Expression of Activation Antigens on T Cells in Rheumatoid Arthritis Patients

The aim of our study was to identify differences in cell surface marker expression between T cells taken from the peripheral blood (PB) of healthy individuals and T cells recovered from inflamed joints of rheumatoid arthritis (RA) patients. Out of 118 monoclonal antibodies (MoAbs) directed against activation antigens on haematopoietic cells, 12 MoAbs recognizing nine distinct surface molecules were selected after a screening procedure to study the expression of the corresponding antigens on T cells from the PB, synovial fluid and synovial tissue of RA patients, and also on T cells from PB and spleens of controls. Using two-colour flow cytometry and immunohistology we found the molecules B-C5, CD39, CD40, CD45 R0, CD54, CD76 and potentially 1D11 to be substantially up-regulated on T cells from various body compartments in RA patients. We thus could determine that the cell surface of T cells in RA patients not only differs in MHC class II expression, but also in a number of other activation-associated cell surface molecules from T cells in healthy individuals.     

Induction of Autoimmune Prostatitis Using Liposomes Is Associated to Peritoneal Cells Activation

PROBLEM: Study and characterization of rat peritoneal cells (PC) involved in the induction of autoimmune prostatitis after the intraperitoneal administration of native extract of accessory glands (RAG) associated with liposomes (RAGL). METHOD OF STUDY: Induction of the autoimmune response in normal recipients by transferring PC or adherent-PC loaded with RAGL (RAGL-PC), but not with PC loaded with empty liposomes (L-PC). Characterization of the morphology, the ultrastructure, and the phenotype of L-PC or RAGL-PC. Study of the respiratory burst by the nitroblue tetrazolium (NBT) reduction assay after stimulation with phorbol myristate acetate (PMA) in both L-PC and RAGL-PC. RESULTS: Liposomes attached to the cell surface of the Mφ were observed by electron microscopy. FACS analyses showed a similar staining pattern with high expression of Ia molecules on L-PC and RAGL-PC compared with controls. PMA-stimulated L-PC or RAGL-PC markedly reduced the NBT compared with controls. CONCLUSION: Our results suggest that the effective uptake of liposomes and the initial activation of PC together with a prolonged stimulatory effect help to disrupt the tolerance state. The present experimental model is an interesting approach to further characterize events associated with antigenic presentation when an autoimmune response is triggered.     

Mechanism of B-Cell Activation and Paralysis by Thymus-Independent Antigens

Normal spleen cells showed a bell-shaped dose response profile when stimulated in vitro with die thymus independent antigen (4 -hydroxy-3,5 dinitiophenyl)acetyl (NNP)-lipopolysaccharide (LPS) with regard to the development of high-avidity plaque-forming cells to NNP The addition of suboptimal concentrations of LPS to cultures stimulated by suboptimal concentrations of NNP LPS resulted in optimal induction of B cells in that affinity fraction Addition of LPS to cultures optimally stimulated by NNP-LPS resulted in paralysis of the specific cells These results are interpreted in terms of the additive effects between the mitogenicity LPS and the mitogenicity of NNP LPS the latter being selectively focused on the specific cells, thus providing further evidence for the 'one nonspecific signal' hypothesis for immune activation of B cells     

Studies of Ia Antigens on Murine Peritoneal Macrophages

Murine peritoneal cells, both induced and noninduced, were examined for Ia antigens by a variety of techniques. Complement-mediated cytotoxicity and indirect immunofluorescence, analyzed by both visual microscopy and the fluorescence-activated cell sorter, detected Ia antigens on the surface of an average of 8% to 15% of cells with the morphologic and functional characteristics of macrophages. Internal radioisotope labeling studies showed that these antigens were actually synthesized by the macrophages. The antigens were borne on molecules which consisted of two coponents with apparent molecular weights of roughly 33,000 and 25,000 daltons. At least some of these molecules existed as a two-chain structure of 58,000 daltons linked by disulfide bonds. Although macrophage Ia antigens appeared to be structurally similar to the Ia antigens found on spleen cells, the radioisotope labeling studies indicated that the quantity of labeled Ia-bearing molecules isolated from peritoneal macrophages was at most 1/15 that found for B lymphocytes. In addition, anti-Ia antisera failed to inhibit the binding of heat-aggregated immunoglobulin to the Fc receptor of macrophage populations, similar to the low levels of Ia antigens found in T-lymphocyte populations. These studies suggest that Ia antigens exist only a subpopulation of peritoneal macrophages. Alternatively, all cells in the population might bear small amounts of Ia antigens with only a fraction having sufficient numbers of molecules to be detected by the assay systems used.     

Interaction of Pneumococcal Antigens with Complement in Rats

Complement activation with pneumococcal antigens was studied both in vitro and after injection of the antigens into rats. Whole pneumococci of various serotypes activated C3-C9 in rat serum treated with ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, although serotypes differed greatly in the extent of activation. Some purified pneumococcal capsular polysaccharides also activated C3-C9 in rat serum, but only when the antigens were present in concentrations of 500 to 1,000 mug/ml. Much of the activation with capsular polysaccharides was eliminated by the use of ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid. Activation of C3-C9 by capsular polysaccharides did not correlate with the level of reactivity observed with whole organisms of the same serotypes. After injection of 5 x 10(9) pneumococci (type 3 or type 4) intravenously into rats, there was a transient decline in serum C3-C9 activity, but there was no decline in C3-C9 levels after intravenous injection of 1,000 mug of type 3 or type 4 capsular polysaccharides. As determined by immunofluorescence, circulating capsular polysaccharide was deposited in several tissues, including the vascular endothelium and glomerular mesangium of the kidney. C3 was not detectable in these deposits, and there was no histological evidence of an inflammatory response. Capsular polysaccharides appear to be only weak activators of complement. Other pneumococcal antigens may be more important in the pathogenesis of hypocomplementemia in pneumococcal infection.     

Mechanism of T-Cell Activation by Mycobacterial Antigens in Inflammatory Synovitis

Earlier studies demonstrated enhanced proliferative responses to an acetone precipitable Mycobacterium tuberculosis (AP-MT) antigenic complex by T lymphocytes from the synovial fluid, compared with the peripheral blood, of patients with inflammatory synovitis, including rheumatoid arthritis. In contrast, decreased proliferation and interleukin 2 (IL-2) production in response to mitogens by synovial fluid lymphocytes from patients with rheumatoid arthritis has been demonstrated. In order to determine if IL-2 was produced in response to AP-MT, the peripheral blood and synovial fluid of patients with inflammatory arthritis were analysed by measuring proliferation and IL-2 production in response to AP-MT and tetanus toxoid. A reduction of IL-2 production relative to proliferation was observed in some, but not all, synovial fluids of patients who responded to the AP-MT. Nevertheless, antibodies to IL-2 as well as interleukin 4 (IL-4), significantly inhibited proliferation of synovial fluid lymphocytes by AP-MT. There was no inhibition by antibodies to interleukin 6 (IL-6). We conclude that AP-MT induced proliferation by synovial fluid lymphocytes is mediated by both IL-2 and IL-4.     

Treatment of Intervertebral Disc Degeneration in Wistar Rats with Mesenchymal Stem Cells

Bulletin of Experimental Biology and Medicine - We studied the effect of erythropoietin on functional properties of mesenchymal stem cells under conditions of oxidative stress and their therapeutic...     

Activation of Complement by Cells Infected with Respiratory Syncytial Virus

The ability of respiratory syncytial virus (RSV)-infected HE_p-2 cells in culture to activate complement was investigated. After incubation of cells with various complement sources and buffer, binding of C3b to surfaces of infected cells was demonstrated by immunofluorescence with a double-staining technique. Nonsyncytial and syncytial (i.e., fused, multinucleated) cells were separately enumerated. Also, lysis of RSV-infected cells was assessed by lactic dehydrogenase release. In this system only RSV-infected cells stained for C3b, and they did so only after incubation with functionally active complement. Blocking of classical pathway activation with ethylenediaminetetraacetic acid diminished the number of infected nonsyncytial cells positively stained for C3b, but had no effect on staining of syncytial cells. Blocking of alternative pathway activation with either zymosan incubation or heat treatment decreased the number of both syncytial and nonsyncytial cells stained for C3b. Decreasing immunoglobulin concentration of the serum used as the complement source also decreased numbers of both cell types stained for C3b. Eliminating specific anti-RSV antibody diminished numbers of both cell types stained for C3b, but staining was not eliminated. Lastly, incubation with functionally active complement markedly increased lactic dehydrogenase release from infected cells. This study demonstrated that RSV-infected nonsyncytial and syncytial cells are able to activate complement by both classical and alternative pathways. Activation of complement by syncytial cells appears to be less dependent on the classical pathway than is activation by nonsyncytial cells, and activation by syncytial cells may require immunoglobulin but not specific antibody. These experiments suggest the possibility of complement activation during respiratory tract infection by RSV. Implications of this are discussed.     

Differential Inhibition by Cyclosporin A Reveals two Pathways for Activation of Lymphokine Synthesis in T Cells

Abstract

Two pathways for the activation of lymphokine synthesis in murine T cell clones and polyclonal T cell blast populations were identified. One was induced by ligands of the T cell receptor (TCR) and led to high production of GM-CSF, IFN-γ, and IL-3. The other was induced by IL-2 and led to production of lower levels of GM-CSF and IFN-γ with relatively little IL-3 synthesis. Cyclosporin A (CsA) markedly inhibited TCR-independent production of lymphokine mRNA and protein at concentrations where IL-2-dependent stimulation of lymphokine production and proliferation was unaffected. Stimulation of lymphokine synthesis by phorbol myristate acetate (PMA) and the Ca²⁺ ionophore ionomycin, or by ionomycin alone, mimicked the TCR-dependent response. PMA on its own was a preferential stimulus for GM-CSF production, but, whereas CsA did not inhibit PMA stimulation of polyclonal T cell blasts, T cell clones displayed a biphasic response in which CsA only inhibited stimulation by high PMA concentrations. The data suggest that Ca²⁺-independent (CsA-resistant) T cell activation induces synthesis of GM-CSF and IFN-γ but is a poor stimulus for IL-3 production. On the other hand, when Ca²⁺-dependent (CsA-sensitive) pathways are activated by TCR binding or by a Ca²⁺ ionophore, production of high levels of all three lymphokines can be induced.     

Trypanosoma cruzi Cytosolic Alkaline Antigens (FI) Induce Polyclonal Activation in Murine Normal B Cells

Several reports have described polyclonal activation in mice acutely infected with Trypanosoma cruzi. The aim of this work was to analyse the participation of one T. cruzi antigenic fraction in this immunological event. The antigen selected was FI, an antigenic fraction of pI 7–9 obtained from T. cruzi cytosol separated by isoelectricfocusing. FI is constituted by molecules with molecular weights of around 60 and 20 KDa. The authors assayed the ability of this antigenic fraction to induce polyclonal activation of spleen mononuclear cells from normal (NSMC) BALB/c mice. NSMC showed a marked lymphoproliferative response measured by ³H-thymidine incorporation after 3 days of culture in presence of FI. The values reached by FI-stimulated cells were 10 times higher than the controls (non-stimulated cells). This effect was dose-dependent. Furthermore, the authors observed that a purified T-cell population in the presence of adherent cells was unaffected by FI. Additionally, in a culture of NSMC, FI stimulated the proliferation of B cells as observed by the increase of the percentage of B220⁺ cells determined by FACS using FITC-conjugated anti-mouse B220. The authors noticed that the percentage of B220⁺Ly1⁺(CD5) populations in the presence of FI did not change with respect to the control (non-stimulated cells), indicating that FI expanded both conventional and CD5⁺ B cells. The isotypic pattern of the antibodies produced after 6 days of culture of NSMC in the presence of FI was predominantly IgM, which reacted with highly conserved antigens such as actin, myosin, myoglobin, thyroglobulin and carbonic anhydrase, but did not react with FI. A slight increase of IgG1 and IgG3 with respect to the control was observed but no changes on the levels of IgG2 was noticed. These results indicate that FI promotes activation, proliferation and differentiation in antibody-secreting cells of normal murine B lymphocytes.     

Characterization of ERK Activation in Human Mast Cells Stimulated by Contact with T Cells

Close physical proximity between mast cells and T cells has been demonstrated in several human conditions. We have identified and characterized a novel mast cell activation pathway initiated by contact with T cells, and showed that this pathway is associated with cytokine release. It has been shown recently that Ras is activated in this pathway. Thus, in the present study we further explore the downstream events associated with Ras activation and cytokine release in human mast cells stimulated by contact with T cells. ERK activation in human mast cells stimulated by either contact with T cells or by crosslinking the FC epsilon receptor was studied. Photobleaching experiments were used to study ERK localization. Enzyme linked immunosorbent assay was used to study the cytokine release by human mast cells. We show that stimulation of human mast cells by contact with activated T cells results is sustained ERK activation. Furthermore, sustained ERK activation in these cells is associated with increased dwell time at the nucleus and with IL-8 release. Interestingly, when mast cells were stimulated by crosslinking the FC epsilon receptor I, ERK activation was transient. ERK activation was associated with a shorter dwell time at the nucleus and with TNF-α release. Thus, retaining ERK in the nucleus might be a mechanism utilized by human mast cells to generate different cytokines from a single signaling cascade.     

Differential Patterns of T-Cell Receptor BV-Specific Activation of T Cells by gp120 from Different HIV Strains

Studies by several groups have suggested that HIV infection in vivo results in a BV-specific alteration of the TCR repertoire and that this might play a role in the pathogenesis of AIDS. Our earlier studies demonstrated tbat both a crude extract of HIV₄₅₁ as well as purified gp160 from HIV₄₅₁ could specifically activate, in vitro , T cells expressing a common set of TCRBV segments (TCRBV3, 12, 14, 15, and sometimes BV17 and 20) in individuals of disparate HLA type. Furthermore, purified gp120 from HIV₄₅₁ was shown to have a similar ability to activate T cells, although with a slightly diiferent TCRBV-specific pattern. In order to determine whether gp120 from other HIV strains could similarly activate T cells in a TCRBV-specific pattern, PBMC from HIV seronegative individuals of disparate HLA type were stimulated with gp120 from three strains of HIV (451, IIIB, and MN). The authors found that gp120 from all three strains activate T cells bearing TCRBV2 and BV3 in nearly every individual. T cells expressing other BV segments are also activated, but this is more variable and appears to be unique to each individual. Furthermore, gp120₄₅₁ and gp120 from HIV_IIIB and HIV_MNdiffer in their ability to activate T cells expressing these other TCRBV segments. These observations suggest that variation in the structure of gp120 and in the genetic and/or environmental background of the individual play an important role in determining which TCRBV segments are'triggered' by gp120. Furthermore, these observations may have important implications for the rate of disease progression in HIV-infected individuals.     

Differential Activation of Dendritic Cells by Toll-like Receptor Agonists Isolated from the Gram-positive Vaccine Vector Streptococcus gordonii

The oral commensal bacterium Streptococcus gordonii has been gathering interest as a candidate live mucosal vaccine delivery vector. S. gordonii has been shown to be capable of activating antigen presenting immune cells in a manner which leads to their activation and maturation, yet the mechanism used by S. gordonii to do so is poorly understood. The aim of this work was to investigate the immunostimulatory components of S. gordonii in inducing murine dendritic cell (DC) activation and maturation. Lipoteichoic acid (LTA), lipoprotein (LP), peptidoglycan (PGN), and DNA were isolated from S. gordonii , and used to stimulate murine DC. Cytokine production and DC surface marker upregulation in response to the bacterial components was quantified by enzyme-linked immunosorbent assay and flow cytometry respectively. The results were contrasted against data obtained from DC derived from MyD88, TRIF [TIR(Toll/Interleukin-1 Receptor)-domain-containing adapter-inducing interferon-beta] or toll-like receptor-2 (TLR-2) knockout mice. The four S. gordonii bacterial components were found to differentially induce cytokine production and surface marker upregulation by murine DC. Activation of DC by both whole S. gordonii cells and the four bacterial components was abrogated in the absence of MyD88, but not in the absence of TRIF. LTA, LP and PGN, but not DNA and whole S. gordonii, required TLR-2 to induce a DC response. The results collectively indicate that S . gordonii activates DC predominantly through a MyD88-dependent and TRIF-independent pathway. This activation can be attributed to multiple immunostimulatory components present within S. gordonii bacterial cells.     

Blood Group Antigens on HeLa Cells shown by Mixed Agglutination

The mixed agglutination reaction has been used for investigating the presence of blood group antigens on the surface of human cervical carcinoma cells (HeLa) cultured for eight years in vitro.

The H antigen was demonstrated in the absence of A and B. The MN-type antigen has been found as well as Tj^a.

Treatment of HeLa cells with ficin greatly enhanced the reaction of anti-H and anti-Tj^a with the corresponding antigens on HeLa cells. The authors failed to show the following antigens: Rh(D) and Rh(c), S, P, Le^a, Le^b, Lu^a and Lu^b.

     

Cytoplasmic Changes in Satellite Cells of Spinal Ganglia Induced by Cisplatin Treatment in Rats

The effects of cisplatin ( cis -DDP) therapeutic treatment on the cytoplasmic compartment of satellite cells (SC) of rat dorsal root ganglia (DRG) were evaluated. Female Wistar rats were treated once a week with i.p. injection of cis -DDP (2 mg/kg) for 9 weeks. Morpho-quantitative changes of the cytoplasmatic organelles in SC cytoplasm from L4-L6 DRG were determined at the electron microscopic level. The quantitative changes in the lysosomal system components called dense bodies (DB) and in the mithocondria were stereologically evaluated. The data from SC were compared to those from the neurons. The cis -DDP treatment induced a great increase in DB and mithocondria volume of SC. Furthermore, the SC sheath showed an increase of the cytoplasmic lamellar expansions responsible of the physical dissociation of SC sheath from the nerve cell body surface. The comparative analysis from SC and neurons showed that the drug affected primarily the SC, supporting the idea that SC could be the initial target of cis -DDP molecule. The alterations of the anatomical relationships between SC and neurons could modify the cell control on extracellular solutions, altering the functional role barrier attributed to SC. It appears that not only the DRG neurons but also and principally the SC were involved in the peripheral neuropathy mechanisms caused typically from therapeutic cis -DDP administration.     

Inhibition of Pseudomonas aeruginosa by Hyperbaric Oxygen: Interaction with Mouse Peritoneal Exudate Cells

High-pressure oxygen (HPO) therapy for Pseudomonas aeruginosa infections of burn wounds has not been as effective as in vitro studies predicted. Mitigation of HPO toxicity for P. aeruginosa by nutrients present at the burn site could explain the lack of in vivo success. Alternatively, HPO-induced depression of host defense mechanisms could negate beneficial effects arising from HPOs known toxicity for P. aeruginosa. Accordingly, mouse peritoneal exudate cells (PEC), preincubated for 24 h in 1 atm of air-CO(2), were used to study the in vitro effects of HPO or air-CO(2) on phagocytosis of P. aeruginosa or sheep erythrocytes (SRBC). Subsequent 2-h exposures of PEC to increasing numbers of bacteria, in an air-CO(2) atmosphere, decreased the percentage of bacteria cleared as well as PEC viability. Similar exposures of PEC to bacteria in an HPO atmosphere prevented the loss of PEC viability and increased bacterial clearance. In control experiments, increasing the number of SRBC relative to PEC decreased the percentage of SRBC cleared without decreasing PEC viability, as determined under air-CO(2); short (2 h) exposure to HPO did not affect SRBC clearance. Microscopic examination of PEC indicated that a 24-h preincubation in HPO decreased the percentage of PEC which could ingest SRBC during subsequent experimental exposures (2 h) to air-CO(2) or HPO. These data suggest that short periods of exposure to HPO promote the ability of PEC to clear pseudomonads by adversely affecting the bacteria. This in turn prevents a pseudomonad-induced depression of PEC viability and function. In contrast, prolonged HPO exposure may be detrimental to phagocytic activity.     

Cytokine Production by Immunocompetent Cells of Peritoneal Fluid in Women with External Genital Endometriosis

The content of some cytokines in the peritoneal fluid and the level of their in vitro production by peritoneal macrophages and lymphocytes were evaluated in women with external endometriosis. The ratio of peritoneal lymphocytes and macrophages in the peritoneal fluid was changed in endometriosis because of increased percentage of lymphoid cells and decreased content of macrophages, in the presence of high absolute counts of both types of cells. The cytokine status of women with endometriosis was characterized by higher levels of IL-1beta, TNF-alpha, and epidermal growth factors both in the peritoneal fluid and supernatants of 24-h cultures of peritoneal macrophages; the content of IFN-alpha remained unchanged. The concentrations of IFN-gamma in the peritoneal fluid did not change in endometriosis, but increased in the supernatants of peritoneal lymphocyte cultures.     

Hydrocortisone Treatment of BCG-Infected Mice Impairs the Activation and Enhancement of Antimicrobial Activity of Peritoneal Macrophages

The present study concerns the effect of hydrocortisone (HC) on the effector functions of Bacillus Calmette Guerin-purified protein derivative (BCG-PPD)-activated macrophages. Such activated macrophages release greater amounts of H2O2 and NO2-, inhibit the intracellular proliferation of T. gondii and kill L. monocytogenes more efficiently than resident macrophages. This activation was not fully expressed by macrophages from BCG-activated mice that had received a subcutaneous injection of HC 2 days before intraperitoneal injection of PPD, since the inhibition of the intracellular proliferation of T. gondii, the release of NO2- and the rate of intracellular killing of L. monocytogenes were lower than in macrophages from BCG-PPD-activated mice. However, treatment with HC did not impair the release of H2O2 by BCG-PPD-activated macrophages. The results show that the treatment of infected mice with HC inhibits their ability to develop adequate intracellular microbicidal mechanisms.