Lysis Of Fibroblasts Research Articles

Stimulation of Fibroblast Proliferation by Insoluble Gadolinium Salts

The purpose of this study was to assess insoluble salts containing gadolinium (Gd(3+)) for effects on human dermal fibroblasts. Responses to insoluble Gd(3+) salts were compared to responses seen with Gd(3+) solubilized with organic chelators, as in the Gd(3+)-based contrast agents (GBCAs) used for magnetic resonance imaging. Insoluble particles of either Gd(3+) phosphate or Gd(3+) carbonate rapidly attached to the fibroblast cell surface and stimulated proliferation. Growth was observed at Gd(3+) concentrations between 12.5 and 125 μM, with toxicity at higher concentrations. Such a narrow window did not characterize GBCA stimulation. Proliferation induced by insoluble Gd(3+) salts was inhibited in the presence of antagonists of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways (similar to chelated Gd(3+)) but was not blocked by an antibody to the platelet-derived growth factor receptor (different from chelated Gd(3+)). Finally, high concentrations of the insoluble Gd(3+) salts failed to prevent fibroblast lysis under low-Ca(2+) conditions, while similar concentrations of chelated Gd(3+) were effective. In conclusion, while insoluble Gd(3+) salts are capable of stimulating fibroblast proliferation, one should be cautious in assuming that GBCA dechelation must occur in vivo to produce the profibrotic changes seen in association with GBCA exposure in the subset of renal failure patients that develop nephrogenic systemic fibrosis.

Potent Alloreactive Effector T Cells Cause Limited Damage to Non-Hematopoietic Tissues Under Non-Inflammatory Conditions, Despite Proper Expression of the Relevant Target Antigens.

Abstract 2454Poster Board II-431Donor T cells specific for minor histocompatibility antigens (mHags) contribute to graft-versus-leukemia (GvL) reactivity and graft versus host disease (GvHD) following HLA-matched stem cell transplantation. Hematopoiesis-restricted mHags may give rise to a more specific GvL effect without GvHD, whereas ubiquitously expressed mHags can be targets for both GvHD and GvL. Interestingly, high frequencies of circulating T cells specific for ubiquitously expressed mHags (e.g. male-specific epitopes, LB-ADIR-1F) have been detected in patients showing strong GvL with no or limited GvHD. Although both hematopoietic and non-hematopoietic cells express the proteins encoding these mHags, the immune effector response in these patients appears to be skewed towards hematopoietic cells. Therefore, we hypothesized that non-hematopoietic tissues are relatively unsusceptible to the cytotoxic effect of these mHag-specific T cells. To test this hypothesis, we developed an in vitro system in which HLA-A2+, ADIR+ primary human fibroblasts were exposed to mHag LB-ADIR-1F or allo-A2 specific CD8+ T cells. ADIR+ EBV-LCL were used as a control for hematopoietic cells. CTL-mediated cytotoxicity was measured using 4, 8 and 20 hours chromium release assays. Whereas all EBV-LCL were killed within 4 hours, 10% lysis of fibroblasts was found after 4 hours, only gradually increasing to 70% lysis after 20 hours in the continuous presence of the CTLs at 10/1 effector/target (E/T) ratios. To analyze whether this inefficient killing was due to low antigen expression by the human primary fibroblasts, we examined cytotoxicity against targets loaded with increasing concentrations of ADIR-peptide. Exogenous loading of fibroblasts with saturating concentrations (10-6M) of ADIR-peptide did not increase the sensitivity to T cell mediated cell death. To exclude that the observed inferior cell killing was due to the number of T cells available for engaging the fibroblasts, E/T ratios were increased to >100/1, but this did not change the poor susceptibility of the fibroblasts to CTL-mediated killing. Since apparently inefficient elimination of non-hematopoietic targets was found despite proper expression of the relevant antigen and the presence of functional T cells, we investigated whether insufficient engagement of T cells during contact with the fibroblasts was the cause of the impaired killing. To analyze this, kinetics of T cell degranulation upon target encounter was measured by CD107a, perforin and granzyme B expression. Although the onset of T cell activation was already seen after 2 hours, stimulation with fibroblasts resulted in degranulation in only a proportion of the T cells (50-60%) after 4hrs, resulting in full activation only after prolonged co-culture. In contrast, stimulation with EBV-LCL resulted in rapid, complete, and profound degranulation (100% of T cells within 2hrs). In concordance with this, T cell activation, as measured by the production of interferon gamma (IFNg) after stimulation with the fibroblasts, was suboptimal as compared to stimulation with EBV-LCL. These data suggest formation of an initial low avidity interaction with fibroblasts under steady state conditions, possibly only leading to efficient T cell engagement and fibroblast killing after a prolonged period of co-culture, resulting in the formation of a local pro-inflammatory environment. To confirm whether susceptibility to mHag-specific T cells was increased under pro-inflammatory conditions, we pre-treated fibroblasts with IFNg. Analysis of the expression of a panel of known adhesion and co-stimulatory molecules revealed that IFNg treatment resulted in a 2-5 fold upregulation of expression of HLA class I, HLA class II and ICAM-1, leading to a more uniform and profound T cell activation. This increased T cell activation resulted in more efficient direct fibroblast killing. In conclusion, despite proper expression of the relevant target antigens, low-avidity interaction between effector T cells and non-hematopoietic tissues results in minimal damage under non-inflammatory conditions. Disclosures:No relevant conflicts of interest to declare.

Roles of the endosymbiont and leishmanolysin-like molecules expressed by Crithidia deanei in the interaction with mammalian fibroblasts

Crithidia deanei is an insect trypanosomatid that harbors a bacterial endosymbiont in its cytoplasm. In this work, we have demonstrated the influence of the endosymbiont on the interaction of C. deanei with mammalian fibroblasts, also implicating the surface leishmanolysin-like molecules of C. deanei in this process. The wild strain of C. deanei expressed a higher amount (2-fold) of leishmanolysin-like molecules in the parasite surface than the aposymbiotic strain. The treatment of parasites with anti-leishmanolysin antibodies or the fibroblasts with purified leishmanolysin-like molecules from C. deanei significantly reduced the association index. The aposymbiotic strain of C. deanei presented interaction rates about 2- and 3-fold lower with fibroblasts than the endosymbiont-bearing counterpart after 1 and 2 h, respectively. However, the association indexes were similar after 3 and 4 h of interaction. Additionally, we observed a 2-fold increase in the association index after 24–96 h of parasite–fibroblast interaction when compared to the interaction process performed for 4 h, irrespective to the presence of the endosymbiont, suggesting that fibroblasts support multiplication and survival of C. deanei. Both parasite strains were able to induce fibroblast lysis. Interestingly, the wild strain led to a 2-fold increase in fibroblasts death in comparison to the aposymbiotic strain after 48–96 h. We also showed that both wild and aposymbiotic biotinylated live parasites recognized the same receptor in the fibroblast cells.

A Comparative Study of Magnetic-Activated Cell Sorting, Cytotoxicity and Preplating for the Purification of Human Myoblasts

Although cultured myoblast transplantation has been extensively studied as a gene complementation approach to muscular dystrophy treatment, clinical success has still been limited. The inability to adequately isolate and purify myoblasts presents a major limitation to the production of sufficient myoblasts for engrafting purposes. This study attempted to purify myoblasts from primary culture by magnetic-activated cell sorting (MACS), complement-mediated cytotoxicity, and a preplating technique. As a result of positive myoblasts selection by MACS, the average percentage of myoblasts in mixed culture was increased from 30.0% to 41.7%. We observed both myoblast lysis and fibroblast lysis after complement-mediated cytotoxicity. Enrichment of myoblasts in mixed culture was found to increase to 83.1% by using the preplating technique. In addition, higher purification (92.8%) was achieved by following the preplating technique with MACS. Thus, preplating in combination with magnetic-activated cell sorting allows for a rapid and effective isolation of myoblasts from human muscle tissue.

Complement expression on astrocytes and astrocytoma cell lines: failure of complement regulation at the C3 level correlates with very low CD55 expression

Primary fetal human astrocytes and an astrocytoma cell line, U373-MG, expressed membrane cofactor protein (CD46), CD59, and low levels of decay-accelerating factor (CD55). Astrocyte CD55 was capable of regulating C3 deposition on the cell surface; albeit at a lower level than primary human fibroblasts. Negligible complement-mediated lysis of primary astrocytes and the U373-MG cell line was observed, even when large amount of astrocyte-specific, complement-activating antibodies were bound to the cells. Blocking the function of CD59 on astrocytes resulted in a > 90% cell lysis, while equivalent lysis of fibroblasts could only be achieved with additional blocking of CD55.

Reduced alveolar macrophage production of tumor necrosis factor during sepsis in mice and men

Tumor necrosis factor (TNF) has been implicated as a major humoral mediator of sepsis and endotoxin shock. TNF is secreted by cells of the reticuloendothelial system, including alveolar macrophages. Alveolar macrophage TNF production has been postulated to play a pathogenetic role in the development of adult respiratory distress syndrome (ARDS) in sepsis. To evaluate alveolar macrophage production of TNF during sepsis and endotoxin shock, we studied the effects of sepsis and/or in vivo lipopolysaccharide on the in vitro production of TNF by pulmonary alveolar macrophages. Human pulmonary alveolar macrophages were obtained by bronchoalveolar lavage from six septic and five nonseptic patients, cultured in the presence or absence of lipopolysaccharide (1 ng/mL), and assayed for TNF activity in a bioassay using fibroblast lysis. A murine model of sepsis was also utilized to study pulmonary alveolar macrophage TNF production under more controlled conditions. Normal mice were given ip injections of either lipopolysaccharide or saline. After 2 hrs, pulmonary alveolar macrophages were obtained and cultured in saline or various concentrations of lipopolysaccharide (0.001 to 10 micrograms/mL). There was no difference in baseline TNF activity, expressed as per cent lysis at 1:10 dilution, between pulmonary alveolar macrophages from control and septic patients (35.7 +/- 5.5% vs. 24.4 +/- 9.3%, respectively) (p greater than .05). However, when stimulated with lipopolysaccharide in vitro, the pulmonary alveolar macrophages from nonseptic patients produced significantly (p less than .01) more TNF (82.8 +/- 3.6%) than did pulmonary alveolar macrophages from patients with the septic syndrome (35.2 +/- 3.8%). Similar findings were obtained using the murine sepsis model. The baseline TNF activity in pulmonary alveolar macrophages from control mice was 22.9 +/- 7.0% (mean +/- SEM) and from lipopolysaccharide-injected mice was 26.8 +/- 3.3% (p greater than .05). Stimulation with 1 ng/mL lipopolysaccharide in vitro produced an increase in TNF activity in both groups, but the increase was greater in the control mice (68.1 +/- 5.7%) than in the lipopolysaccharide-injected mice (47.5 +/- 5.3%) (p less than .01). When the murine pulmonary alveolar macrophages were stimulated with higher concentrations of lipopolysaccharide (0.1 to 10 micrograms/mL), pulmonary alveolar macrophages from lipopolysaccharide-injected mice produced less than 25.5% of the TNF produced by pulmonary alveolar macrophages from control mice. These studies indicate that sepsis and endotoxin injection result in a rapid decrease in the ability of pulmonary alveolar macrophages from both humans and mice to produce and secrete TNF in response to lipopolysaccharide. We speculate that a downregulation of TNF production or of macrophage responsiveness to lipopolysaccharide has occurred. These results suggest that sustained TNF production by macrophages is not required for lung injury in sepsis.

Kupffer cell tumor necrosis factor-α production is suppressed during liver regeneration

Mammalian liver regeneration following resection invokes intrinsic hepatic responses which result in rapid tissue repair. The role of soluble immune cytokines in this phenomenon is not known. The capacity of Kupffer cells (KC) from regenerating liver to produce the potent cytokine TNF-α was evaluated. Twenty-four hours after 70% partial hepatectomy (PHx) or sham operation, Kupffer cells were harvested from collagenase-digested Wistar-Furth rat livers and purified (>95% by phagocytosis) by adherence. Following overnight culture with or without the cyclooxygenase inhibitor indomethacin (10 μ M), 5 × 10 5 KC were repleted with fresh media with or without 2.5 μg/ml lipopolysaccharide (LPS). Supernatant TNF-α activities (units/ml) were measured using the L929 fibroblast lysis assay. With LPS, sham KC TNF-α levels were significantly higher ( P < 0.001) than those for PHx KC. Indomethacin significantly increased PHx KC TNF-α levels, but did not affect those for sham KC, suggesting autoregulation by arachidonic acid cyclooxygenase metabolites following PHx. We conclude that KC TNF-α production is suppressed following PHx by a mechanism apparently regulated by eicosanoid metabolism. During the stress of hepatic regeneration, a coordinated limitation of excessive TNF-α responses by PHx liver KC may naturally protect the host.

Effects of irrigation fluid on human menisci: An experimental comparison of water, normal saline, and glycine

A technique has been developed to demonstrate the macroscopic morphology of collagen fiber arrangement in human menisci. It was observed that 0.9% normal saline solution removed the ground substance of the meniscus, leaving intact collagen bundles. This suggests that meniscal damage may occur as a direct result of using normal saline for irrigation during knee arthroscopy. Experiments were conducted using demineralized water and 1.5% glycine for comparison. The disruption of collagen fiber binding in the meniscus by normal saline is time and temperature dependent. Glycine solution causes fibroblast lysis and a lesser degree of collagen alteration. Demineralized water can cause edema in the fibroblast but no notable change in collagen fibers. None of the irrigation solutions tested has proved to be completely safe. Demineralized water is not the ideal solution but is preferable to normal saline or glycine solution, especially when it can be used in conjunction with electrosurgery.

Lentivirus-induced interferon inhibits maturation and proliferation of monocytes and restricts the replication of caprine arthritis-encephalitis virus

In this study, we investigated the effect of a lentivirus-induced interferon (LV-IFN) on the interaction of caprine arthritis-encephalitis virus and its host cell, the monocyte-macrophage. LV-IFN was produced in culture supernatant 48 h after adding fresh goat lymphocytes to caprine arthritis-encephalitis virus-infected goat macrophages. The culture supernatant contained IFN activity at a titer of 1:360 as assayed by inhibition of vesicular stomatitis virus-induced lysis of fibroblasts. LV-IFN inhibited in vitro monocyte proliferation and maturation of monocytes to macrophages. Nevertheless, treated monocytes produced prostaglandin E2, a cytokine generally produced by activated macrophages. By inhibiting the maturation of monocytes to the more permissive macrophage, LV-IFN indirectly downregulated virus replication. The cytokine also had a direct inhibitory effect on virus gene expression in already mature macrophages. In these cells, LV-IFN blocked the viral life cycle at the level of transcription. Finally, LV-IFN blocked fusion between infected macrophages and highly permissive goat synovial membrane cells. By restricting macrophage maturation, viral replication, and cell fusion, LV-IFN may downregulate the net rate of virus replication in vivo. These functions may contribute to the persistence of the virus in the host by reducing the expression of the viral genome.

Altered oxidative metabolic responses in vitro of alveolar macrophages from asymptomatic cigarette smokers.

Superoxide anion (O2) release by alveolar macrophages (AM) from young asymptomatic cigarette smokers was greater than that by AM from age-matched nonsmokers. Greater O2 release by AM from cigarette smokers was observed before and after stimulation by bacteria or phorbol myristate acetate (PMA). In contrast, oxygen uptake and glucose (1(-14)C) oxidation by unstimulated or stimulated AM from cigarette smokers was the same as that by AM from non-smokers. The selective increase of O2 release by AM from cigarette smokers was not due to a lack of O2 scavenging agent within the cells, since intracellular superoxide dismutase (SOD) was increased in AM from smokers. The potential importance of enhanced O2 release by AM from cigarette smokers was confirmed by demonstrating that lysis of fibroblasts induced by AM from smokers was completely prevented by addition of SOD and catalase.

Microcytotoxicity assay for cell-mediated immunity enumerating residual target number by 86Rb

Lymphocyte-mediated lysis of target cells grown as monolayers in microtiter wells is readily quantitated by an assay measuring the 86Rb incorporated after attaining isotopic equilibrium. Lysis of fibroblasts by allogeneic lymphocytes sensitized by skin grafts and of tumor cells by syngeneic spleen cells sensitized by intraperitoneal tumor inoculation were readily detected. Weakly cytolytic lymphocytpopulations can be assayed by increasing incubation times to 48 h or longer. A potential problem, 86Rb incorporation by lymphocytes sticking to residual target cells, was controlled by comparing 86Rb incorporation by targets incubated with non-immune lymphocytes. Results by 86Rb incorporation were identical to those determined by microscopic counting or 51Cr release. 86Rb incorporation assays should be considered as an alternate to 51Cr release techniques, especially in those experimental systems where the cytolytic potential of a lymphocyte population is so low that lysis can be detected only after long incubation times and/or when the spontaneous release of 51Cr is prohibitively high.

Generation of Cytotoxic T Lymphocytes during Coxsackievirus B-3 Infection

Abstract The production of cytotoxic cells in the spleens of adult male BALB/c mice infected with Coxsackievirus B-3 has been examined. An in vitro 51Cr release assay was used to measure cytotoxic activity against virus-infected and uninfected neonatal syngeneic fibroblasts. Cytotoxicity of immune spleen cells against virus-infected targets was detected on the 3rd day after infection, reached a peak on day 7, and then declined to low levels by days 12 and 14. Spleen cells obtained 3 and 5 days after infection also exerted cytotoxicity against uninfected fibroblasts, but by the 7th day there was little or no reactivity against uninfected target cells, although activity against infected fibroblasts was maximal at this time. Reciprocal assays performed by using Coxsackie and vaccinia viruses provided evidence of virus specificity of the cytotoxic reaction. When spleen cells were obtained 7 days after infection, the Coxsackievirus-immune population was not cytotoxic for vaccinia-infected fibroblasts, and the vaccinia-immune population was not cytotoxic for Coxsackievirus-infected targets, although each immune cell preparation caused significant lysis of fibroblasts infected with the homologous virus. Additional studies showed that primary mouse or hyperimmune rabbit anti-Coxsackieviral serum could not block immune spleen cell cytotoxicity or induce complement-mediated lysis of in fected targets. The findings indicate that Coxsackievirus infection results in surface membrane alterations, but no evidence was obtained that antiviral antibody could react with the infected cells.

Restriction by H-2 gene complex of transfer of cell-mediated immunity to Listeria monocytogenes.

RESULTS from three distinct experimental systems indicate that, except for lymphocytes reacting to alloantigens, interaction between sensitised thymus-derived lymphocytes (T cells) and other somatic cells occurs only in syngeneic or semiallogeneic systems. First, histoincompatible T cells do not function as helpers for antibody-forming cell precursors (B cells)1,2. Second, maximal proliferation of lymphocytes (presumably T cells) resulting from exposure in vitro of sensitised guinea pig lymphocytes to antigen-pulse macrophages is observed only when the two cell types share major histocompatibility antigens3. Third, cell-mediated lysis of fibroblasts or macrophages infected with lymphocytic choriomeningitis (LCM) virus, a function of specifically sensitised T cells, occurs only when lymphocyte and target cells share at least one set of H-2 antigenic specificities4,5. The phenomenon is unaffected by differences either in minor histocompatibility antigens, or at the M locus.

The development of hypersensitive lymphocytes in cell culture.

An in vitro cell-mediated immune response to pokeweed mitogen (PWM) is described. Rat lymphocytes were stimulated by PWM, by phytohemagglutinin (PHA), and by concanavalin A (ConA). In the presence of PWM only a fraction of the lymphocytes underwent blastogenesis. This was in contrast to the apparent total blastogenesis obtained in response to PHA or ConA. When blast cells derived from each of the mitogens were plated on rat fibroblast monolayer in the absence of mitogen they differentiated into a distinct type of lymphocyte termed "secondary lymphocyte." Addition of mitogens to cultures of these lymphocytes resulted in a retransformation to blast cells. The secondary lymphocytes were tested for their ability to effect lysis in the presence of each of the three mitogens. In. the presence of PWM, lysis of fibroblasts produced by PWM-lymphocytes was considerably more efficient than lysis obtained by ConA- or PHA-lymphocytes. No difference in effect on target fibroblasts was obtained when the three types of secondary lymphocytes were tested in the presence of either PHA or ConA. The stimulating action of PWM on lymphocytes was shown to be immunologically specific. No such specificity was found in the case of PHA or ConA. The results are interpreted to indicate that PWM combines with cell membranes and acts on the lymphocytes as a "transplantation antigen." Lymphocytes capable of responding to "PWM-transplantation antigen" transform to blast cells capable of specifically lysing PWM-conjugated fibroblasts. In the absence of the mitogen, PWM-induced blast cells differentiate to lymphocytes hypersensitive to PWM.

The lysis of fibroblasts by lymphocytes sensitized in vitro: Specific antigen activates a nonspecific effect

The antigenic specificity of a lymphocyte antifibroblast cellular immune reaction was studied in vitro. Rat lymphocytes were sensitized in vitro against foreign rat or mouse fibroblasts. Sensitized lymphocytes were then transferred to target monolayers of fibroblasts antigenically identical with, or unrelated to, the sensitizing fibroblasts. Injury to target cells was measured by the release of a radioactive label ( 51Cr). It was found that injury to antigenically unrelated target fibroblasts consistently occurred, although injury to antigen-specific fibroblasts was usually three- to sixfold greater. To test the ability of specific antigen to activate the lymphocyte cytolytic mechanism, target monolayers were prepared which contained mixtures of unlabeled antigen-specific and 51Cr-labeled antigenically unrelated fibroblasts. It was found that the interaction of sensitized lymphocytes with antigen-specific fibroblasts augmented the cytolytic effect against unrelated fibroblasts. The role of specific antigens in activating cytolysis was further studied by incubating target fibroblasts with specific antiserum. Antibodies were found to inhibit lymphocyte-mediated cytolysis in two distinct ways: they prevented antigen-dependent activation of lymphocytes by binding to specific fibroblast antigens, and they acted as a physical barrier to contact between activated lymphocytes and target cells. These results suggest that the destruction of target fibroblasts by sensitized lymphocytes proceeds in two stages. The first stage appears to be immunospecific and comprises the activation of the cytolytic effector by an interaction between lymphocytes and specific antigens. The second stage—the action of the cytolytic effector mechanism itself—does not seem to be immunospecific. Activated lymphocytes appear to be capable of injuring any fibroblasts, including syngeneic, with which close contact is made.