antibody.

Animals↗
PubMed1985

Immunomodulatory and antiproliferative effect of recombinant alpha, beta, and gamma interferons on cultured human malignant squamous cell lines, SCL-1 and SW-1271.

Two different human malignant squamous cell lines (SCL-1 and SW-1271) and normal human foreskin fibroblasts were treated with recombinant human alpha, beta, and gamma interferons. HLA-DR expression was induced in a concentration-dependent fashion only on the SCL-1 cells treated with recombinant human gamma interferon (r-IFN-gamma) (10(2)-10(3) U/ml). No HLA-DR expression was observed with alpha or beta interferon on either malignant squamous cell line, nor with gamma interferon on SW-1271 cells. All three interferons reduced the number of malignant cells growing in culture but had no effect on the fibroblasts. There was a concentration-dependent growth-inhibitory response of the malignant cells by the interferons (dose range 1-10(3) U/ml; 7.1 X 10(-12) M to 7.1 X 10(-9) M). The SCL-1 cells were 10(2) more sensitive (based on weight) to the antiproliferative effects of gamma interferon than alpha or beta interferon. A brief (30-min) exposure of the SCL-1 cells to r-IFN-gamma (10(2) and 10(3) U/ml) produced approximately the same inhibition of cell growth as continuous exposure over a 2-week period. The SW-1271 cells were equally sensitive to alpha, beta, and gamma interferons. However, the maximal inhibitory effect on SW-1271 cells was less than that observed for the SCL-1 cells. Combining beta and gamma interferon resulted in cytotoxicity with SCL-1 cells and additive cytostatic effect on the SW-1271 cells. These additional malignant cell lines with their different sensitivities to alpha, beta, and gamma interferons may prove useful in studying the mechanisms of action of various interferons.

Carcinoma, Squamous Cell↗
PubMed1985

Enrichment of murine and human Langerhans cells with solid phase immunoabsorption using pan-leukocyte monoclonal antibodies.

Using a solid phase immunoabsorption (panning) technique, we have employed pan-leukocyte monoclonal antibodies to enrich and deplete murine and human Langerhans cells from cell suspensions of normal skin. Langerhans cell-enriched fractions contained 80-99% mononuclear cells, almost all of which had the ultrastructural features of Langerhans cells. These results are comparable to those achieved by panning for human Langerhans cells with anti-Leu-6(T6) antibody. Similarly, less than 1% of these cells were detectable in Langerhans cell-depleted fractions and such fractions were incapable of stimulating allogeneic lymphocytes in the skin cell-lymphocyte reaction. We conclude that panning with pan-leukocyte antibodies is an effective means of enriching or depleting Langerhans cells from heterogeneous skin cell suspensions and can yield results similar to those achieved with more Langerhans cell-specific reagents such as anti-Leu-6(T6). These findings are of particular significance to the enrichment and depletion of murine Langerhans cells since they express no known correlate of the human Leu-6(T6) antigen.

Animals↗