Cytokines in the control of beta-2 microglobulin release. II. In vivo studies with recombinant interferons and antigens

The influence of in vivo application of recombinant interferon-alpha 2c (IFN-alpha 2c) and recombinant interferon-gamma (IFN-gamma) on beta-2 microglobulin levels was studied in eight patients with chronic myelogenous leukaemia or advanced renal cell carcinoma. Data indicated enhanced beta-2 microglobulin biosynthesis in close temporary association with injection of both types of interferons. The influence of in vivo stimulation by allogenic leukocytes and the influence of renal allografts or cytomegalovirus infection on serum beta-2 microglobulin and IFN-gamma levels were also studied. Increased beta-2 microglobulin concentrations were observed again in each of these clinical situations and were closely associated with enhanced endogenous interferon production. From these in vivo data and the in vitro data presented in the preceding publication, (1) we conclude that endogenous interferon levels are crucial for the regulation of beta-2 microglobulin release in vivo.     

In situ immunocytochemical staining method for haemopoietic cells grown in vitro

Characterization and enumeration of haemopoietic cells grown in vitro is routinely performed either on unstained cultures or on cultures stained by cytochemical agents. This report describes a novel method for the immunocytochemical analysis of cells grown in plasma clots. The stain is performed in situ by subjecting the whole plasma clot in the culture dish to the staining procedure. The growth of early haemopoietic progenitor cells was monitored in cultures from normal human peripheral blood and proliferating progenitor cells were identified with an anti-human transferrin receptor monoclonal antibody followed by a two-layer immunoperoxidase stain. The number of transferrin receptor positive clusters demonstrable after 5 days of culture was similar to the number of haemopoietic colonies detectable after 12 days of culture.     

In vitro histamine and serotonin release studies in atopic dermatitis.

6 patients suffering from severe atopic dermatitis with high serum IgE were investigated. 3 of the patients had elevated plasma histamine levels (1.5-2.0 ng/ml). Compared to 9 nonatopic normal volunteers, the patient showed increased in vitro histamine release from peripheral leukocytes after stimulation with iothalamate and methacholine: while there was no significant histamine release at a methacholine concentration of 10(-4) M in normals, 4 of the patients with atopic dermatitis showed measurable histamine release under these conditions in vitro. The uptake of radiolabeled serotonin by platelets in vitro was decreased in 2 of the patients. There was no significant difference in serotonin release induced in vitro by different concentrations of thrombin, epinephrine and methacholine; 2 patients showed an increased platelet release reaction after iodipamide stimulation. It is concluded that a general tendency to release vasoactive mediators, even after 'nonimmunologic' stimulation, might play a role in the pathogenesis of atopic dermatitis.     

Serum-free culture of enriched murine haemopoietic stem cells. I: Effect of haemopoietic growth factors on proliferation.

Using a population of cells highly enriched for multipotential day 12 spleen colony forming cells (CFU-S) (termed the FACS-BM population), and a serum-free culture system, the requirements for development of multipotential cell have been investigated and compared to previous results using serum containing cultures. In both serum-free and serum supplemented cultures interleukin-3 (IL-3) was a potent colony stimulating factor, although it was more effective in serum free conditions. However, colony stimulation by granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage-colony stimulating factor (M-CSF) was markedly reduced in the absence of serum. Significantly, the ability of interleukin-1 (IL-1) and granulocyte-colony stimulating factor (G-CSF) to synergise with these two growth factors was retained in serum-free conditions, indicating that these growth factors act directly on the FACS-BM without serum co-factors. Furthermore synergistic interactions between IL-3 plus IL-1, and IL-3 plus M-CSF were only manifest in serum-free conditions. The significance of these results in relation to the ability of these growth factors to act directly on multipotential cells is discussed.     

Nitrogranulogen-dependent inhibition of antibody synthesis. I. In vitro studies in man.

Nitrogranulogen causes a comparable suppression of immunoglobulin synthesis by human lymphocytes in vitro as a standard alkylating agent, 4-hydro-peroxycyclophosphamide. Mixed lymphocyte culture reaction-elicited T and B cell responses are especially sensitive to the agent. The immunosuppressive activity of NTG is enhanced by a thymic hormone (TFX).     

In vitro histamine release induced by radiocontrast media and various chemical analogs in reactor and control subjects

The factors governing in vitro basophil histamine release induced by radiocontrast material (RCM) were evaluated by the use of two RCMs (diatrizoate and metrizamide) and three structural analogs of RCM (benzoic acid, diaminobenzoic acid, and 3-acetamidobenzoic acid) in basophil histamine-release studies. Diatrizoate was chosen because it is a commonly used RCM and is routinely administered as a hypertonic drug (958 mOsm/kg or greater). Metrizamide is a newly synthesized RCM and is routinely administered in isotonic form (approximately 280 mOsm/kg). The analogs were used in hope of identifying any structure-function relationship that might exist between RCMs and the activation of a proposed basophil membrane receptor responsible for the induction of de granulation. In addition, results in a control group of normal subjects were compared with those in a group of patients who had experienced anaphylactoid reactions to intravenous RCM. Basophils from both groups were incubated with diatrizoate and metrizamide to assess their relative sensitivity to these drugs. Both metrizamide and diatrizoate induced in vitro histamine release. Therefore hypertonicity is not an absolute requirement for RCM-induced in vitro basophil de granulation. Although there was a trend far reagents without a prosthetic group at position 5 on the benzene ring (especially 3-acetamidobenzoic acid) to induce more release of histamine than reagents with such a prosthetic group, differences between these reagents did not reach statistical significance. Therefore no clearcut structure-function relationship could be demonstrated. “Reactor” subjects released significantly-larger percents of their intracellular histamine than did “nonreactors” (p < 0.05). Degranulated cells retained their ability to exclude trypan blue, an observation suggesting that RCM-induced histamine release does not involve cell death.     

In vitro studies on the release of mitogenic factor by sensitized human lymphocytes

In order to demonstrate mitogenic activity released by human lymphocytes, supernatants from “primary cultures” (sensitized cells together with antigen) were transferred to “secondary cultures” (autologous or homologous sensitized or non-sensitized lymphocytes). It could be shown that sensitized human lymphocytes excrete a soluble mitogenic factor after exposure to antigen which induces DNA synthesis in secondary cultures. The production of this mitogenic factor was immunologically specific. Factor production could also be demonstrated in an autologous system; therefore the effect of mitogenic factor is independent of histocompatibility antigens. Maximal production of mitogenic activity was obtained after 24 h of primary culture. The amount of activity produced was dependent on the dose of antigen added. An effect of mitogenic factor on secondary cultures was already visible after 48–60 h; the maximal response, however, was seen after 120–136 h of incubation. The kinetics of DNA synthesis obtained were comparable to kinetics induced by specific antigen in short term lymphocyte cultures. DNA synthesis in secondary cultures was dependent on the amount of supernatant added. Since the addition of further antigen to different volumes of supernatants did not lead to an enhanced factor activity, the operation of mitogenic factor was not dependent on the amount of antigen present. Supernatants obtained after 3 days of primary culture, tested on autologous lymphocytes, exhibited a lower response in most instances when compared with the reconstituted controls. This effect may be due to an inhibitor produced by the cells.     

Alpha-interferon induces enhanced expression of HLA-ABC antigens and beta-2-microglobulin in vivo and in vitro in various subsets of human lymphoid cells.

The effect of cloned alpha-interferon (alpha-IFN) on the in vitro and in vivo expression of HLA-ABC antigens and beta-2-microglobulin (beta-2-m) on subpopulations of human lymphoid cells was studied by flow cytometry. Mononuclear cells isolated from patients and cell cultures were labelled with saturating amounts of FITC conjugated monoclonal anti-HLA-ABC or anti-beta-2-m. Phycoerythrin conjugated monoclonal antibodies were simultaneously used for the selection of T lymphocytes. T helper lymphocytes, T suppressor lymphocytes, B lymphocytes and monocytes. In vitro, alpha-IFN induced a significant increase of beta-2-m on all subsets investigated. The increase was more pronounced on B lymphocytes (64%) and monocytes (69%) than on T lymphocytes (39%) (P less than 0.01). Also the pretreatment level of beta-2-m was found to be higher on B lymphocytes (0.64 arbitrary units (a.u.)) and monocytes (0.65 a.u.) than on T lymphocytes (0.24 a.u.) (P less than 0.001). In vivo, the expression of both HLA-ABC antigens and beta-2-m was studied in three patients 24 h after administration of 50 x 10(6) units alpha-IFN/m2 i.m. HLA-ABC antigens were significantly (P less than 0.05) increased on all subsets investigated, except for T suppressor lymphocytes. The increase in beta-2-m only reached significance on T lymphocytes. T helper lymphocytes and monocytes (P less than 0.02). At 48 h after administration of alpha-IFN, expression of HLA-ABC antigens and beta-2-m approached pretreatment levels. Enhanced expression of HLA-ABC antigens and beta-2-m could be reinduced by treatment of alpha-IFN on day 7.     

A sandwich method of enzyme immunoassay. III. Assay for human beta-2 microglobulin compared with radioimmunoassay

A sandwich enzyme immunoassay has been developed to measure human beta-2 microglobulin (beta 2m) in bilogical fluids. beta 2m is first bound by specific antibody covalently coupled to microcrystalline cellulose. The solid phase is washed and reincubated with glucose oxidase-labeled anti-beta 2m antibody. After washing, the enzymic activity of the solid phase is measured by incubation with the appropriate chromogenic substrate. The OD is directly related to the quantity of beta 2m to be measured. A second sandwich assay has also been developed which uses plastic microplates coated with the IgG fraction of rabbit anti-beta 2m serum. Reproducible results are obtained in the range 2-150 and 0.75-48 microgram/l respectively. These tests detect 17 fmoles of beta 2m. Assays of 27 sera showed good agreement between these two enzyme immunoassay methods and two radioimmunoassays.     

In vitro IgE-secreting cells in man I. Mitogen-independent and -dependent subpopulations

In vitro IgE secretion by atopic and normal peripheral-blood lymphocytes was examined in culture with pokeweed mitogen or Staphylococcus aureus strain Cowan-I (StaCw) or without mitogen. IgE secreted in culture supernatants was measured with double antibody radioimmunoassay. Enumeration of IgE-secreting cells was made by a protein-A plaque assay. IgE was detected in increasing quantities in supernatants of cultured lymphocytes without mitogen up to the 12th day. IgE-plaque-forming cells were formed by the lymphocytes in large numbers on days 4–7 in cultures with mitogen. These results suggest that not only mitogen-independent but also mitogen-dependent subpopulations may exist in the IgE-secreting cells.     

In vitro studies on the adjuvanticity of Brucella fractions.

Two Brucella fractions, the murein-linked fraction PI and the murein-free fraction SF, behave as in vitro adjuvants for primary anti-sheep erythrocyte responses: added to Mishell and Dutton-type cultures of spleen cells from B6/DB F1 mice they significantly enhance the number of direct anti-sheep erythrocyte PFC observed on day 5. They exert both nonspecific, polyclonal activating effects and antigen-dependent specific adjuvanticity. These two functions, however, differ in their dose responses and in their cellular requirements and can therefore be dissociated. Thus, polyclonal activation requires high doses of the "adjuvant fraction," is enhanced by adherent-cell depletion, and is not impaired by T-cell depletion. Specific adjuvanticity, on the other hand, requires lower doses of the adjuvant fractions (very high doses are in fact suppressive) and is T-cell and adherent-cell dependent. Moreover, adjuvanticity can be transferred to unstimulated spleen cells (or restored in adherent-cell-depleted populations) by PI- or SF-stimulated adherent cells or by the filtered supernatants of such cultures; adjuvant-soluble factors are therefore involved in the phenomena of adherent, T- and B-cell cooperation required for the adjuvanticity of Brucella fractions.     

Injectable microparticle-gel system for prolonged and localized lidocaine release. I. In vitro characterization

Current treatment protocol for postoperative pain is to infuse anesthetic solution around nerves or into the epidural space. This clinical practice is beset by the short duration of the anesthetic effect unless the infusion is continuous. Continuous infusion, however, requires hospitalization of the patients, thereby increasing medical costs. In addition, it also causes systemic accumulation of the drug. We reported herein a novel treatment for the postoperative pain by applying to the surgical site a biodegradable microsphere-gel system for prolonged and localized release of encapsulated anesthetic drugs. This lidocaine-containing biodegradable poly(D,L-lactic acid) (PLA) microsphere system, although being established previously by other investigators, was hindered by a burst release and a followed rapid release of the drug within several hours in vitro. In this article, we demonstrated that by a step-by-step modification of the formulation, prolonged release of lidocaine, up to several days in vitro, could be achieved. Differential scanning calorimetry revealed a lower glass transition temperature for these lidocaine-loaded microspheres comparing to that of lidocaine-free microspheres. This decreased Tg explained for the tendency of the lidocaine-loaded microspheres to physically fuse at higher temperatures. In vitro studies showed that microspheres, when loaded with 35% lidocaine, yielded a threefold increase in the degradation rate. The molecular weight of PLA of the drug-loaded microspheres was reduced by 50% within a period of 1 month. Based on the results (of prolonged lidocaine release and rapid PLA microsphere degradation), this lidocaine-loaded PLA microsphere system could offer a simple solution to the treatment of postoperative pain.     

The effect of synthetic double-stranded polyribonucleotides on haemopoietic colony-forming cells in vitro

Addition of low concentrations of the synthetic double-stranded polynucleotides Poly I—Poly C and Poly AU to stimulated cultures of normal mouse bone marrow increased the number of macrophage colonies in the cultures. Double strandedness of the polynucleotides was essential for activity and they were active without the α-globulin which was previously shown to be a necessary co-factor in colony potentiation by some antigens. The evidence suggested that polynucleotides acted at the cell surface to make colony forming cells more responsive to colony stimulating factor.     

In vitro biosynthesis of complement factor I by human endothelial cells.

We have studied the secretion of the complement regulatory protein factor I by human umbilical vein endothelial cells (HUVEC). Northern and Western blot analysis and biosynthetic labeling experiments indicate that HUVEC secrete factor I at very low levels in basal conditions and that this secretion is significantly enhanced by interferon-gamma. Analysis of the proteolytic inactivation of C3b by HUVEC supernatants show that factor I is secreted in a functional form and can promote the specific proteolytic inactivation of C3b to iC3b. Together with previous studies establishing the secretion of complement factor H by HUVEC, this work demonstrates that the endothelial cell is able to secrete in its environment two complement regulatory proteins, factor I and factor H, which can mediate the degradation of C3b to iC3b. The secretion of factor I by HUVEC provides a useful in vitro model to analyze the modulation of this secretion and may be relevant to the local deposition of iC3b at the surface of the endothelium during the inflammatory reaction.     

In vitro andin vivo studies of radiographic contrast media-induced histamine release in pigs

Routine clinical use of radiographic contrast media (RCM) causes adverse reactions in some patients. To elucidate the mechanisms of these reactions bothin vitro andin vivo studies are necessary. In this study, RCM-induced histamine release from isolated mast cells was compared with thein vivo release of histamine and cardiovascular symptoms using a porcine model. The 2 non-ionic preparations examined (Solutrast^® and Ultravist^®) released little or no histamine from the 4 cell types tested (porcine pulmonary, cardiac, hepatic, and renal mast cells). The 4 ionic preparations (Angiographin^®, Hexabrix^®, Rayvist^®, and Telebrix^®) caused histamine release from most of the cell suspensions. In almost all cases, the cardiac mast cells were the most sensitive followed by the hepatic mast cells. All 4 RCM testedin vivo produced elevated plasma histamine levels in some animals. The highest incidence was observed using the ionic, high osmolal Rayvist^® (6 of 12 animals), followed by the non-ionic RCM with the lowest osmolality Ultravist^® (4 of 12 animals).In vivo, mechanisms in addition to direct histamine release may also be involved in RCM-induced adverse reactions, since low osmolal, non-ionic RCM can cause elevated plasma histamine levels withoutin vitro release. The susceptibility of cardiac mast cells to RCM-induced histamine release suggests that patients undergoing e.g. coronary angiography may be especially at risk for an adverse reaction.     

In vitro studies on the trafficking of dendritic cells through endothelial cells and extra-cellular matrix

Dendritic cells (DC) are antigen presenting cells (APC) with the unique ability to initiate an immune response. Immature DC are localized in peripheral tissues where they exert a sentinel function for incoming antigens (Ag). After Ag capture and exposure to inflammatory stimuli DC undergo maturation and migrate to regional lymph nodes where the presentation of antigenic peptides to T lymphocytes takes place. Thus their correct functioning as APC involves localization in tissues and trafficking via the lymph or blood to lymphoid organs. In the present study we have investigated the ability of DC to interact in vitro with human vascular endothelial cells (EC) and extracellular matrix (ECM). DC are differentiated from monocytes by in vitro exposure to GM-CSF and IL-13 for 7 days. In adhesion assays a considerable proportion of DC binds to resting EC monolayers and this adhesion is inhibited by anti-CD11a and CD11b, but not anti-CD11c mAbs. Binding to a natural ECM, derived from cultured EC involves VLA-4 and VLA-5 integrins. In a transmigration assay, 10% of input cells are able to cross the EC monolayer in the absence of exogenous stimuli. The amount of DC transmigrated through a monolayer of EC was increased of 2-3 fold by C-C chemokines RANTES, MIP1alpha, and MIP-1beta. Most importantly, in view of the trafficking pattern of these cells, a significant proportion of DC can migrate in a reverse transmigration assay, i.e. across the endothelial basement membrane and subsequently, across endothelial cells. Upon exposure to immune or inflammatory signals peripheral DC undergo maturation and migration to lymphoid organs. Functional maturation is associated with loss of responsiveness to chemokines present at sites of inflammation (e.g. MIP1alpha, MIP1beta and RANTES) and acquisition of a receptor repertoire which renders these cells responsive to signals which guide their localization in lymphoid organs (e.g. MIP3beta). A better understanding of the molecular basis of DC trafficking may provide molecular and conceptual tools to direct and modulate DC localization as a strategy to upregulate and orient specific immunity.