Radioactive labelling and characterization of the products of activated mouse lymphocytes.

For chemical characterization of the products of activated lymphocytes a radioactive double-label technique was developed which allows one to distinguish those products synthesized either de novo or in increased amounts by the stimulated culture. Spleen cells from Balb/c mice were cultured in serum-free medium in the presence or absence of concanavalin A and simultaneously labelled with radioactive leucine. Optimal culture conditions were established by determining parameters such as cell density, mitogen concentration, and kinetics of protein synthesis following stimulation. Combined supernatants of stimulated and unstimulated cultures each labelled with either [3H]leucine or [14C]leucine were fractionated on Sephadex G-75. Materials derived from control or stimulated supernatants both yielded a qualitatively similar radiolabelled profile. The isotope ratio of stimulated to nonstimulated culture, however, showed a broad peak at KD 0--.35 (approx. mol. wt 75000-20000) which was further analyzed by isoelectric focusing. Pools of every two fractions were focused in polyacrylamide gels at pH 3.5-10. By determining the isotope ratio, the isoelectric point, and the KD (mol wt), it was possible to distinguish at least 24 molecules which had been produced only, or in greater degree, by the stimulated culture.     

Activation of human B lymphocytes. IX. Modulation of antibody production by products of activated macrophages.

Human monocytes, after in vitro activation by mixed lymphocyte culture (MLC) supernatants produce a monokine (MK) that enhances the plaque-forming cell (PFC) response of pokeweed mitogen (PWM)-stimulated human B lymphocytes. Technical conditions and kinetics of MK production were established. Irradiation of monocytes (5000 rads) does not abolish MK production but heat-killed cells are unable to release the factor. Highly T cell-depleted monocyte populations still produced the PFC-enhancing factor. The same MK has an inconsistent enhancing effect on the PFC responses of lipopolysaccharide (LPS) and nocardia water-soluble mitogen (NWSM)-stimulated B cells. Other macrophage activators such as LPS, phytohemagglutinin (PHA), and latex particles failed to induce consistently the liberation of the PFC-enhancing MK. The target cell for the MK activity on PWM-stimulated B cells appears to be the B lymphocyte itself. These studies demonstrate that soluble monocyte products can have substantial modulatory effects on human B cell function.     

Plasminogen activator production by human monocytes. I. Enhancement by activated lymphocytes and lymphocyte products.

Human monocytes, but not nylon wool column-nonadherent lymphocytes, produce plasminogen activator. The activity is found only in association with intact cells. Exposure of monocytes to activated lymphocytes or to lymphokine-rich supernatants enhances monocyte plasminogen activator production. The assay allows assessment of baseline and activated human monocyte function.     

Experimental uveitis induced by products of activated lymphocytes: Intraocular effects of rhodopsin-induced lymphokines

Immunization of strain 13 guinea pigs by footpad injection of bovine rhodopsin in complete Freund's adjuvant produces experimental autoimmune uveitis (retinopathy) with retinal pathology characterized by destruction of the retinal photoreceptor cell layer, as we reported previously. Since rhodopsin-induced EAU can be passively transferred by immune cells but not antirhodopsin antibody, the present studies were conducted to determine if soluble products of activated lymphocytes (PAL) could cause the retinal pathology seen in experimental uveitis. These studies, reported here, show that intravitreal injection of supernatant factors generated by guinea pig lymph node cells specifically activated in vitro with rhodopsin or purified protein derivatives of tuberculin or nonspecifically activated with the mitogen concanavalin A produce uveitis in the normal guinea pig eye. The PAL produced mononuclear cell infiltration in the vitreous and a retinopathy with loss of retinal photoreceptor cells. Inflammatory cell infiltrates were found in the retina but not found in the anterior segment of the eye. Control lymphocyte supernatants did not produce retinal pathology. In guinea pigs sensitized to rhodopsin-complete Freund's adjuvant, intravitreal injection of the PAL produced a mononuclear vitreal infiltrate, disruption of the photoreceptor cell layer, necrosis of the inner retina, and a granulomatous infiltrate in the choroid with damage to the retinal pigment epithelium. Injection of control supernatants into the rhodopsin-complete Freund's adjuvant sensitized guinea pig eye did not produce inflammation and the retinal photoreceptor cell layer remained intact. Results from our studies indicate a role for the PAL along with other, yet undefined, factors in the initiation of autoimmune uveitis and in the autoimmune pathology of the retina.     

Cytotoxic activity of lymphocytes. VIII. Lymphotoxin activity in cell-free extracts of activated lymphocytes.

Cell-free extracts of human lymphocytes activated by PHA contain a cytotoxin that kills mouse L cells. Such cells elaborate two different kinds of toxins into the culture supernatant fluids, called α- and β-lymphotoxin (-LT), which differ in size, stability, and antigenicity. The amount of intracellular toxin is 1 to 24% of that found in supernatants of different tonsil donors. Equal amounts of intracellular toxin appear in both microsomal fraction (100,000g pellet) and soluble supernatant fractions of the cell-free extracts (CFE). The toxin can be solubilized from the membrane by digestion with papain or extraction with a nonionic detergent, but not by repeated sonication. The molecular weight of both the microsomal and soluble cellular cytotoxin is 45,000 ± 5000. The intracellular toxin differs from the extracellular toxins secreted by the same cells in two major characteristics: one, although its size approximates that of supernatant β-LT (and is smaller than the 76,000 M_r α-LT), antibody-inhibiting α-LT but not β-LT inhibits both the microsomal and soluble CFE-LT. Two, the intracellular LT does not display the charge heterogeneity so characteristic of supernatant α-LT. Supernatant α-LT and CFE-LT are similar in their patterns of inactivation by heating to 80 °C and treatments with sodium dodecylsulfate (SDS), guanidine, proteases, and heavy metal ions, and are similarly unaffected by treatment with 8 M urea, N-ethylmaleimide, and sodium periodate. These results suggest that the single polypeptide intracellular LT is the precursor of the more complex secreted α-LT molecule.     

Splenic suppressing factor: purification and characterization of a factor suppressing thymidine incorporation into activated lymphocytes.

Suppressing activity upon the mitogen-activated lymphocytes was found in the supernatant (SUP) from the culture of mouse spleen, high-density subpopulation of thymocytes, and peritoneal exudate cells. Suppressing factor was obtained from the non-stimulated lymphocytes cultured for 24 to 36 hr with or without serum. Suppressing activity in the SUP was observed in the incorporation of 3H-thymidine, 3H-uridine, and 3H-leucine into Con A-activated lymphocytes or in the proliferation of L cells. Suppressing factor partially purified by Sephadex G-25 column chromatography was a heat-stable and dialyzable substance(s). Further purification and isolation of this factor by two-dimensional thin layer chromatography revealed that this was thymidine and thymidine monophosphate. The suppression in 3H-thymidine incorporation was attributed to the dilution effect of cold thymidine released from cultured lymphocytes.     

The effects of soluble products of activated lymphocytes and macrophages on blastocyst implantation events in vitro

The purpose of this study was to investigate the effects of soluble products of activated lymphocytes and macrophages on mouse blastocyst attachment and trophoblast outgrowth in vitro. Hatched blastocysts were incubated with medium alone, supernatant fluids from mixed lymphocyte cultures (MLC), and with individual human and murine lymphokines and monokines in fibronectin-coated wells. Cultures were assessed at 24, 48, and 72 h for blastocyst attachment and trophoblast outgrowth. Both human and murine MLC supernatant fluids significantly enhanced trophoblast outgrowth in vitro. The cytokine, interleukin-1 beta (Il-1 beta), at a concentration of 10(3) U/ml, inhibited blastocyst attachment but significantly enhanced trophoblast outgrowth of attached blastocysts. Granulocyte, macrophage-colony-stimulating factor (GM-CSF) at a concentration of 250 U/ml significantly inhibited blastocyst attachment, while gamma interferon (gamma-IFN) at a concentration of 2.5 x 10(3) U/ml significantly inhibited trophoblast outgrowth and caused degenerative morphological changes in these cells. The results of this study indicate that products of activated immune cells may either facilitate or impede implantation events depending on the types of predominant cytokines present, their concentration(s), and the timing of their secretion relative to embryonic development.     

Interaction of thymus lymphocytes with histoincompatible cells. IV. Mixed lymphocyte reactions of activated thymus lymphocytes

CS7BL-activated CBA T cells (T.TDL) were obtained by thoracic duct cannulation of (CBA × C57BL)F₁ mice 4 days after heavy irradiation and injection of CBA thymus cells. T.TDL behaved differently from the TDL of normal CBA mice in unidirectional mixed lymphocyte culture in a number of respects: (a) the response of T.TDL was directed specifically against C57BL antigens, whereas normal TDL responded to both C57BL and BALB/c antigens; (b) the response of T.TDL was rapid but transient compared to that of TDL; (c) whereas only approximately 3% of TDL synthesized DNA specifically in response to C57BL antigens, as many as 25% of C57BL-activated T.TDL responded to these antigens. Evidence is presented which suggests that the T.TDL have a very limited capacity to proliferate. Most of the cells which responded to antigen synthesized DNA without subsequently entering mitosis.     

IL-12 augments antigen-dependent proliferation of activated T lymphocytes.

Ag-dependent T cell activation requires multiple transmembrane signals including activation of Ag-specific T cell receptor in combination with signals delivered through cytokine receptors. IL-12 is a heterodimeric cytokine involved in the regulation of NK and T lymphocyte responses. In examining the role of IL-12 in T cell activation, we found a direct relationship between Ag stimulation and IL-12-induced proliferation. Unlike IL-2, which induced proliferation of CTL either in the presence or absence of a CD3/TCR co-signal, IL-12 mediated proliferation of CTL only when the cells were recently co-stimulated with alloantigen or solid-phase anti-CD3 antibody. After culture in the absence of alloantigen or anti-CD3 for 7 to 14 days, these CTL lost the ability to proliferate to IL-12 alone. Under these conditions, however, IL-12 synergized with low-dose IL-2 to induce CTL proliferation. Restimulation with alloantigen or solid-phase anti-CD3 restored the ability of the CTL to proliferate to IL-12 alone. Not all Ag signals resulted in IL-2 independent proliferation to IL-12. For example, CTL with specificity for influenza matrix peptide proliferated best when co-cultured with peptide Ag presented on self MHC and a combination of IL-2 and IL-12. This evidence suggests that IL-12 may be useful in expanding an Ag-specific T cell population, as the culture of CTL with IL-12 and low-dose IL-2 leads to proliferation only in response to an Ag co-signal.     

Phenotypic characterization of murine tumor-infiltrating T lymphocytes.

Tumor infiltrating lymphocytes (TIL) can be isolated from solid tumors and selectively expanded in long term culture with IL-2 and autologous irradiated tumor. Such long term cultured cells express anti-tumor activity in vitro, mediate the regression of established tumor in murine models of cancer, and have been used for the treatment of cancer in humans. We have characterized freshly isolated mouse Thy-1+ TIL populations, as well as long term TIL cultures, from several different C57BL/6 (B6) tumors. Freshly isolated Thy-1+ TIL include both CD4+ and CD8+ cells, as well as cells bearing NK markers. These cells are predominantly TCR alpha beta+, with a smaller population of TCR gamma delta+ cells. The TCR alpha beta+ cells expressed a broad distribution of V beta phenotypes that was statistically different from that expressed in normal B6 splenic Thy-1+ cells or CD8+ cells, presumably reflecting in vivo selection in the host anti-tumor response. NK cells are present in these tumors at a greater frequency than noted in splenic T cells. Cultured TIL populations rapidly became exclusively Thy-1+/CD8+/CD4- and TCR alpha beta+/gamma delta-. Individual long term TIL populations initially expressed multiple V beta products, but rapidly restricted their V beta expression, frequently expressing a single dominant V beta. The identity of this dominant V beta varied among different TIL lines, but the overall representation of V beta phenotypes in these cultures was statistically different from that seen in Thy-1+ or CD8+ splenocytes. No statistical difference was noted between lines derived from antigenically distinct tumors. The selection of tumor specific T cells in vitro is therefore not reflected in any simple predominance of V beta usage. The complexity of TCR usage in the anti-tumor response may result from the involvement of multiple alpha- and beta-chain regions in the response to a single antigenic determinant, or may reflect multiple antigenic determinants expressed on a single syngeneic tumor.     

Accumulation of activated lymphocytes in liver of endotoxin treated rabbits.

Intravenous administration of endotoxin in doses, 1.0 mg/kg, 0.1 mg/kg or 0.01 mg/kg caused activation (in the sense of nucleolar RNA synthesis) of the lymphocytes in peripheral blood, kidney and liver in rabbits. The evaluation of the ratio of lymphocyte count to the number of organ tissue cells leads to the conclusion that the accumulation of endotoxin activated lymphocytes occurs in liver.     

Phenotypical characterization of lymphocytes infiltrating regressing papillomas.

Papillomavirus-induced lesions often regress spontaneously in both humans and animals. Papilloma regression is deemed to be due to a cell-mediated immune response, the nature of which is still ill defined, and is accompanied by immune cell infiltrates. To gain further information on the nature and role of the immune cells present in regressing papillomas, we have analyzed biopsies of papillomas induced in the soft palate of cattle by bovine papillomavirus type 4 (BPV-4) and have phenotypically characterized and quantified the lymphocytes present in these lesions. Eleven papilloma biopsies and seven biopsies of noninfected palate were analyzed for the presence of activated CD4+, CD8+, and gamma delta(WC1+) lymphocytes. We found large numbers of lymphocytes in the subepithelial derma of papillomas but not in normal palate tissue; these cellular masses consisted predominantly of CD4+ lymphocytes, with only a few CD8+ and gamma delta(WC1+) lymphocytes, generally positioned at the periphery of these masses. All three subtypes of lymphocytes were found interdigitated with the cells of the basal layer both in papillomas and in normal palate tissue, but while basal layer CD8+ and gamma delta(WC1+) T cells were detected with similar frequencies in papillomas and uninfected palate, basal layer CD4+ T cells were much more frequent in papillomas. CD4+, CD8+, and gamma delta(WC1+) lymphocytes were found in the suprabasal layers of papillomas, but the CD8+ and gamma delta(WC1+) T cells were more numerous and had migrated further into the differentiating keratinocytes of the papilloma fronds than the CD4+ T cells. We conclude that T-cell infiltration is characteristic of regressing BPV-4 papillomas, that CD4+ lymphocytes are specifically and massively recruited into the regressing papillomas, and that although all three lymphocyte subsets can penetrate the papilloma, only the CD8+ and gamma delta(WC1+) lymphocytes are able to migrate into the fronds. These results suggest that all three lymphocyte subsets have an important role to fulfill during natural regression of papillomas.     

Relationship between ANS fluorescence and electrokinetic potential of activated lymphocytes.

An immune response was induced in vivo on C3H/He ♂ mouse strain with Bovine Serum Albumin (BSA), or Sheep Red Blood Cells (SRBC). The membrane fluorescence changes of activated splenic lymphocytes were studied two weeks after the injection of antigens. Experiments were performed with the hydrophobic fluorescent probe: 1-anilino-8-naphthalene sulphonate (ANS). The kinetic studies further indicated that the course of fluorescence changes may considerably vary depending on antigens. Their fluorescence intensities were lower than control values. A maximum decrease of fluorescence was recorded on days 1, 6 and 9 after immunization with BSA-stimulated lymphocytes. SRBC-stimulated lymphocytes exhibited a maximum ANS fluorescence decrease on days 4 and 9 after immunization. These fluorescence phenomena would be in an inverse relationship with the electrokinetic surface potential of activated lymphocytes, as assessed by the electrophoretic mobility analysis (EPM). Some parameters affecting the ANS fluorescence in T and B cells are discussed. Quantification of hydrophobic sites in splenic cells would indicate that forces other than the hydrophobic ones may also be involved in the dye-binding changes following immune activation.     

Physical characterization of cyclosporine binding sites in lymphocytes.

The binding of cyclosporine to human peripheral blood lymphocytes (PBLs) was studied by measuring the fluorescence emission spectrum and lifetime of the fluorescent and immunosuppressive cyclosporine derivative dansyl-cyclosporine (DCs). The emission maximum and fluorescence lifetime of DCs were characterized in several solvents. The fluorescence emission maximum and lifetime of DCs increased at a high dielectric constant. The fluorescence lifetime decay curve of DCs was a monoexponential function in all solvents tested. Fluorescence micrographs of lipid vesicles and erythrocytes labeled with DCs exhibit uniform staining patterns, whereas PBLs show heterogeneous DCs labeling. DCs exhibits a relatively low emission maximum (490 nm) in erythrocyte membranes. Such an emission maximum is characteristic of a hydrophobic environment. DCs in PBLs also has a low emission maximum (484 nm). The lifetime of DCs in PBLs required two exponential terms to properly fit the lifetime decay curve and could not be attributed to light scattering. One short component (4.7 +/- 1.0 ns) and a second long component (18.5 +/- 1.0 ns) were resolved from the DCs fluorescence decay curves. Time-resolved anisotropy of DCs in PBLs revealed that the labeled drug was present in an anisotropic environment, consistent with at least some DCs being bound to a membrane. These fluorescence studies suggest that DCs interacts with multiple and/or heterogeneous sites in peripheral blood lymphocytes.     

Further characterization of Renibacterium salmoninarum extracellular products.

Renibacterium salmoninarum, the agent of bacterial kidney disease in salmonids, releases high concentrations of extracellular protein in tissues of infected fish. The extracellular protein consists almost entirely of a 57-kDa protein and derivatives of degradation and aggregation of the same molecule. The 57-kDa protein and its derivatives were fractionated into defined ranges of molecular mass. Separated fractions continued to produce degradation and aggregation products. One-dimensional electrophoretic separation of extracellular protein revealed a number of proteolytically active bands from > 100 to approximately 18 kDa associated with various 57-kDa protein derivatives in the different molecular mass fractions. Two-dimensional separation of extracellular protein showed that continued degradation and aggregation, similar both in location and behavior to some of the 57-kDa protein derivatives, was also displayed by the proteolytically active bands after their separation. Effects of reducing agents and sulfhydryl group proteinase inhibitors indicated a common mechanism for the proteolytically active polypeptides characteristic of a thiol proteinase. The results suggested that the 57-kDa protein and some of its derivatives undergo autolytic cleavage, releasing a proteolytically active polypeptide(s) of at least 18 kDa. Soluble polysaccharide-like material also was detected in extracellular products and tissue from infected fish. Antiserum to the polysaccharide-like material cross-reacted with O-polysaccharide of the fish pathogen Aeromonas salmonicida, suggesting some structural similarity between these polysaccharides. The polysaccharide and the proteolytic activity associated with the 57-kDa protein derivatives should be investigated with respect to the pathogenesis of R. salmoninarum infections.     

Interactions between B lymphocyte subpopulations. Augmentation of the responses of resting B lymphocytes by activated B lymphocytes

Mouse B lymphocytes were fractionated from normal T lymphocyte-depleted spleen cell populations using discontinuous percoll gradients and were stimulated with rabbit F(ab')2 anti-mouse mu-specific antibodies (anti-mu) plus the supernatant of Con A-stimulated rat spleen cells (SN) as a source of lymphokines. The responses of small (mean volume 120 mu 3), dense (greater than 1.087 specific gravity), resting (least spontaneous thymidine incorporation) B lymphocytes were augmented by irradiated (4000 rad), larger (mean volume greater than 170 mu 3), less dense (less than 1.081 specific gravity), activated (greater spontaneous thymidine incorporation) B lymphocytes. Proliferation was augmented 2- to 4-fold and polyclonal antibody-forming cell responses three- to sixfold. Maximal augmentation of the responses of 5 X 10(4) resting B cells was obtained with 10(4) activated B cells. Augmenting activity was specific for activated B lymphocytes in that responses were not augmented by irradiated thymocytes, T lymphoblasts, macrophages, or additional supernatant. B lymphocytes activated in vitro by LPS or anti-mu also had augmenting activity. Augmentation of responses was maximal only when activated B lymphocytes were added simultaneously with anti-mu. The interaction between activated and resting B lymphocytes did not appear to be genetically restricted. Interestingly, the augmenting activity of activated B cells could be reconstituted by a combination of supernatant and cell membranes from these cells but not by either alone, suggesting that two components are required, one soluble and the other membrane-bound. Thus, a functional interaction has been demonstrated between B lymphocyte subpopulations which differ in their state of activation, and this interaction appears to involve a novel mechanism of action.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. I. Expression of the antigen on monocytes and activated lymphocytes

It has been shown previously that HBJ127 and HBJ98 monoclonal antibodies raised against a human bladder cancer cell line, and B3 monoclonal antibody against a rat bladder cancer cell line recognized unique cell surface antigens abundant in proliferating cells of the corresponding species. Distribution of the antigens and kinetics of the appearance on human and rat lymphoid cells were examined by means of flow cytometry. Rat macrophages and human peripheral blood monocytes were stained strongly with the B3 and HBJ127 monoclonal antibodies, respectively. With regard to lymphocytes, the expression of the B3-defined antigen on rat lymphocytes was found to have a negative correlation with the maturation of the lymphocytes; the antigen was most abundant in bone marrow cells, less abundant in thymocytes, and least abundant in spleen, lymph node, and peripheral blood lymphocytes. Similarly, the HBJ127-defined antigen on human peripheral lymphocytes was negligible. On activation with Con A or alloantigens, however, both rat and human T lymphocytes did strongly express these antigens. Activation of human or rat B cells with lipopolysaccharide also resulted in the augmented expression of these antigens. Kinetics studies revealed that the antigen expression was readily manifested within 12 hr on activation of rat or human T cells with Con A, was augmented progressively with culture time, and reached a plateau within 36 hr. This somewhat earlier appearance of these antigens apparently preceded the manifestations of the IL 2 receptor (Tac antigen) and the augmented DNA synthesis. The B3-defined antigen on Con A-stimulated T cells was more rich on the lymphocytes in S and G2/M phases than those in G1 phase, and the expression was not significantly affected by the addition of hydroxyurea, but was moderately inhibited by the addition of sodium butylate. These results suggest that the appearance and expression of the B3-defined antigen and probably also those of the HBJ127/HBJ98-defined antigen are correlated with lymphocyte activation and subsequent progression through the cell cycle.