Nitric oxide production by coelomocytes of Asterias forbesi.

Vertebrate mononuclear phagocytes produce a plethora of molecules involved in host defense. Among the most potent are the reactive oxygen and nitrogen intermediates. Coelomocytes from invertebrates subserve many of the same functions. In order to determine whether invertebrate phagocytes employ reactive nitrogen intermediates, we investigated the effect of various nonspecific stimulators and invertebrate interleukin (IL)-1alpha- and beta-like molecules on nitric oxide (NO) production. Elevated NO release by stimulated coelomocytes was seen after 24 h. Incubation of stimulated coelomocytes in the presence of arginine analogs inhibited NO release. When invertebrate IL-1-like molecules were added to the coelomocytes, they stimulated the release of NO. Western blot analysis using a polyclonal rabbit antiserum to murine NO synthase detected a band at approximately 125 kDa. These data indicate that coelomocytes are capable of producing and releasing NO and that NO is a chemical mediator that has been conserved as a host defense weapon of phagocytes through evolutionary time.

Characterization of an IL-6-like molecule from an echinoderm (Asterias forbesi).

Our previous studies have demonstrated the presence of two of the three vertebrate inflammatory cytokines in invertebrates, namely, interleukin (IL)-1 and tumour necrosis factor (TNF). We studied the coelomic fluid of the echinoderm, Asterias forbesi, to determine whether it contained the third inflammatory cytokine, IL-6. Coelomic fluids were concentrated and then fractionated by a combination of gel sieve chromatography and preparative isoelectric focusing. The M, observed for the invertebrate IL-6-like molecule was approximately 30,000 and the pI was 5.5. Western blot analysis using a polyclonal rabbit antiserum to human IL-6 detected a band at approximately 30,000 Da. The invertebrate IL-6 was inhibited by an antiserum to human IL-6 when used in the B9 assay. Finally, the coelomocytes were found to be capable or releasing the IL-6-like molecule as early as 12 h following stimulation by LPS. These results, together with our other data, show that echinoderms possess correlates of all three vertebrate inflammatory cytokines.

Nonprotein antigens of Borrelia burgdorferi.

Preparative thin-layer chromatograms of chloroform-methanol extracts of Borrelia burgdorferi (B31) sonicates showed four fractions (Rf values of 0.84, 0.81, 0.66 and 0.61) that stained with iodine vapors, orcinol, or phospray, suggesting the presence of lipid-, carbohydrate-, and phosphorus-containing compounds. Sera from patients with Lyme disease showed IgM or IgG antibody reactivity to hydrophobic fractions, designated F1 and F2, in both early and late stages of the disease. Lack of constitutive amino acids in these fractions was shown by protein, amino acid, and peptide detection analyses. Sera from patients with syphilis, systemic lupus erythematosus, and antiphospholipid syndrome reacted to one or both of the fractions. Adsorption of sera from Lyme disease patients with intact B. burgdorferi resulted in significantly different pre- and postadsorption patterns of reactivity by whole cell ELISA, whereas adsorption with F1 and F2 resulted in similar pre- and postadsorption patterns. These fractions may not be present in aqueous whole cell or whole cell lysate ELISA antigens or in immunoblots.

Invertebrate cytokines. III: Invertebrate interleukin-1-like molecules stimulate phagocytosis by tunicate and echinoderm cells.

Phagocytosis is the predominant defense mechanism of invertebrates. Here we show that phagocytosis by echinoderm bladder amoebocytes and tunicate granular amoebocytes can be enhanced by invertebrate interleukin-1-like molecules. As little as 5 ng/ml of invertebrate interleukin-1 produced a significant stimulation of echinoderm and tunicate amoebocyte phagocytosis. Stimulation of phagocytosis by echinoderm interleukin-1-like molecules was inhibited by antisera to vertebrate interleukin-1. Invertebrate interleukin-1 also acted as an opsonin when preincubated with erythrocytes or yeast. In addition, the cellular mechanisms of invertebrate phagocytosis were studied using pharmacologic agents to inhibit echinoderm amoebocyte phagocytosis. The energy requirements and involvement of cellular cytoskeletal elements in phagocytosis by bladder amoebocytes were similar to those of mammalian macrophages. These results demonstrate a role for interleukin-1 in invertebrate host defense mechanisms.

Purification and immunological characterization of a major low-molecular-weight lipoprotein from Borrelia burgdorferi.

Borrelia burgdorferi resembles gram-negative bacteria in having both cellular and outer membranes. We previously showed that a lipopolysaccharide (LPS)-like material could be extracted from B. burgdorferi with phenol-chloroform-petroleum ether (PCP). The PCP extract of B. burgdorferi exhibited biological activity in several in vitro assays (e.g., mitogenicity, pyrogenicity, and cytokine release). These activities suggested the presence of endotoxin. The PCP extract of B. burgdorferi, however, also contained a small amount of protein. Preliminary studies showed that monoclonal antibody prepared against this protein inhibited the mitogenic activity of the PCP extract toward murine spleen cells. The current study was therefore undertaken to characterize this protein and to establish methods for its separation from the LPS. The PCP-extracted protein consisted of a single, low-molecular-weight lipoprotein (apparent M(r), 10,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (SDS-PAGE). By protein analysis, it accounted for 2% of the dry weight of defatted cells, thus making it a major constituent of the spirochete. It was purified from the LPS by initial extraction into 10% Triton X-100 followed by immunoaffinity chromatography in the presence of detergent. On removal of the LPS, the purified lipoprotein formed aggregates stable to SDS-PAGE which were detectable on Western blots (immunoblots) probed with either the monoclonal antibody or polyclonal antiserum. From a plot of the aggregate molecular weight versus aggregate size, a monomer molecular weight of 7,500 was obtained. Indirect immunofluorescence with the monoclonal antibody showed that the lipoprotein was exposed at the surface of the spirochete in only a small percentage of cells. The lipoprotein was present in several strains of B. burgdorferi but absent in other Borrelia spp., treponemes, and gram-negative human pathogens, indicating species specificity.

Invertebrate cytokines II: release of interleukin-1-like molecules from tunicate hemocytes stimulated with zymosan.

Conditioned media and cell extracts from tunicate hemocytes that had been cultured with a variety of antigenic stimulants were tested for interleukin-1 (IL-1)-like activity. Media conditioned by hemocytes stimulated with zymosan significantly increased the proliferative and phagocytic activities of tunicate hemocytes. SDS-PAGE indicated that these biological activities were associated with the adaptive release of tunicate IL1-like (tunicate IL-1) molecules by stimulated hemocytes. The data suggest that tunicate IL1 molecules are expressed in response to selected antigenic stimuli. Such responses may form the basis for nonclonal, inducible immune reactions among phylogenetically primitive animals.

Invertebrate cytokines: tunicate cell proliferation stimulated by an interleukin 1-like molecule.

Tunicate pharyngeal cells include lymphocyte-like cells and granular amoebocytes. They are involved in the specific allogeneic and phagocytic reactions of tunicates. Little is known about their regulation or control. A tunicate interleukin 1 (IL-1)-like fraction is shown to stimulate the proliferation of these cells in vitro. This fraction, designated tunicate IL-1 beta, was isolated from tunicate hemolymph by gel filtration and chromatofocusing chromatography. Mitogenic responses to tunicate IL-1 beta were dose dependent and could be eliminated rapidly by removing tunicate IL-1 beta from culture medium. A second tunicate hemolymph fraction had no effect on tunicate cell proliferation even though it exhibited IL-1-like activity in a mouse thymocyte proliferation assay. Phytohemagglutin did not act synergistically with either fraction. These data are discussed in terms of the function and evolution of IL-1-like molecules in invertebrates.

Cytokines and the pathogenesis of neuroborreliosis: Borrelia burgdorferi induces glioma cells to secrete interleukin-6.

Lyme disease is a multisystemic disease caused by a tickborne spirochete, Borrelia burgdorferi. Neuroborreliosis is characterized by intrathecal production of antibodies specific for the spirochete. This suggests that spirochetal infection of the central nervous system produces conditions that support the maturation of B lymphocytes to immunoglobulin-secreting cells. Interleukin 6 (IL-6) stimulates B cell differentiation into antibody-secreting cells. The present study was undertaken to determine whether B. burgdorferi can stimulate cells of central nervous system origin to secrete IL-6. C6 rat glioma cells cultured with spirochetes induced secretion of IL-6 activity. Peak stimulation was achieved at 24 h with 25 spirochetes per glioma cell. Glioma cells were also stimulated to produce IL-6 by interleukin 1 and tumor necrosis factor. That very few spirochetes are found in Lyme disease patients suggests that biologic amplification factors derived from the organism or the host, or both, are responsible for the pathogenesis of this disease. IL-6 can now be added to the growing list of such factors.

Purification and biochemical characterization of an invertebrate interleukin 1.

Interleukin 1 (IL-1) is a major immunoregulatory protein released by macrophages with many host defense related properties. That IL-1 has been found in the invertebrates attests to its importance in homeostasis. The first step in comparing the vertebrate protein to its invertebrate correlate is to purify the protein to study. We have purified to homogeneity IL-1 isolated from the coelomic fluid of the starfish Asterias forbesi. The IL-1 had isoelectric points of 7.4, 5.4 and 4.8. The pI 4.8 species had a molecular weight of 22,000 and the pI 7.4 and 5.4 species both had Mr of 17,000. Higher Mr forms were also found. These molecules were biologically active in the human melanoma A375 cytotoxicity assay for IL-1, and were also able to stimulate murine dermal fibroblast proliferation, protein synthesis, and PGE2 production. The pI 4.8 and 5.4 forms were purified to homogeneity and the amino acid composition was determined. The pI 4.8 and 5.4 species were purified more than 200-fold to specific activities of 3 x 10(6) and 1 x 10(6) units mg-1, respectively. The pI 7.4 form was isolated and partial N-terminal sequence analysis was performed. The similarities of molecular weight, isoelectric points and biological properties between vertebrate and invertebrate IL-1 show that it is an important, evolutionarily stable host defense molecule.

Primitive cytokines: harbingers of vertebrate defense.

The evolution of the immune system has awarded cytokines a key role as coordinators of the immune response. The exquisite action of cytokines in fine tuning and controlling the response raises the question of whether or not these molecules have been highly conserved through evolution. Gregory Beck and Gail Habicht have isolated and characterized two major cytokines, interleukin 1 and tumor necrosis factor, from invertebrates. In this article, they speculate on the possible function of these molecules and on the existence of other cytokines in invertebrates.

Mediators, initiating the inflammatory response, released in organ culture by full-thickness human skin explants exposed to the irritant, sulfur mustard.

Mediators released from injured human skin that initiate the inflammatory response have not been adequately identified. Organ culture of full-thickness skin explants enables us to do so, because injury to the skin can be made in vitro, eliminating the rapid leakage of serum and infiltration of leukocytes that occur in vivo. In our studies, the military vesicant sulfur mustard (SM) (10 microliters of a 0.01 to 1.0% dilution) was topically applied to injure the epidermis of the explant. Then, the explants were cultured in small Petri dishes, usually for 18 h at 36 degrees C, and the organ-culture fluids were assayed for various inflammatory mediators. We found that the culture fluids from SM-exposed and control explants contained similar amounts of angiotensin-converting enzyme, trypsin-like and chymotrypsin-like proteases, acid phosphatase, beta-glucuronidase, beta-galactosidase, lysozyme, deoxyribonuclease, ribonuclease, interleukin 1, and lactic dehydrogenase. However, the culture fluids from SM-exposed explants contained increased amounts of histamine and plasminogen-activating activity, and often prostaglandin E2, when compared to culture fluids from control explants. After 3 to 4 d in culture, full-thickness human skin explants, when exposed to 0.2% SM (but not when exposed to 1.0% SM), sometimes showed separation of the epidermis and increased collagenase activity (i.e., hydroxyproline release). Thus, histamine (from local mast cells), and prostaglandin E2 and plasminogen-activating activity (probably from both mast cells and epidermal cells) are apparently involved in early mediation of the inflammatory response.

Isolation, preliminary chemical characterization, and biological activity of Borrelia burgdorferi peptidoglycan.

Peptidoglycan (PG), an essential cell wall polymer of most bacteria, has been isolated from many species of spirochetes. Our interest in the host response to Borrelia burgdorferi led us to isolate and characterize its PG. Extracted cells were solubilized with warm 1% SDS followed by digestion with proteases. Amino acid analysis of the isolated PG demonstrated the presence of alanine, glycine, glutamic acid, and ornithine as occurs in other spirochetes and bacteria. Intense erythematous reactions were observed after id injection of 10 micrograms of PG into normal human skin. PG was not mitogenic for human peripheral blood mononuclear cells. Murine splenocytes of certain strains responded to the PG, but only at concentrations of 25 micrograms/ml or more. PG stimulated macrophages to produce interleukin 1. Sixteen micrograms of PG injected iv into rabbits produced biphasic fevers. These observations on the in vitro and in vivo activities associated with the cellular components of the B. burgdorferi spirochete give further insight to how a small number of invading organisms can cause a multisystemic disease such as Lyme disease.

Interaction of C1q with its receptor on cultured cell lines induces an anti-proliferative response.

The receptor for human C1q (C1qR) is expressed on a wide variety of somatic cells, including cultured cell lines of different lineages such as Raji, Daudi, Wil2WT, U937, and Molt4. In this report, we present evidence which shows that culturing of C1qR-expressing cell lines with C1q inhibits their growth. When each of the different cell lines were cultured for 5 days with or without various concentrations (5-50 micrograms/ml) of micro-filtered (0.22 micron) C1q, cell proliferation was inhibited in a dose-dependent manner with maximal inhibition (90%) occurring at a concentration of 50 micrograms/ml at Day 4 of culture. This anti-proliferative effect of C1q was inhibited when 30 micrograms/ml of F(ab')2 anti-C1q was included in the culture with C1q while the antibody alone did not have any effect. The specificity of this interaction was further substantiated by the finding that neither macromolecular C1, or subcomponents C1r and C1s, nor human or murine IgG nor IgM had any inhibitory activity when cultured with these cell lines. That this C1q-induced inhibition of cell growth is mediated by C1qR was deduced from experiments in which (i) culturing of cells in the presence of two IgM monoclonal antibodies II1/D1 and II1/B5, directed against the C1q-binding site of C1qR resulted in the inhibition of cell growth while nonimmune murine IgM did not, and (ii) the collagenous portion of C1q (c-C1q) which contains the intact, C1qR-binding domain was also capable of inhibiting cell proliferation in a manner similar to intact C1q. The effect of C1q was not cytotoxic but cytostatic since the number of dead cells in the C1q-treated cultures was not significantly different than that in the untreated cells (5% vs 4%), a figure which represents the normal wear and tear of tissue culture conditions. On the basis of these findings we propose that the C1qR alone or in conjunction with other cellular factors may function as a molecule which supports cell growth. Upon ligand binding, however, the ligand-receptor interaction may suppress postreceptor events which are necessary for cell proliferation.

Invertebrate cytokines: the phylogenetic emergence of interleukin-1.

Cytokines are polypeptides released by activated vertebrate blood cells which have profound effects on other blood cells and which have hormone-like properties affecting other organ systems as well. In recent years a wide variety of these mediators has been isolated and characterized. Many of these molecules have subsequently been cloned and expressed in E. coli. The tremendous importance of these proteins to host immune and non-specific defense systems along with the striking similarities of their properties among different species suggested to us that cytokines may have been proteins that have been conserved through evolution. Investigations of the evolution of cytokines will help us decipher the complex cellular, humoral and molecular interactions that regulate host defenses. Studies of the invertebrates will shed light on the phylogenetic emergence of these molecules as well.

Isolation of interleukin 1 from joint fluids of patients with Lyme disease.

To investigate the role of interleukin 1 (IL-1) in Lyme arthritis we assayed synovial fluids (SF) for the presence of IL-1 activity. Crude SF from patients with Lyme disease showed IL-1-like activity. Chromatography of joint fluids revealed activity at 15-20,000 daltons. Two populations of cells were grown, which produced significant IL-1 activity when stimulated with the Lyme disease spirochete or its lipopolysaccharide. IL-1 activity from SF or stimulated cells was neutralized with an antihuman IL-1 antibody. Our results suggest IL-1 is important in the pathogenesis of Lyme arthritis, and is similar to other arthritides.

Effect of interleukin 1 on the induction and maintenance of B cell tolerance in vitro.

The effects of interleukin 1 (IL-1) on induction and maintenance of immune tolerance in vitro have been studied by using splenocytes from C57BL/6 male mice. B cell tolerance to the hapten trinitrophenol (TNP) was induced with TNP-human gamma globulin (HGG) and the cells were challenged with TNP-Ficoll. To determine the tolerance (or immunity), antibody concentrations in the supernatant fluids were measured by using a TNP-specific ELISA assay. Partially purified murine IL-1 abrogated tolerance induction, and when it was added at challenge phase, it also abrogated tolerance. In addition, partially purified IL-1 converted TNP-HGG from a tolerogen into an immunogen without any additional exposure to antigen. Similar results were obtained when recombinant human IL-1 alpha was used in place of partially purified natural IL-1. IL-1 is most likely acting directly on B cells rather than through the agency of T cells because purified B cells failed to become tolerant in the presence of IL-1. Studies of IL-1 production by antigen- or tolerogen-stimulated splenocytes or purified B cells showed that only antigen could elicit IL-1 production in these cells. That tolerance abrogation is unique to IL-1 is suggested by studies which show that TNF, IL-2, and INF gamma, alone, in combination with each other, or in combination with subeffective concentrations of IL-1 failed to effect tolerance abrogation.

Adult Ixodes dammini on rabbits: development of acute inflammation in the skin and immune responses to salivary gland, midgut, and spirochetal components.

Rabbits exposed to female Ixodes dammini (both uninfected and infected with Borrelia burgdorferi) or injected with B. burgdorferi showed an acute inflammatory response in the skin. Granulocytes and monocyte-histiocytes were the predominant infiltrating cells. Spirochetes were detected in the tick feeding cavities in the deep dermis. The inflammatory process was accompanied by polyclonal antibody responses to tick salivary gland components. Western blots showed that immune rabbit serum reacted with proteins of molecular masses of 8, 24, and 36-41 kilodaltons in both unengorged and engorged tick salivary glands. Additional reacting bands in the immunoblot of the engorged salivary gland indicated that new antigenic components of the salivary gland are synthesized during engorgement. Rabbits did not produce antibodies to tick midgut components. Murine monoclonal antibody 11G1 detected outer surface protein A of B. burgdorferi in immunoblots of midguts from unengorged ticks, faintly in engorged salivary gland, and seldomly in unengorged salivary gland, findings suggesting that the spirochete is transmitted to the host via tick saliva during the later stages of feeding.

Characterization of interleukin-1 activity in tunicates.

1. Eight North American species of tunicates were examined for the presence of interleukin-1 (IL-1) like activity. 2. The tunicates studied produce molecules with readily detectable lymphocyte activation factor (LAF) activity. 3. G50 column chromatography separated molecular species that were directly mitogenic for thymocytes (mol. wt greater than 50,000) from those that were comitogenic in an IL-1 assay (mol. wt 20,000). 4. Tunicate fractions with LAF activity induced increased vascular permeability in rabbit skin. 5. Tunicate LAF activity was neutralized by polyclonal anti-human IL-1 antisera. 6. These data further support the conclusion that IL-1 is an ancient and functionally conserved molecule.