Soluble IL-1 receptor type I binds to human dermal fibroblasts and induces calcium flux.

Soluble cytokine receptors appear to modify ligand concentrations by stabilizing ligands or by specifically inhibiting interactions of ligands with their membrane-bound receptors. Here we describe a new function of the soluble interleukin-1 receptor type I (IL-1sR I). This receptor induced a transient rise of intracellular free calcium concentration in human dermal fibroblasts in a dose-dependent fashion. Mobilization of calcium by IL-1sR I was abolished in the presence of an equimolar concentration of IL-1 receptor antagonist (IL-1ra). Neutralizing antibodies against IL-1beta also abolished calcium mobilization stimulated with IL-1sR I indicating that IL-1beta is involved. IL-1sR I bound with high affinity (Kd 1-2 nM) to the fibroblasts. In addition, IL-1sR I enhanced expression of IL-6 and IL-8 mRNA. The observation that IL-1sR I can act as a ligand and agonist for membrane IL-1 extends the concept of the ligand-receptor functions of both IL-1 and IL-1sR I and adds a new dimension to the cytokine network.

Delivery to cancer cells of antisense L-myc oligonucleotides incorporated in fusogenic, cationic-lipid-reconstituted influenza-virus envelopes (cationic virosomes).

Antisense oligodeoxy-nucleoside phosphorothioates (OPTs) of L-myc were encapsulated into reconstituted influenza-virus-A envelopes (virosomes). The envelopes of the virosomes consisted of a single positively charged (cationic) lipid bilayer. Binding of cationic virosomes to cellular receptors that are membrane glycoproteins or glycolipids containing terminal sialic acid is mediated by the hemagglutinin glycoprotein (HA) of the influenza virus. After internalization through receptor-mediated endocytosis, cationic virosomes fuse efficiently with the membranes of the endosomal-cell compartment, and as a consequence the encapsulated OPT are delivered to the cell cytoplasma. Examination by fluorescence microscopy of the cellular uptake of cationic virosomes containing fluorescein-labeled OPT showed rapid and efficient incorporation of virosomes. Addition of cationic virosomes (75-150 microl) containing antisense L-myc OPT in the picomolar range to small-cell-lung-cancer (SCLC) cell cultures that expressed highly the L-myc oncogene led to strong inhibition of thymidine incorporation in a concentration-dependent manner. Virosome-entrapped sense L-myc OPT and random-order OPT had only minimal effects on the thymidine uptake. Cells of SCLC cell line NCI-H82 expressing a very low level of L-myc were not affected by antisense-L-myc virosomes. In Western-blot analysis, expression of L-myc protein was suppressed in the antisense-virosome-treated NCI-H209 cells but not in untreated control NCI-H209 cells. These results suggest that cationic virosomes may have great potential as an efficient delivery system for antisense oligonucleotides in cancer therapy.

Autologous versus allogeneic T cell-stimulated IL-6 production by dermal fibroblasts.

T cells adhere to human dermal fibroblasts (HDF). This cellular interaction leads to a pronounced secretion of the proinflammatory cytokines IL-6 and IL-8 via a juxtacrine stimulation induced by HDF-associated IL-1. Upon stimulation, fibroblasts express various surface proteins such as MCH-I molecules, which may interact with corresponding receptors on T cells. The present study was conducted to further investigate the mechanism of this complex interaction with regard to the secretion of IL-6 in cocultures of T cells and HDF. IL-6 was time- and dose-dependently upregulated in such cocultures. Spatial separation of the cells by microporous membranes resulted in a 90% reduction of IL-6 secretion, but when cells had limited cell contact IL-6 secretion was increased again. Allogeneic cocultures of T cells and HDF showed increased capacity of IL-6 stimulation as compared to autologous cultures. Our results suggest that MHC-I/T cell receptor interaction modulates IL-6 secretion in allogeneic and autologous cocultures.

Juxtacrine stimulation of cytokine production in cocultures of human dermal fibroblasts and T cells.

Adhesion of T cells to fibroblasts activates cells to produce cytokines, either by direct cell contact and/or soluble factors. A cell-associated form of IL-1 beta on fibroblasts might act through a cell contact mediated fashion. To test this hypothesis we analysed the activation of T cells and human dermal fibroblasts (HDF) in coculture experiments. Elevated levels of IL-1 beta, secreted by T cells as well as IL-6 and IL-8, mainly produced by HDF, were found in supernatant fluids of cocultured cells. IL-1 beta mRNA expression was induced in T cells as well as in HDF. While in HDF IL-1 beta remained cell-associated, T cells were activated to produce and secrete soluble IL-1 beta and IL-6. IL-1 beta and possibly other soluble factors increased IL-6 production by fibroblasts. These effects could be mainly attributed to CD8+ T cells. Our results suggest, that IL-1 beta, produced as a cell-associated cytokine by human dermal fibroblasts, acts as a juxtacrine molecule to stimulate T cells. Such a cellular cooperation, could be a powerful mediator in inflammatory response and possibly in wound healing.

Proliferation and differentiation of cultured human follicular keratinocytes are not influenced by biotin.

In humans and in animals, biotin deficiency causes pathological changes in the skin and its appendages. High doses of biotin may also have beneficial effects on skin, hair and fingernails in humans and animals with normal biotin status. Therefore, we investigated the effects of low and high concentrations of biotin on proliferation and differentiation of cultured outer root sheath cells from human hair follicles as an in vitro model for skin. The activities of biotin-dependent carboxylases were measured to evaluate the biotin status of the cells. In monolayer cultures of outer root sheath cells, proliferation and expression of the differentiation-specific keratins K1 and K10 were not influenced by extremely low concentrations of biotin (<2 x 10(-10) mol/l) or by pharmacological doses of biotin (10(-5) mol/l). Biotin deficiency of the cells was confirmed under the former condition by demonstrating decreased activities of the mitochondrial carboxylases. In organotypic cocultures of outer root sheath cells and dermal fibroblasts, in which stratified epithelia resembling epidermis were developed, the biotin concentration had no effect on the expression of all tested epidermal differentiation markers, including the suprabasal keratins K1 and K10, the hyperproliferation-associated keratin K16, involucrin and filaggrin.

Epstein-Barr virus subtype distribution in angioimmunoblastic lymphadenopathy.

The tissues of 16 patients bearing a T-cell lymphoma of angioimmunoblastic lymphadenopathy type (AILD-TCL) were investigated for the distribution of Epstein-Barr virus (EBV) subtypes 1 and 2. EBV-association had been proven in these cases by polymerase chain reaction (PCR) for EBV-DNA, in situ hybridization (ISH) for EBV-encoded small nuclear RNAs (EBER) and immunohistology for EBV-encoded latent membrane protein (LMP). PCR and EBER-ISH produced mostly identical results, but some cases were positive with only one of the 2 methods employed. LMP was detected in a few large cells of 8/13 cases. Twelve cases were investigated for the distribution of EBV subtypes. One case contained EBV genome of subtype 2, 3 cases contained subtype 1 and 4 cases contained both subtypes. Four cases could not be typed. These findings suggest that in AILD, as in AIDS-associated lymphomas and lymphomas of the lethal midline granuloma type, subtype 2 of EBV may occur, perhaps in relation to an immunodysfunction developing progressively in these patients.

Association of the subtype 2 of the Epstein-Barr virus with T-cell non-Hodgkin's lymphoma of the midline granuloma type.

Lethal midline granuloma (LMG) is associated with Epstein-Barr virus (EBV). The latter has at least two subtypes with different biological properties. The subtypes can be identified by their genomic configuration. Using EBV-RNA (EBER) in situ hybridization and EBV polymerase chain reaction (PCR), we have looked for the presence of EBV in six LMGs and six non-Hodgkin's lymphomas (NHLs) located in the nasopharyngeal region, and determined the subtype of EBV. Six of six LMGs were positive by PCR and EBER in situ hybridization, whereas NHLs were either negative or, in three of six cases, showed few EBER-positive cells considered to be nonneoplastic lymphocytes. The subtype 2 was found in LMG lesions of three of six patients; the remaining three of six patients with LMG had the generally occurring subtype 1. The results indicate that the association of EBV with NHL may depend more on tumor type than on its localization. The occurrence of the rare subtype 2 in LMG may relate to a covert immune defect.

Soluble factors from human hair papilla cells and dermal fibroblasts dramatically increase the clonal growth of outer root sheath cells.

Depending on environmental influences, follicular outer root sheath (ORS) cells in vivo can differentiate either towards interfollicular keratinocytes or, as demonstrated in the rat vibrissa, hair matrix cells. Crucial regulators of both their proliferation and differentiation are the mesenchymal cells of the respective tissues. The interactions of human ORS cells with human hair papilla cells (HPC) or human dermal fibroblasts (HDF) were studied using a two-chamber model separating the two cell types either by a microporous membrane or additionally by a medium layer. The results of 3H-thymidine incorporation studies indicated that ORS cell growth was markedly enhanced in co-culture with either HPC or HDF, the highest stimulatory effect resulting when ORS cells were in close association with the mesenchymal cells. No correlation was found between ORS cell proliferation and IL-6 production in the co-culture system, thus pointing to the secretion by HPC and HDF of growth-promoting soluble factors that are different form IL-6 as well as from EGF, bFGF and insulin present in the culture medium.

Increased levels of inflammatory cytokines in human skin lymph derived from sodium lauryl sulphate-induced contact dermatitis.

A superficial peripheral lymph vessel draining the skin of the upper and medial part of the foot was cannulated on the lower leg of six healthy human volunteers. After 2 days an irritant contact dermatitis was induced by application of 10% sodium lauryl sulphate to the area of skin drained by the lymph vessel. Three days later the spontaneously regressing skin reaction was treated with clobetasol propionate. Lymph was collected twice daily for 7 days, and the levels of various cytokines (IL-1 alpha, IL-1 beta, IL-2 and soluble IL-2 receptors, IL-6, IL-8, TNF-alpha, GM-CSF) were determined by ELISA technique. In the majority of the volunteers all cytokines examined were detected in several lymph samples, with the exception of IL-1 alpha and IL-8. In parallel with the clinical symptoms of the contact dermatitis the levels of IL-6 and TNF-alpha increased 8-10-fold, whereas for IL-1 beta, IL-2, IL-2 receptors, and GM-CSF there was a delayed, 2-3-fold increase. These results suggest that cytokines, in particular IL-6 and TNF-alpha, may actively participate in the immunological reactions in the skin and in the regional lymph nodes during contact dermatitis.

Co-culture of human keratinocytes on post-mitotic human dermal fibroblast feeder cells: production of large amounts of interleukin 6.

A large synthesis of human IL-6 was demonstrated in co-cultures of human keratinocytes on post-mitotic human dermal fibroblast (HDF) feeder layers. Immunoreactive IL-1 beta could be detected in the co-cultures and the addition of rabbit anti-IL-1 beta antibodies to the co-cultures considerably reduced the IL-6 synthesis, suggesting that it was induced by endogenous IL-1 beta. Addition of saturating concentrations of IL-1 beta to HDF feeder layers as well as to subcultures of keratinocytes induced in both similar but moderate IL-6 production. Conditioned medium from keratinocyte cultures induced IL-6 secretion in HDF feeder cells, whereas the conditioned medium from HDF feeder layers led to only minimal increase of keratinocyte IL-6 production. The co-cultures of keratinocytes on HDF feeder layers produced much larger amounts of IL-6 than the sum of the IL-6 produced by the feeder cell and keratinocyte cultures after the addition of IL-1 beta. The co-cultures of keratinocytes with HDF feeder layers separated by a permeable membrane in a two-chamber system produced significantly lower amounts of IL-6 than the unseparated co-cultures. These findings indicate that a direct cell contact between keratinocytes and feeder cells is involved in the overproportioned increase of IL-6 production and secretion into the medium.

Immunocytochemical evidence of progesterone receptors in human meningiomas.

The presence of progesterone receptors in meningioma tissue is demonstrated by use of highly specific monoclonal antibodies against the rabbit progesterone receptors which cross-react with human progesterone receptors in breast cancer cells, thus giving evidence of the existence of genuine progesterone receptors in human meningiomas.

Endocrine manipulation of meningiomas with medroxyprogesterone acetate. Effect of MPA on growth of primary meningioma cells in monolayer tissue culture.

Intracranial meningiomas from patients treated with medroxyprogesterone acetate as well as from untreated patients were studied in monolayer tissue culture with trials of in vitro hormonal modulation with medroxyprogesterone acetate. The following conclusions were drawn from investigations which comprise 37 cell culture assays: (a) tissue cultures of meningiomas inherit the disadvantages of loss of the progesterone receptor and frequent transformation to cells resembling fibroblasts after three to four passages. For these reasons, drug testing as well as the establishment of cell cultures that exhibit the characteristics of meningioma are impeded; (b) the progesterone receptor-content of the solid tumors does not reflect the response to medroxyprogesterone acetate-therapy in vitro; (c) medroxyprogesterone acetate-pretreated meningiomas showed sufficient in vitro growth in 38%, and untreated meningiomas grew well in 56% of the cases; (d) medroxyprogesterone acetate-induced inhibition or delay of growth was observed in 35%. These findings have resulted in criticism with respect to the value of meningioma tissue cultures for trials of hormonal manipulation and it is thought that another method, which consists of immunostaining of cycling cells, and has been tested in another study, may be superior to cell culture assays with respect to evaluation of the effect of hormonotherapy in meningiomas. Medroxyprogesterone acetate holds an interesting position because it reduces cell growth in some meningiomas in vitro.

Hormonotherapy of meningiomas with medroxyprogesterone acetate. Immunohistochemical demonstration of the effect of medroxyprogesterone acetate on growth fractions of meningioma cells using the monoclonal antibody Ki-67.

The effect of medroxyprogesterone acetate (MPA) on growth fractions of ex vivo meningiomas is demonstrated in using the Ki-67 monoclonal antibody in three cases of meningiomas operated on in two stages and in meningioma specimens from a group of eight patients operated on in one single stage after MPA therapy. Growth fractions in samples from five meningioma patients not treated with MPA were determined for comparison. In the three cases of two-stage operation of the tumors, the percentage of Ki-67-positive cells in meningioma tissue was lower by a factor of 6, 5, and 3, respectively, after MPA therapy. In meningioma specimens from patients receiving no MPA therapy, Ki-67-positive cells were present in 1.02 +/- 0.48%; in samples from MPA-treated tumors the percentage of Ki-67-positive cells was 0.41 +/- 0.40 (different at p less than 0.02 [Wilcoxon's test]). In comparison to our previously published data on untreated meningiomas analyzed for progesterone receptors (PR), MPA significantly reduced the PR activity. There was no obvious correlation between PR activity and potential suppression of the tumor growth fraction. It is concluded that MPA is attractive because it reduces the growth fractions of most meningiomas and might be suitable for adjuvant hormonotherapy.

Short-lasting accumulation in osteoid bone seams of radioactive iron injected as citrate into mice.

The possible role in vivo of osseous structures in binding radioactive iron injected as a low-molecular-weight complex was studied in mice, using combined autoradiography and histomorphometry on sections of undecalcified, plastic-embedded femur epiphyses/metaphyses. A single intraperitoneal injection of 10 microCi 59Fe (1.2 micrograms Fe) per animal as citrate within 3 hours led to a preferential accumulation of this metal in the osteoid mineralized tissue interphase (osteoid seams) of bone. Within the next 2 days the labeling intensity in this localization diminished markedly to approximate levels of the bone marrow and calcified bone. The bulk of the injected radioiron was utilized according to known erythrokinetics. Findings suggest a direct entry of "free," ie, not transferrin-bound, iron into osteoid seams and its consecutive rapid removal from this site.

Endocrine manipulation of meningiomas with medroxyprogesterone acetate. Effect of MPA on receptor status of meningioma cytosols.

Fifteen patients with intracranial or spinal meningiomas have been treated with the semisynthetic progestational agent medroxyprogesterone acetate (MPA, Depo-Provera) prior to surgical removal of the tumors in order to investigate the influence of MPA on the progesterone receptor (PR) status of meningioma cytosols. MPA acted as a competitive binder to meningioma-PR: The mean PR values were 15.6 fmol/mg protein (range 0-69) and 338.3 fmol/g tumor (range 0-1190), respectively. In comparison, mean PR values of our untreated meningioma series (n = 58) were 54.9 fmol/mg protein (range 0-586) and 2813 fmol/g tumor (range 0-17,168), respectively. In cases of two-stage resection of meningiomas MPA significantly decreased PR activity in the cytoplasm of meningioma cells. We conclude that MPA binds to meningioma PRs, however, its effect on the growth rate of meningiomas has still to be elucidated.

Estrogen and progestin receptors in acoustic and spinal neurilemmomas. Clinicopathologic correlations.

Estradiol and progestin receptors were studied in 20 patients with neuraxial Schwann cell tumors, and their presence was correlated to the clinicopathologic features and the amount of preoperative corticosteroid therapy. Based on an arbitrary cutoff value of 200 fmol per gram of tumor as indicative of a positive receptor value in breast cancer, 4 and 13 of the neurilemmoma tissue samples could be considered as positive for estrogen and progesterone receptors, respectively. Whereas there was no convincing correlation between the estrogen and progestin receptor activity and the age, sex, or menopausal status of the patients, overweight patients had significantly higher estrogen and progestin binding values. The correlation between the amount of preoperative prednisone therapy and the amount of [3H]estradiol and [3H]promegestone binding revealed no dose relationship. Correlating [3H]estradiol and [3H]promegestone content with the histologic type of the schwannomas (Antoni types A and B, respectively), we were not able to draw conclusions, because of the predominance of Antoni type A over Antoni type B tissues in our material. The necessity of nuclear receptor assays, ligand specificity testing, and in vitro studies is stressed as a prerequisite for answering the questions whether neurilemmomas contain genuine sexual steroid hormone receptors and whether these receptors are regulated via an estrogen-estrogen-receptor system as is the case in classical sexual steroid hormone target tissues.

Enhancement of formylmethionyl-leucyl-phenylalanine-induced directional locomotion of human neutrophils by serum low-density lipoproteins.

Human low-density lipoproteins (LDL) significantly increased neutrophil locomotion in a gradient of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), while LDL alone had no effect on either directional or random locomotion. The enhancing effect on directional locomotion was constantly observed at FMLP concentrations of 10(-8) M and 5 X 10(-8) M after addition of 50 microgram LDL/ml.

Modulation of concanavalin A-induced lymphocyte stimulation by human low-density lipoproteins.

Spleen lymphocytes and T cells were stimulated by concanavalin A in the presence of various concentrations of human low-density lipoproteins (LDL). The proliferative responses were measured by [14C]thymidine incorporation. Low LDL doses (5-50 microgram/ml) significantly enhanced the stimulation of splenic lymphocytes and T cells. Further, LDL had the capacity to partially relieve the suppression produced by supraoptimal doses of concanavalin A.