The comparative characteristics of the immunomodulating properties of protein A from Staphylococcus aureus of different origins

The immunomodulating properties of highly purified staphylococcal protein A and its analog obtained by gene engineering techniques have been compared with those of commercial preparations. The comparison has shown that the differences observed in this investigation may be explained by the presence of admixtures of staphylococcal nature in commercial preparations. The preparations of highly purified staphylococcal and recombinant protein A stimulate humoral immune response and the processes of phagocytosis and do not show mitogenic activity with respect to T cells. The conclusion on the identity of the immunomodulating activity of the preparations of natural and recombinant protein A has been made.     

Stimulation of chicken lymphocytes by T- and B-cell mitogens.

Cultures of chicken spleen, peripheral blood, thymus, and bursal lymphocytes were tested for mitogenic stimulation by phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), trypsin, and insulin. Spleen and blood leukocytes were stimulated by both the lectins and LPS, and also to some degree by trypsin and insulin as judged by increased incorporation of [³H]thymidine into acid-insoluble material. This was observed in cultures incubated in serum-free medium as well as in the presence of foetal bovine serum or autologous plasma. Thymus cells were reproducibly stimulated by high concentrations of PHA. No significant responses were obtained in bursal cell cultures with any of the compounds tested. Removal of cotton wool-adherent cells from the spleen cell suspensions resulted in a subpopulation of cells which were stimulated by PHA but showed little response to ConA, PWM, or LPS. This procedure did not remove surface immunoglobulin-bearing cells from the original suspension. Both these enriched spleen lymphocytes and the unfractionated spleen, blood and thymus leukocyte cultures were effectively stimulated by a partially purified PHA but with a highly purified PHA preparation only at very high concentrations. These and other results suggest that the mitogenic components in crude PHA preparations are different for chicken and human or mouse cells.     

Temperature sensitivity of interleukin-dependent murine T cell proliferation: Q2 mapping of the responses of peanut agglutinin-negative thymocytes

Recent studies in our laboratory have shown that IL 1-dependent mitogenic activity is present in apparently homogeneous preparations of rabbit endogenous pyrogen. This finding suggested that the mitogenic and pyrogenic activities of this molecule might serve a common goal. Initial studies on the temperature dependence of interleukin-dependent thymocyte mitogenesis suggested that high temperature sensitivity was associated with the action of IL 1 but not that of IL 2. The present study has used an expanded range of temperatures and refined tissue culture conditions to further examine the relative temperature dependence of thymocyte mitogenesis due to IL 1 or IL 2. Both the pI 5 and pI 7 species of rabbit IL 1 evoke highly temperature-sensitive responses from mouse thymocytes in the presence of a suboptimal dose of PHA and from peanut agglutinin-negative (PNA-) thymocytes in the absence of PHA. IL 2 also evokes a highly temperature-sensitive response from unseparated thymocytes in the presence of PHA. However, in the absence of PHA, vigorous responses by either unseparated or PNA- thymocytes to IL 2 alone lack strong temperature sensitivity. The temperature-dependent responses of both unseparated and PNA- thymocytes to either IL 1 or IL 2 have been analyzed by Q2 mapping, a determination of the temperature intervals most sensitive to temperature changes. By using this mode of analysis, we have found that IL 1 and IL 2 generate distinct Q2 maps, and that PHA transforms the shape of the IL 2-derived Q2 map but not that of IL 1. The possible significance of the temperature sensitivity of IL 1- and IL 2-driven reactions is discussed with respect to the biological functions of inflammation and fever.     

Comparison of mitogenic responses of young and old rhesus monkey T cells to lectins and interleukins 2 and 4

Purified T cells from rhesus monkeys, like human T cells, do not show a significant mitogenic response to lectins or PMA, but when combined with PMA or accessory cells, PHA and Con A induce a vigorous mitogenic response. This response is strongly impaired in purified T cells from old rhesus monkeys compared to young T cells, from 56 to 72%, and parallels results obtained with T cell preparations containing accessory cells. Likewise, purified T cells do not respond to interleukin 2 (IL-2) or IL-4, but in the presence of PMA, a significant mitogenic response occurs in the young but not the old T cells. This response is augmented by accessory cells, but is still very deficient in the old T cells. These results show that the IL-2 independent activation of T cells triggered by IL-4, like the conventional IL-2 activation, is age impaired. The deficient response to IL-2 implies an age-related deficiency in IL-2 receptor as well in aged rhesus T cells, and may account for the less effective response of the old cells to calcium ionophore (+PMA) activation. The use of purified T cells in these studies obviate the influence of accessory cells, and thus simplify interpretation.     

Reduction of cell lysate viscosity during processing of poly(3-hydroxyalkanoates) by chromosomal integration of the staphylococcal nuclease gene in Pseudomonas putida

Poly(3-hydroxyalkanoates) (PHAs) are biodegradable thermoplastics which are accumulated by many bacterial species in the form of intracellular granules and which are thought to serve as reserves of carbon and energy. Pseudomonas putida accumulates a polyester, composed of medium-side-chain 3-hydroxyalkanoic acids, which has excellent film-forming properties. Industrial processing of PHA involves purification of the PHA granules from high-cell-density cultures. After the fermentation process, cells are lysed by homogenization and PHA granules are purified by chemical treatment and repeated washings to yield a PHA latex. Unfortunately, the liberation of chromosomal DNA during lysis causes a dramatic increase in viscosity, which is problematic in the subsequent purification steps. Reduction of the viscosity is generally achieved by the supplementation of commercially available nuclease preparations or by heat treatment; however, both procedures add substantial costs to the process. As a solution to this problem, a nuclease-encoding gene from Staphylococcus aureus was integrated into the genomes of several PHA producers. Staphylococcal nuclease is readily expressed in PHA-producing Pseudomonas strains and is directed to the periplasm, and occasionally to the culture medium, without affecting PHA production or strain stability. During downstream processing, the viscosity of the lysate from a nuclease-integrated Pseudomonas strain was reduced to a level similar to that observed for the wild-type strain after treatment with commercial nuclease. The nuclease gene was also functionally integrated into the chromosomes of other PHA producers, including Ralstonia eutropha.     

Analysis of the mitogenic effect of fetuin preparations on arterial smooth muscle cells: the role of contaminant platelet-derived growth factor

Fetuin, a major protein of fetal calf serum, partially purified by the method of Pedersen, stimulated growth of aortic smooth muscle cells. More highly purified fetuin preparations stimulated growth less than Pedersen fetuin, as previously described for other cell types, suggesting that this activity is due to a contaminant. Recently bovine alpha 2-macroglobulin or "Embryonin" has been proposed as the mitogenic component of crude fetuin preparations. We found that active fetuin preparations did contain alpha 2-macroglobulin that stimulated smooth muscle cell growth. However, alpha 2-macroglobulin purified directly from platelet-poor bovine plasma or fetuin purified from Pedersen fetuin by gel filtration lacked appreciable mitogenic effect on smooth muscle cells. Since alpha 2-macroglobulin can bind platelet-derived growth factor (PDGF), and since highly acidic fetuin might bind the very basic PDGF molecule non-specifically, we measured the PDGF content of various fetuin preparations and found a good correlation between the PDGF content and mitogenic activity. Gel filtration experiments demonstrated that in Pedersen fetuin PDGF occurred both free, and in association with alpha 2-macroglobulin. We conclude that the principal mitogenic component for smooth muscle cells in crude fetuin preparations is PDGF, since purified bovine alpha 2-macroglobulin or fetuin do not appreciably affect growth of these cells. These results help to resolve a long-standing controversy regarding the nutrition of cultured cells. In addition, we suggest that before alpha 2-macroglobulin or "Embryonin" is accepted as a bona fide growth factor for a given cell type, the role of contamination with PDGF should be assessed.     

Isolation of multiple biologically and chemically diverse species of epidermal growth factor

We have analyzed several lots of epidermal growth factor (EGF) purified from murine submaxillary glands including "receptor grade" EGF from Collaborative Research and EGF from Boehringer Mannheim Biochemicals. New England Nuclear uses "receptor grade" EGF to produce 125I-labeled EGF. Though these reagents are reported to be homogeneous, we found them to be a mixture of six species. A method was developed to separate this mixture into its component parts. The individual components were chemically characterized and tested for biological potency. N-terminal sequence analysis of the unfractionated EGF-mixture reveals three different sequences starting with residues 1, 2, or 3 of the mature peptide. Each component exhibited different degrees of mitogenic and EGF receptor binding activity indicating that the N-terminal region contributes to the biological response. The species representing the complete EGF peptide is the most active species in all biological assays. A rapid method for purification of homogeneous complete EGF from commercial EGF preparations is described.     

Reduction of Cell Lysate Viscosity during Processing of Poly(3-Hydroxyalkanoates) by Chromosomal Integration of the Staphylococcal Nuclease Gene in Pseudomonas putida

Poly(3-hydroxyalkanoates) (PHAs) are biodegradable thermoplastics which are accumulated by many bacterial species in the form of intracellular granules and which are thought to serve as reserves of carbon and energy. Pseudomonas putida accumulates a polyester, composed of medium-side-chain 3-hydroxyalkanoic acids, which has excellent film-forming properties. Industrial processing of PHA involves purification of the PHA granules from high-cell-density cultures. After the fermentation process, cells are lysed by homogenization and PHA granules are purified by chemical treatment and repeated washings to yield a PHA latex. Unfortunately, the liberation of chromosomal DNA during lysis causes a dramatic increase in viscosity, which is problematic in the subsequent purification steps. Reduction of the viscosity is generally achieved by the supplementation of commercially available nuclease preparations or by heat treatment; however, both procedures add substantial costs to the process. As a solution to this problem, a nuclease-encoding gene from Staphylococcus aureus was integrated into the genomes of several PHA producers. Staphylococcal nuclease is readily expressed in PHA-producing Pseudomonas strains and is directed to the periplasm, and occasionally to the culture medium, without affecting PHA production or strain stability. During downstream processing, the viscosity of the lysate from a nuclease-integrated Pseudomonas strain was reduced to a level similar to that observed for the wild-type strain after treatment with commercial nuclease. The nuclease gene was also functionally integrated into the chromosomes of other PHA producers, including Ralstonia eutropha.     

Human chorionic gonadotropin: effects of crude and purified preparations on lymphocyte responses to phytohemagglutinin and allogenenic stimulation.

The factors that prevent maternal immunologic rejection of the histoincompatible fetus are not understood. High levels of human chorionic gonadotropin (HCG) are present in the placenta, and several reports have noted suppresion of mitogen-induced lymphocyte transformation when cultures were supplemented with crude preparations of HCG. Purified HCG and multiple lots of crude HCG obtained from different suppliers were examined for their ability to suppress lymphocyte transformation produced by phytohemallutinin (PHA) or allogeneic stimulation. Crude preparations of HCG produced suppression of the lymphocyte stimulation induced by low doses of PHA, but the suppression could be overcome completely by increasing the PHA dose. The purified preparations of HCG produced no suppression of lymphocyte responses, even at the lower PHA dose. Purified HCG did not give a dose-related suppression of allogeneic lymphocyte responses, and crude lots of HCG gave highly variable results. One lot of crude HCG produced spontaneous stimulation of lymphocytes. Isoelectric focusing of HCG preparations demonstrated multiple bands, and lymphocyte suppression may be secondary to these additional unidentified proteins. The failure of pruified HCG to suppress lymphocyte responses makes it unlikely that the absence of maternal rejection of the fetus is due to high placental levels of HCG.     

Brucella abortus lipopolysaccharide is mitogenic for spleen cells of endotoxin-resistant C3H/HeJ mice.

Preparations of lipopolysaccharide (LPS) from rough and smooth strains of Brucella abortus were mitogenic for spleen cells of athymic nude mice, C3H/HeAU mice, and the endotoxin-resistant C3H/Hej mice. The mitogenic response induced by crude smooth-LPS (f5) was greater than that produced by purified smooth-LPS (f5p); however, the dose-response curves were similar for both preparations. The mitogenic activity of mouse spleen cells to both f5 and f5p was higher than that produced by stimulation with purified rough-LPS. The dose-response curves with rough-LPS were also qualitatively different from those produced with the preparations of smooth-LPS.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Isolation and partial characterization of mitogenic factors from cementum.

Cementum is the mineralized structure through which soft connective tissues are attached to the teeth. It is a unique calcified tissue characterized by a low metabolic turnover, lack of blood supply, and presence of very few cells. However, it contains substances that influence the biological activities of fibroblasts of adjacent soft tissues. We have partially characterized cementum proteins that have mitogenic activity toward fibroblasts. Cementum was harvested from bovine teeth, and mitogenic factors were extracted in 0.5 M CH3COOH. Heparin-Sepharose chromatography separated the mitogenic activity into a major and a minor fraction eluted by 0.5 and 2.0 M NaCl, respectively. The distribution of cementum mitogens in heparin-Sepharose fractions was different from that of alveolar bone and other bones. The cementum mitogenic factor eluting with 2.0 M NaCl from a heparin-Sepharose column was shown to be basic fibroblast growth factor (bFGF) on the basis of inhibition by anti-bFGF antibody and Western blots. The 0.5 M NaCl fraction was purified by HPLC with use of a combination of a DEAE-3W column followed by TSK-250 and C18 columns. NaDodSO4-polyacrylamide gel electrophoresis revealed that the purified fraction contained two protein bands with Mr 22,000 and 19,000, and mitogenic activity was associated with the Mr 22,000 species. The activity of this mitogen, designated as CGF, was potentiated by small quantities of plasma-derived serum or epidermal growth factor. It was heat resistant, but was destroyed by reduction. Assays of CGF preparations revealed that they contained no detectable platelet-derived growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacterial endotoxins: Comparison of mitogenic,polyclonal, antibody-inducing and toxicity activities

The mitogenic effects on mouse spleen lymphocytes were determined in a large series of commercially available and laboratory-prepared lipopolysaccharides (LPS) obtained fromEscherichia, Salmonella, Serratia andShigella species; part of these LPS preparations was chemically modified prior to testing. In order to establish whether the degree of mitogenic activity corresponds with other biological effects of these preparations, polyclonal activity, capability to induce specific antibody formation and toxicity were determined for selected LPS's with different mitogenic effects. Some of the detoxication procedures used succeeded in reducing the toxicity of LPS while preserving its high mitogenic activitione of the Fe-detoxified preparations of LPS (from the R-form ofShigella dysenteriae serovar 1) exhibited a medium-degree efficacy in all parameters studied. Generally, there was no correlation between the degree of mitogenic activity and the polyclonal and antibody-inducing activities, but in some instances polyclonal activity did correlate with the antibody-inducing activity.     

Phaseolus vulgaris L. var. HUR 15--a potential indigenous source for commercial PHA preparation.

Mitogenic potential of the partially purified lectin from P. vulgaris isolated by ammonium sulphate precipitation was assessed by lymphocyte transformation test (LTT) and was compared with three commercially available phytohemagglutinins (PHA). The blast inducing capacity and mitotic index analysis revealed that this preparation has potential to be used in the indigenous commercial production of PHA which is routinely used for human chromosomal studies from the peripheral blood culture at, at present, is imported.     

Steroid sensitivity of the PHA and PWM responses of fractionated human lymphocytes in vitro

The in vitro PHA and PWM responses of various fractions of human peripheral lymphocytes were tested for sensitivity to water-soluble prednisolone. Removal of cells having the capacity to phagocytize carbonyl iron particles or cells having a tendency to adhere to plastics increased the steroid sensitivity of both the PHA and the PWM responses. T and B cell-rich preparations were obtained by passing cell suspensions, depleted of macrophages-monocytes as described above, through anti-Ig labelled bead columns or by a rosette centrifugation technique. The mitogenic response of cell suspensions enriched for B cells were more steroid resistant than suspensions enriched for T cells.     

Physicochemical and immunochemical properties of carcinoembryonic antigen (CEA) from different tumour sources.

The physical, chemical and immunochemical properties of carcinoembryonic antigen (CEA) purified from hepatic metastases of eight tumours, originating in the colon (6), stomach (1) and lung (1), have been examined. Differences were observed in the overall molecular charge, and also in the carbohydrate composition of the different preparations (both total % carbohydrate, and mole % of the individual sugars). Negligible differences in amino acid composition were found. Gel filtration analysis of these CEA preparations and an additional four partially purified preparations (from pancreatic, hepatic, breast and oesophageal tumour tissues) revealed a single CEA-active peak of similar molecular weight (about 200,000-300,000 daltons) in all preparations. Radioimmunoassay data for the twelve CEA preparations indicated that all preparations contain the same antigenic determinants, as detected by our antiserum, but that there are differences in the expression of these determinants in different preparations.     

Role of iron in transferrin-dependent lymphocyte mitogenesis in serum-free medium

Transferrin can partially replace serum requirement for the mitogenic activity of both PHA and the oxidizing mitogen, neuraminidase and galactose oxidase, whereas several other proteins are inactive. Catalase enhances the potentiating effect of transferrin, indicating that transferrin stimulates hydrogen peroxide production by the cell preparations. Iron is required for transferrin to replace serum, since prior addition of desferrioxamine to medium or cell cultures eliminates the potentiating effect of transferrin on lymphocyte mitogenesis. In addition to the possibility that iron is required for nutritional purposes, transferrin-mediated activation of oxidative metabolism may have a stimulatory effect on lymphocyte activation.     

Mechanisms of action of “lymphocyte-activating factor” (LAF): IV. Differential stimulation of T lymphocytes by induced macrophage enzymes (catheptic carboxypeptidase B and serine proteases)

PHA and concanavalin A are unable to induce DNA synthesis in a substantial proportion of highly purified rat lymph node T cells in serum-free culture. Addition of dialyzed macrophage supernatant (LAF or TAF) to these mitogen-stimulated cells, even as a 12-hr pulse starting at 14 hr, gives a stronly potentiated response, while LAF (TAF) alone is nonmitogenic. Thus mitogen provides a first signal and LAF (TAF) a second signal in time. General protease inhibitors (trasylol, soybean trypsin inhibitor, and especially ?-aminocaproic acid) markedly inhibit both the low response to mitogen alone and the LAF effect, while certain more narrowly specific inhibitors (phenylmethylsulfonyl fluoride, tosyl-lysl-chloromethyl ketone, iodoacetate) do not. This finding suggests that the LAF effect involves protease action. The LAF effect has been shown to parallel the Carboxypeptidase B content of various LAF preparations and can be mimicked by commercial pancreatic Carboxypeptidase B. A lesser LAF effect is also induced by such serine proteases as trypsin, chymotrypsin, and plasmin, none being mitogenic alone. Thus LAF (TAF) may represent the combined action of several enzymes. These findings are contrasted with the demonstrated ability of many serine proteases to stimulate DNA synthesis, i.e., to serve as first signal, in B cells. LAF (TAF) acts synergistically with commercial carboxypeptidase B. This implies that they may act on different target sites in the cell membrane and that triggering of an adequate number of sites is required for an effective second signal. LAF (TAF) interaction with T cells is not cell cycle specific since activity is absorbed by unstimulated cells and by cells at various times after mitogen exposure.     

Lipopolysaccharide interactions with lysozyme differentially affect lipopolysaccharide immunostimulatory activity

The effect of complex formation between lysozyme and lipopolysaccharide (LPS) on the immunostimulatory activities of LPS have been investigated in vitro. Three prototype immunostimulatory activities were examined: B-lymphocyte proliferation, B-lymphocyte differentiation and macrophage production of lymphocyte-activating factor activity. Different effects of lysozyme were noted, depending upon the structure of the LPS, even though previous studies have established that all LPS preparations readily bind lysozyme. Both Re-LPS- and lipid-A-dependent immunostimulatory activities were readily inhibited by lysozyme in a dose-dependent fashion. In contrast, S-LPS and Ra-LPS were unaffected in their immunostimulatory activities by lysozyme. These differences were not the result of quantitative differences in LPS binding of lysozyme, or effects of lysozyme on overall binding of LPS to target cells. These data suggest that the factors which dictate the initial interactions between LPS and lymphoreticular cells may not be identical for all LPS preparations and/or purified lipid A.     

Activation of human T lymphocytes by 12-O-tetradecanoylphorbol-13-acetate: role of accessory cells and interaction with lectins and allogeneic cells

Stimulatory effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on human T lymphocytes have been investigated. TPA was found to stimulate highly purified T cells (obtained by a three-step isolation procedure involving plastic adherence, nylon wool passage and Ig-anti-Ig column passage) in the absence of accessory cells (stimulation index of 5 to 10), whereas phytohemaglutinin (PHA) and concanavalin A (Con A) did not. This response was, however, increased by the addition of autologous adherent cells. Addition of TPA, but not adherent cells, induced T-cell proliferation in response to the nonmitogenic lectin, wheat germ agglutinin (WGA), while both adherent cells and TPA restored T-cell proliferation to mitogenic lectins such as PHA and Con A. Furthermore, TPA greatly increased the mixed-lymphocyte response of purified T cells to otherwise nonstimulating allogeneic cells such as T lymphocytes or tumor cells from some patients with chronic lymphocytic leukemia. These results suggest that TPA can directly act on human T cells to render them reactive to a variety of stimuli.