The phylogenetic structure of the genus Acinetobacter

Abstract A N-acetyl-D-galactosamine (GalNAc) specific bacterial lectin-like substance from Eikenella corrodens 1073 (EcLS) was found to have potent mitogenic activity when cultured with splenocytes from BALB/c mice. The results indicated that B lymphocytes are the major cell type responding to EcLS. The mitogenic activity of EcLS was dose-dependent, and the optimal concentration was around 5 μg/ml. The mitogenic activity did not appear to be due to a bacterial endotoxin, as GalNAc inhibited the mitogenic activity of EcLS, but did not inhibit the activity of lipopolysaccharide isolated from E. corrodens . EcLS stimulated murine B lymphocytes not only to proliferate, but also to differentiate into antibody-secreting cells, as demonstrated by the production of immunoglobulin by B lymphocytes stimulated with EcLS. These findings suggest that EcLS is a novel lectin that not only induces B lymphocyte proliferation, but also differentiation.     

Time-dependent inhibition of tuberculin-induced lymphocyte DNA synthesis by a serine protease inhibitor

Diisopropylfluorophosphate (DFP), a group-specific irreversible inhibitor of serine proteases, has been shown to exert time-dependent inhibition of DNA synthesis of lymphocytes stimulated by three different B lymphocyte mitogens: purified protein derivative of tuberculin (PPD), endotoxin protein (EP), and lipopolysaccharide (LPS). The time-dependent inhibition profile found in B lymphocytes is absent in concanavalin A (Con A)-stimulated T lymphocytes. Structural analogs of DFP, which have lost the phosphorylating ability, are not inhibitory. Inhibition of DNA synthesis by DFP is reversible in the first 8 hr of mitogenic stimulation. Maximal and irreversible inhibition by DFP occurs around the 16th hour of stimulation. These data support the postulate that a mitogenesis-linked protease, or proteases, in B lymphocytes is absent in the resting cells but is made available several hours before the initiation of DNA synthesis in the late G1 phase of the cell cycle.     

Altered surfactant protein A gene expression and protein metabolism associated with repeat exposure to inhaled endotoxin

Chronically inhaled endotoxin, which is ubiquitous in many occupational and domestic environments, can adversely affect the respiratory system resulting in an inflammatory response and decreased lung function. Surfactant-associated protein A (SP-A) is part of the lung innate immune system and may attenuate the inflammatory response in various types of lung injury. Using a murine model to mimic occupational exposures to endotoxin, we hypothesized that SP-A gene expression and protein would be elevated in response to repeat exposure to inhaled grain dust and to purified lipopolysaccharide (LPS). Our results demonstrate that repeat exposure to inhaled endotoxin, either in the form of grain dust or purified LPS, results in increased whole lung SP-A gene expression and type II alveolar epithelial cell hyperplasia, whereas SP-A protein levels in lung lavage fluid are decreased. Furthermore, these alterations in SP-A gene activity and protein metabolism are dependent on an intact endotoxin signaling system.     

Endotoxin contamination of ovalbumin suppresses murine immunologic responses and development of airway hyper-reactivity

The reversible airway hyper-reactivity (AHR) of asthma is modeled by sensitizing and challenging mice with aerosolized ovalbumin. However, the C57BL/6 murine strain does not display the large increase in circulating IgG and IgE antibodies found in human atopy and asthma. We found that commercial ovalbumin was contaminated with lipopolysaccharide (LPS) in amounts sufficient to fully activate endothelial cells in an in vitro assay of the first step of inflammation. Desensitization of TLR4 by LPS pretreatment suppressed the inflammatory effect of ovalbumin. The presence of LPS was occult, because it does not require serum presentation and, like the LPS of Salmonella minnesota, was not suppressed by polymyxin B. Purified ovalbumin did not activate endothelial cells in vitro; however, endotoxin-free ovalbumin was far more effective than commercial material in stimulating IgE production and respiratory dysfunction in a C57BL/6 murine model of AHR. Moreover, endotoxin-free ovalbumin induced lung inflammation with alveolar enlargement and destruction in a histologic pattern that differed from the changes caused by commercial, endotoxin-contaminated ovalbumin. Reconstitution of purified ovalbumin with S. minnesota LPS decreased lung inflammation, decreased changes in lung function, and suppressed anti-ovalbumin antibody production. We conclude endotoxin contaminates ovalbumin preparations and that endotoxin co-administration with the ovalbumin antigen creates a state of tolerance in a murine model of AHR. Co-exposure to endotoxin and antigen occurs in humans through organic dusts, so murine models of AHR may reflect the clinical situation, but models based on commercial ovalbumin do not accurately reflect the effect of protein antigen alone on animal physiology.     

Activation of protein kinase C from B lymphocytes by lipid A

The effect of lipid A, a residue of the lipopolysaccharide molecule, on protein kinase C from B lymphocytes has been studied. Lipid A activates and promotes the translocation of protein kinase C from the soluble to the particulate membrane fraction in a cell-free system reconstituted with purified enzyme and membranes isolated from B lymphocytes. These results demonstrate that the activating effect of lipopolysaccharide on protein kinase C from B cells is due to the lipid moieties of this molecule.     

Inhibitors of protein kinase C block activation of B lymphocytes by bacterial lipopolysaccharide

Activation of murine splenic B lymphocytes (B cells) by bacterial lipopolysaccharide (LPS) was found to be markedly inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), two potent inhibitors of protein kinases. The higher sensitivity of DNA synthesis, RNA synthesis and protein N-glycosylation activity to H-7, relative to H-8, strongly supports the proposal that protein kinase C plays a critical role in the activation of B cells. A kinetic study on the time of addition of H-7 indicated that protein kinase C promoted the activation process continuously after the addition of LPS.     

Endotoxin-induced T lymphocyte proliferation

The lymphocyte response to endotoxin (LPS) has been attributed largely to the action of this agent as a polyclonal activator of B lymphocytes. In this study we found that a cloned murine interleukin 2-dependent cytotoxic T cell line, CT 6, proliferates in response to LPS, thus providing the first evidence that T cells can be stimulated directly by LPS. The response was dose and time dependent and was blocked by polymyxin B, an inhibitor of LPS-induced mitogenesis. The fact that this is a cloned T cell line, free of other potentially contaminating lymphoid cell types, precludes the possibility that this proliferation is due to contaminating B lymphocytes or is mediated by macrophage-derived products such as interleukin 1. Moreover, highly purified splenic T lymphocyte populations (purified by negative/positive selection or by a rigorous column purification procedure) contain a small subpopulation (approximately 3%) of T cells that proliferate in response to LPS. This population is missing in the endotoxin-hyporesponsive C3H/HeJ mouse. As was observed in the CT 6 line, proliferation of splenic T cells in response to LPS was inhibited by polymyxin B. Furthermore, treatment of LPS-stimulated T cells with anti-T cell antibodies plus complement blocks the uptake of 3H-thymidine by these cultures. Exogenous interleukin 1 failed to stimulate the T cell cultures comparably to LPS and therefore cannot account for the degree of stimulation observed. These findings support and extend previous findings that suggested a role for an endotoxin-sensitive T cell population in the induction of certain responses, such as LPS-induced adjuvanticity of the lymphocyte-dependent LPS induction of macrophage procoagulant activity.     

Ly-4.2: a cell membrane alloantigen of murine B lymphocytes. III. In vitro studies

The alloantigenic specificity Ly-4.2 is present on a restricted population of murine lymphocytes which have previously been shown to have some of the properties generally ascribed to B lymphocytes, both with regard to distribution and function. In the study reported herein, the effect of anti-Ly-4.2 and anti-Thy-1.2 (θ) antisera have been examined in various in vitro systems. (a) T cell-mediated lysis of ⁵¹Cr-labeled P815-X2 target cells by immune allogeneic peritoneal exudate cells is inhibited by anti-Thy-1.2, but not affected by the anti-B (Ly-4.2) reagent. (b) Antibody-dependent lymphocyte-mediated lysis of ⁵¹Cr-labeled sheep red cells was only slightly inhibited by anti-Ly-4.2 and anti-Ig antisera, and not at all by anti-Thy-1.2 antisera, indicating that this type of cell lysis is mediated by neither T (Thy-l⁺) nor B (Ly-4.2⁺,Ig⁺) cells. (c) The response of lymph node lymphocytes to various mitogens was affected thus: PHA, completely inhibited by anti-Thy-1.2 but not by anti-Ly-4.2; Con A, largely inhibited by anti-Thy-1.2, and slightly by anti-Ly-4.2; PWM (pokeweed mitogen), partially inhibited by both antisera; E. coli endotoxin lipopolysaccharide, greatly inhibited by anti-Ly-4.2 but only slightly by anti-Thy-1.2. The findings demonstrate that anti-Thy-1.2 reacts predominantly with T cells and anti-Ly-4.2 with B cells.     

B lymphocyte receptors and polyphosphoinositide degradation

Resting B lymphocytes can be activated and induced to proliferate by antibodies against their antigen receptors (anti-lg). We demonstrate an early increase in the level of [3H]inositol trisphosphate in [3H]inositol-labeled murine B cells, which suggests breakdown of phosphatidylinositol bisphosphate by phospholipase C. In line with this, the level of [3H]1,2-diacylglycerol was also elevated after incubation of [3H]arachidonic-acid-labeled B cells with anti-Ig. Anti-lg also caused a rapid increase in the level of cytosolic Ca2+ in B cells. In contrast, two other polyclonal B cell activators, lipopolysaccharide and phorbol myristate acetate, failed to induce any of these effects. Our results suggest that anti-lg may induce B cell growth via phosphoinositide degradation and Ca2+ mobilization, and that phorbol myristate acetate, and possibly lipopolysaccharide, bypass these initial events.     

Differentiation of B lymphocytes in C3H/HeJ mice: the induction of Ia antigens by lipopolysaccharide.

The lipid A moiety of bacterial lipopolysaccharide (LPS) elicits several types of responses in murine B lymphocytes. First, lipid A induces the nonproliferative expression of cell surface antigens in more immature cell types. Second, lipid A induces a mitogenic response in more mature B cell types. Lipid A induces the expression of Ia antigens on bone marrow cells from C3H/DiSn but not C3H/HeJ mice. The Ia-inducible cells possess surface immunoglobulin. Agents that elevate intracellular levels of adenosine 3',5'-cyclic monophosphate (cyclic AMP) induce the appearance of Ia antigens on B lymphocytes from both C3H/HeJ and C3H/DiSn mice, suggesting that lipid A exerts its inductive effects by increasing cyclic AMP levels in cells. In contrast to what is observed by using other strains of mice, mature B lymphocytes from C3H/HeJ mice do not support a mitogenic response to lipid A. The subpopulation of B lymphocytes in C3H/HeJ mice that normally respond mitogenically to LPS not only appear to lack an LPS-response mechanism utilized in the mitogenic pathway, but they lack the LPS-response pathway of the immature B cell types. A lipid A-bound protein (LAP) induces both the expression of Ia and a mitogenic response in the different subpopulations of B lymphocytes from C3H/HeJ and C3H/DiSn mice. The genetic defect in C3H/HeJ mice that limits responses to lipid A may be associated with a receptor that is normally expressed on many different cell types.     

Cloning of a complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homologous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19

The human B1 (CD20) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In order to determine whether this structure is also expressed by murine B cells, cDNA clones that encode the mouse equivalent of the B1 molecule were isolated. The longest murine cDNA clone isolated, pmB1-1, contained a 1.4-kb insert with an 873 base pair open reading frame that encodes a protein of 32 kDa. The predicted mouse B1 protein contains three hydrophobic domains that may span the membrane four times and shares a 73% amino acid sequence homology with the human B1 protein. The pmB1-1 cDNA probe was used to examine mB1 mRNA expression. Northern blot analysis indicated that pmB1-1 hybridized with two mRNA species of 2.3 and 3.0 kb that were expressed only in murine spleen lymphocytes, in B lineage cell lines representing mature B cells, and were weakly expressed in one of two plasmacytoma cell lines. pmB1-1 failed to hybridize with RNA isolated from murine T cell lines, thymus, and nonlymphoid tissues. Southern blot analysis indicated that mB1 was encoded by a single copy gene. In situ hybridization localized the mB1 gene to chromosome 19 band B, a region that also contains the genes that encode the Ly-1, Ly-10, and Ly-12 Ag. These results suggest that only B cells express this heretofore undescribed murine cell-surface protein that is structurally homologous with the membrane-embedded human B1 Ag.     

An anti-endotoxin peptide that generates from the amino-terminal domain of complement regulatory protein C1 inhibitor

C1 inhibitor (C1INH), a complement regulatory protein, prevents endotoxin shock via a direct interaction of the amino-terminal domain with gram-negative bacterial lipopolysaccharide (LPS). Importantly, the cleaved, inactive C1INH still is an anti-endotoxin effector indicating the anti-endotoxin peptide that generates from the amino-terminal domain of C1INH. In this study, we first identified that a cleaved fragment within the major part of the amino-terminal domain in in vitro proteolytic analysis of C1INH had an ability to bind to LPS. We synthesized several peptides overlapping the C1INH cleaved fragment. Among these synthetic peptides, a 13-mer derivative peptide at position from 18 to 30, named N2((18-30)), exhibited the most powerful anti-endotoxin activity in vitro, enlightening that it was most strong at binding to LPS, inhibiting the interaction of LPS with LPS-binding protein (LBP), blocking LPS binding to CD14(+) cells, and suppressing production of tumor necrosis factor (TNF)-alpha by murine macrophages, RAW 264.7. In the murine endotoxin shock model, the peptide N2((18-30)) protected mice from LPS-induced lethal septic shock by inhibiting macrophage activation. These data indicate that the peptide N2((18-30)) derived from the amino-terminal region of C1INH is anti-endotoxin.     

A B Lymphocyte Mitogen Extracted from a Fungus Peziza vesiculosa1

A water-soluble mitogen was extracted with hot-water from the fruiting bodies of a fungus, Peziza vesiculosa, collected in the wild. The active substance, named vesiculogen, was able to stimulate selectively murine B cells because mitogenic activity was observed in the spleen cell cultures of congenitally athymic nude mice, but not in the thymus cell cultures. The possibility that the mitogenicity of vesiculogen was due to lipopolysaccharide was denied completely by the following evidence: 1) lipopolysaccharide in vesiculogen was undetectable (less than 0.001% in the Limulus test), 2) vesiculogen was able to stimulate strongly DNA synthesis of spleen cells from C3H/HeJ mice, and 3) the mitogenic activity of vesiculogen was not inhibited by polymyxin B. Vesiculogen increased antigen-nonspecifically the number of direct plaque forming cells to sheep erythrocytes, horse erythrocytes, and trinitrophenylated-horse erythrocytes. This result shows that vesiculogen acts as a polyclonal B cell activator on murine spleen cells.     

Characterization of a spontaneous murine B cell leukemia (BCL1). II. Tumor cell proliferation and IgM secretion after stimulation by LPS.

A spontaneous BALB/c B lymphocyte leukemia could be stimulated in vitro by the polyclonal B cell activator lipopolysaccharide (LPS) and the conditions for activation were studied. Spleen cells or peripheral blood lymphocytes from tumor-bearing animals responded by increased DNA synthesis and the peak of activation occurred earlier than with normal mouse spleen cells. Tumor cells harvested from the spleen, but not from the peripheral blood, could be induced by LPS to secrete IgM. Direct demonstration that the response was due to tumor cell activation and not that of contaminating normal B lymphocytes was provided by karyotype analysis and by immunoprecipitation, which showed the restriction of light chains on secreted IgM molecules to the lambda isotype.     

Effect of platinum compounds on murine lymphocyte mitogenesis.

Polyclonal activation of T and B lymphocytes by concanavalin A and lipopolysaccharide mitogens, respectively, was used to study the effect of iv injection of the following platinum compounds: cis-dichlorodiammine platinum, dichloro 1,2 benzenediamine N,N' platinum, cis-dichlorobis(cyclohexylamine) platinum, and cis-dichlorobis(cyclopentylamine) platinum, on murine T and B splenic lymphocytes. The results indicated that platinum compounds were more toxic to T-lymphocyte function than to B-lymphocyte function. These results were from both single-dose-time-course analysis and dose-response studies.     

Recognition of EBV plasma membrane protein expressed on murine cells after gene transfer

The immune response to B lymphocytes infected with Epstein-Barr virus (EBV) prevents their overgrowth in normal humans. A murine model is now described for analyzing the T cell immune response to Epstein-Barr virus genes expressed in murine lymphoblasts by gene transfer. In mice, a 60,000 dalton virus-encoded protein characteristically found in the plasma membrane of latently infected human lymphocytes readily induces both proliferative and cytolytic T lymphocytes specific for both the EBV protein and murine major histocompatibility proteins. Longterm cultures of L3T4+ cells, some of which were cytolytic, were found to be restricted by H-2I-Ed and the latent membrane protein. Similarly, Lyt-2+ cells were cytolytic and were restricted by H-2Ld and the lymphocyte membrane protein gene product. The similarity in murine and human effector cell responses suggests that this is a useful experimental model, and the EBV latent infection membrane protein may be an important antigen in the immune restriction of growth transformed latently infected lymphocytes.     

Characterization of a spontaneous murine B cell leukemia (BCL1). I. Cell surface expression of IgM, IgD, Ia, and FcR.

The surface marker expression of a spontaneous B lymphocyte leukemia discovered in a BALB/c mouse (BCL1) was examined and found to include a subset of markers known to occur on normal B lymphocytes. The tumor cells bore surface Ig that included both mu- and delta-chains associated with the lambda light chain. Alloantigens coded for within the murine MHC, including H-2D, H-2K, and I-region products, were identified on the tumor cells. Although normal B lymphocytes are thought to express products coded for within both the I-A and I-E subregions, the BCL1 expressed only normal amounts of I-E subregion products. In addition, the H-2 and Ia antigens revealed by 2-dimensional gel electrophoresis exhibited an abnormal pattern of post-translational modifications. The Fc, but not the complement-receptor, was present on the surface of tumor cells. The presence of IgD, Ia antigens, and the responsiveness to lipopolysaccharide (see subsequent paper) have led us to postulate that the BCL1 tumor represents a later differentiative stage than murine B lymphocyte tumors previously described.