Influence of Lactobacillus on the migration activity of macrophages

Influence of Lactobacillus on the migration activity of intact macrophages during their cultivation with serum and supernatant of cells from Peyer's patches and spleen that were obtained from mice of CBA line orally sensibilized with lactobacteria was studied. Data about stimulation of production of factor inhibiting migration of macrophages by immunocompetent cells and development of delayed-hypersensivity reaction are obtained. Mechanisms of complex influence of lactobacteria on cell factors of innate and adaptive immunity are discussed.     

Role of lymphokines in regulation of macrophage differentiation

The regulatory mechanism of guinea pig lymphokines was investigated in regard to differentiation of myeloid cells to macrophages. The Ml-cell line, established from a myeloid leukemia of an SL-strain mouse, was induced to differentiate in vitro into mature macrophages possessing Fc receptors and the ability to phagocytize latex particles by treatment with crude lymphokines. Both concanavalin A- and antigen-induced lymphokines showed the differentiation-inducing factor (D factor) activity. However, macrophage migration inhibitory factor/ macrophage activation factor (MIF/MAF) purified by an immunoadsorbent column with anti-MIF antibody had no such an activity. The D-factor activity was detected in the lymphokine preparation that was not retained on the immunoadsorbent column. In contrast, colony-stimulating factor (CSF) was adsorbed to the immunoadsorbent column, and could be recovered in the purified MIF/MAF preparation. These findings suggest that the molecular entity of D factor is distinct from MIF/ MAF and CSF. A culture supernatant of guinea pig peritoneal macrophages activated with MIF/ MAF (CSF) exhibited strong D-factor activity. However, the supernatant possessed rather reduced CSF activity as compared to that of the original MIF/MAF (CSF) preparation. Thus, MIF/MAF may play an important role in macrophage differentiation by regulating the production of D factor or CSF from macrophages.     

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity II. Analysis of the macrophage-replacing activity of the LPS-stimulated macrophage culture supernatant in the antigenic activation of purified immune lymphocytes

A requirement for the cooperation of macrophages (adherent cell population) in the process of the antigenic activation of immune lymphocytes for the production of migration inhibitory factor (MIF) has been demonstrated previously. It was found, in the present study, that the culture supernatant of peritoneal macrophages, which had been pulse-stimulated with a bacterial lipopolysaccharide (LPS), could be substituted for live macrophages. Fractionation of the supernatant by gel filtration revealed its activity in the fraction of 15,000–100,000 MW and the activity was completely abolished by heat treatment at 85°C for 30 min. These results distinguished the nature of the active component from that of LPS which was found to be present in a trace amount in the supernatant and strongly suggested the presence of a factor(s) derived from macrophages which effects, in some way, the process of the antigenic activation of immune lymphocytes. Our experiment showed that the cooperating function of macrophages was inhibited by the treatment of macrophages with vinblastine. It may be that vinblastine affects the release of an active factor(s) from macrophages although other possibilities remain that the inhibition resulted from its effects on other functioning processes of macrophages.     

Complement proteins and macrophages. 1. Quantitative estimation of factor B produced by mouse peritoneal macrophages

Cobra venom factor was used for the detection of factor B synthesized by mouse peritoneal macrophages. This method was shown to be specific for factor B assay by neutralization by antimouse factor B antibody. The amount of factor B in the culture supernatant, assessed by this method, was found to be dependent on the medium used for cultivation of macrophages. The addition of 25% L cell-conditioned medium to minimal essential medium (LCM-MEM) enhanced the production of factor B and also of lysozyme. Kinetic analysis in LCM-MEM showed that factor B produced by 6 x 10(4) cells/cm2 increased up to 72 hr and reached a plateau at 96 hr. The amounts of factor B and lysozyme produced in LCM-MEM depended upon the number of macrophages. Production of factor B was completely inhibited by 1 microgram of cycloheximide per ml and was restored by its removal.     

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. III. A soluble factor released from LPS-stimulated peritoneal adherent cells effective in antigenic activation of sensitized lymphocytes.

Antigen-induced production of migration inhibitory factor (MIF) by sensitized lymphocytes requires macrophages to effectively stimulate lymphocytes with soluble antigen in vitro. The present study showed that macrophage-depleted lymphocytes of sensitized guinea pigs could be activated with antigens when the culture supernatant of peritoneal adherent cells pulse-stimulated with a macromolecular fraction of bacterial lipopolysaccharide (LPS) was added to the lymphocyte culture. The apparent macrophage-replacing activity was found in the fraction which emerged slightly ahead of serum albumin upon gel filtration of the culture supernatant, and the activity was shown to be destroyed by heating at 65 °C for 30 min or by trypsin digestion. These results appeared to show that the activity was due to a protein component, most probably released from macrophages. Two-step culture experiments revealed that the soluble factor should be present in the early stage of the culture to activate the macrophage-depleted immune lymphocytes with antigen, as well as in the later stage when the presence of antigen in the medium is no longer required. Furthermore, the factor was shown to act in the activation of a T-cell-enriched fraction of immune lymphocytes. The factor appeared to be playing some essential role in making an antigenic stimulus effective for the activation of immune lymphocytes.     

Upregulation of tumor necrosis factor-alpha expression by trans10-cis12 conjugated linoleic acid enhances phagocytosis of RAW macrophages via a peroxisome proliferator-activated receptor gamma-dependent pathway

The aim of this study was to examine whether tumor necrosis factor (TNF)-alpha expression in the phagocytic activity of RAW macrophages by trans10-cis12 (10t-12c) conjugated linoleic acid (CLA) is associated with peroxisome proliferator-activated receptor gamma (PPARgamma) activation. 10t-12c CLA induced the TNF-alpha expression in RAW macrophages. Phagocytic activity of naive RAW macrophages was increased either by recombinant mouse (rm) TNF-alpha or by culture supernatant from 10t-12c CLA-treated RAW macrophages. This phagocytic activity was inhibited by addition of anti-rmTNF-alpha polyclonal antibody (pAb). 10t-12c CLA also increased the level of PPARgamma protein and mRNA in RAW macrophages. When naive RAW macrophages were incubated with the culture supernatant from RAW macrophages treated with 10t-12c CLA plus GW 9662, a PPARgamma antagonist, their phagocytic activity was significantly inhibited. In addition, GW 9662 antagonized the effect of 10t-12c CLA in stimulating TNF-alpha expression. These results suggest that 10t-12c CLA modulates the phagocytic activity of RAW macrophages by upregulating TNF-alpha expression via a PPARgamma-dependent pathway.     

Migration of putative progenitor T cells in response to thymus-derived chemotactic factors

Progenitor T cells reach the thymus through the circulation from hematopoietic organs and then migrate toward the site of differentiation in the thymus. The mechanism that regulates such intrathymic migration is not well understood. In order to clarify this mechanism, in vitro chemotactic activity for murine thymocytes was assayed in the extracts and culture supernatants of thymic tissue elements. A potent thymocyte chemotactic activity was found in the extract and culture supernatant from Ig-, Ia- thymic stromal cells. Peanut agglutinin-positive (PNA+1), Thy 1+, TL-, Lyt 1+2-, L3T4- thymocytes, Ig-, Thy 1- bone marrow cells, and mononuclear cells of spleen and peripheral blood, but neither B cells nor lymph node cells, were chemotactically attracted by the factor(s). The chemotactic activity was found in none of the following materials tested: the extract and culture supernatant of thymocytes, culture supernatant of lymph node stromal cells, normal mouse serum, and zymosan-activated serum. The chemotactic activity was found in three molecular fractions by gel chromatography. The activity in all three fractions was destroyed by trypsin digestion or by heating at 56 degrees C for 30 min. These results suggest that Ig-, Ia- thymic stromal cells but not thymocytes secrete a chemotactic factor(s) for progenitor T cells with three molecular species. The factor is considered to play an important role in the migration of intrathymic progenitor T cells into the site of differentiation.     

Tumor necrosis factor alpha (TNFalpha)-like factor produced by macrophages in rainbow trout, Oncorhynchus mykiss

The presence of a Tumor Necrosis Factor alpha (TNFalpha)-like molecule has been suggested in fish by biological assays and biological and antigenic cross-reactivities with human TNFalpha. In the present study, whether rainbow trout macrophages produce TNFalpha was examined. Murine recombinant TNFalpha (m-rTNFalpha) was used as the standard mammalian TNFalpha. The supernatants were harvested from trout macrophage culture stimulated with lipopolysaccharide (LPS) and then passed through a Polymyxin B column to remove LPS. Results show that trout macrophage culture supernatants exhibit TNF-like activities. The supernatants significantly enhanced neutrophil migration and macrophage respiratory burst activity as assessed by NBT reduction test. The supernatants were also highly cytotoxic to murine L929 cells, which are known to be sensitive to mammalian TNFalpha. The biological activities of TNF in the trout macrophage culture supernatant was determined as 2.6 U ml(-1) in the presence of actinomycin D. This indicates biological cross-reactivity of trout TNFalpha-like factor on mammalian cells. Moreover, these activities were inhibited by a rabbit anti-mTNFalpha antibody. These results suggest that rainbow trout macrophages produce a TNFalpha-like factor that is similar to the mammalian TNFalpha in functions.     

Production of a factor inhibiting tumor cell migration by spleen cells from tumor-bearing mice

A factor inhibiting tumor cell migration was found in the culture supernatants of spleen cells of BALB/c mice transplanted with sarcoma cells 20 days before or injected with killed sarcoma cells 14 days before, when the spleen cells were stimulated in vitro for 2 or 24 hr with a 3 M KCl extract of the sarcoma. This factor did not inhibit the migration of guinea-pig macrophages or of theophylline-pretreated sarcoma cells. When the supernatants were fractioned on Sephadex G-100 columns, active fractions with molecular weights lower than 14,000 were distributed broadly.     

Protein-mediated adhesion of Lactobacillus fermentum strain 737 to mouse stomach squamous epithelium

The mechanism of adhesion of Lactobacillus fermentum strain 737 to mouse stomach squamous epithelium was investigated. Adhesion inhibition tests involving chelators, monosaccharides, periodate and concanavalin A and the use of bacteria grown in the presence of tunicamycin failed to clarify the adhesive mechanism. Washed bacterial cells had reduced adhesive capacity, except in the presence of spent broth culture supernatant fraction or cell washings. Spent culture supernatant fractions of erythrosine-supplemented broth did not enhance adhesion of washed cells. The adhesion-promoting factor(s) in the spent broth culture supernatant fractions and cell washings bound to both bacterial and epithelial cell surfaces, but did not promote adhesion of two other Lactobacillus strains which were not of mouse origin, thereby indicating host specificity for the adhesion-promoting activity. Chemical characteristics of the adhesion-promoting factor were determined by pretreatment of the dialysis retentate of spent broth culture supernatant fractions with proteolytic enzymes, concanavalin A-Sepharose or periodate before the adhesion assay. The adhesin was non-dialysable, pronase-sensitive, heat sensitive at 100 degrees C, had no affinity for concanavalin A-Sepharose and contained no carbohydrate groups active in the adhesion process. The protein profiles of dialysis retentates of spent broth culture supernatant fractions after bacterial growth in the absence and presence of erythrosine were determined by 2-dimensional SDS-PAGE. Gel filtration by HPLC was used for purification of an adhesion-promoting fraction. The host-specific adhesion of L. fermentum strain 737 was mediated by a protein, with an Mr of 12-13000, that was not detectable in cells grown in the presence of erythrosine. A model for the mode of binding of the adhesin to host epithelia and bacterial surfaces is proposed.     

Soluble suppressor of T cell proliferation in primary in vitro response to murine minor histocompatibility antigens

Normal mouse lymphocytes are not capable of mounting a primary cytotoxic T cell response to Mls encoded, non H-2, allodeterminants, although a strong lymphoproliferative response is observed in primary MLR between Mls incompatible cells. In this study it is reported that in the supernatant of primary cultures between AKR macrophages and CBA/H lymphocytes (H-2 identical, incompatible for Mls and other minor antigens) a suppressor of T cell proliferation in MLR is detected. By contrast, a suppressor is not detected in supernatants from primary cultures between BALB/C macrophages and CBA/H lymphocytes (H-2 incompatible, Mls identical), B10.BR macrophages and CBA/H macrophages and CBA/H lymphocytes (syngeneic) suggesting that the production of the suppressor factor occurs only when an Mls incompatibility exists. The suppressive activity of the Mls incompatible culture supernatant upon MLR between incompatible macrophages and lymphocytes is neither antigen specific nor Mls or H-2 restricted, nor is it due to an irreversible toxic effect on T lymphocytes or macrophages. The inhibition of T cell proliferation could be explained by inhibition of IL 2 production, by blocking its union to T cells or by a combination of both effects. Our findings could help explain previous observations that lymphocytes from mice preimmunized with Mls incompatible cells have a depressed proliferative response as well as depressed cytotoxicity against alloantigens.     

Differing macrophage and lymphocyte roles in resistance to Legionella pneumophila infection.

Similar to guinea pig macrophages and human monocytes, macrophages from the peritoneal cavity of thioglycolate pretreated A/J mice are permissive for growth of Legionella pneumophila. In contrast, macrophages from BDF1 mice are not permissive for L. pneumophila. Lymphocytes from A/J and BDF1 mice proliferated in response to Legionella Ag but guinea pig lymphocytes did not. Also, splenocyte cultures from A/J mice treated with either Con A or Legionella vaccine produced supernatants which induced A/J macrophages to restrict Legionella growth, but guinea pig splenocyte culture supernatants obtained after stimulation with L. pneumophila vaccine did not induce Legionella growth restriction activity by guinea pig macrophages. Murine rIFN-gamma but not rIFN-alpha markedly inhibited growth of Legionella in A/J mouse macrophages and monoclonal anti-IFN-gamma antibody neutralized the anti-Legionella activity of culture supernatants from A/J mouse splenocytes responding to Legionella Ag. From these data, IFN-gamma appears to be an important factor in anti-Legionella activity of Ag-activated mouse splenocyte culture supernatants. Cyclosporin A, when given to either A/J or BDF1 mice, reduced the proliferation responses of splenocytes to T cell mitogens and also decreased the IFN production of A/J spleen cells to Legionella Ag. In addition, drug treatment decreased the resistance of A/J mice to Legionella infection as shown by an increase in the number of viable bacteria in the liver. However, injection of drug treated mice with lymphokine-rich splenocyte culture supernatant reconstituted the resistance of these animals. These results suggest an important role for lymphocyte activation and lymphokine production in the resistance of A/J mice to Legionella infection. The greater resistance of BDF1 mice, however, may result from nonpermissive macrophages and responsive lymphocytes. In the case of guinea pigs, susceptibility to Legionella infections may result from both the permissive nature of the macrophages and the relatively unresponsive nature of the lymphocytes in these animals.     

The purification and biological activity of a macrophage-derived factor with interleukin 1-like activities from guinea pigs

A macrophage-derived factor with interleukin 1-like activity was purified from culture supernatant of muramyl dipeptide-stimulated peritoneal exudate macrophages of guinea pigs. Starting with serum-free culture supernatant, the purification was carried out by gel permeation chromatography, affinity chromatography on procion red agarose, removal of carry-over serum proteins by Sepharose-coupled antibodies against bovine serum proteins, anion exchange chromatography and hydrophobic chromatography. The purified sample potentiated the phytohemagglutinin-induced thymidine uptake of thymocytes with a 50% effective concentration of 9.6 X 10(-11) M. The sample showed a single band in polyacrylamide gel electrophoresis, and a 65 kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis by silver staining. A single peak of activity was detected by thymocyte assay at the position corresponding to the stained band in both of the electrophoretic analyses. The purified factor had activities to potentiate the antigenic activation of sensitized T cells for the production of a lymphokine, macrophage migration inhibitory factor, and also the proliferative response of sensitized T cells to antigen. Thus, the 65 kDa factor has activities to modulate various T cell responses in guinea pigs such as interleukin 1 does in other species. The molecular relationship of the 65 kDa macrophage factor to interleukin 1 remains to be determined.     

Molecular regulation of MHC class III (C4 and factor B) gene expression in mouse peritoneal macrophages

To study the molecular regulation of C4 and factor B synthesis in mouse peritoneal macrophages, mouse C4 cDNA clones isolated from an H-2d haplotype liver cDNA library, and a previously described mouse factor B cDNA clone, pBmB2 (9), were used to assess quantitative and qualitative differences in C4 and factor B mRNA in resident and elicited cells. The C4 clones that were isolated, pBmS2 (1 Kb) and pBmS10 (0.9 Kb), overlap and together span a 1.5 Kb coding region of mouse pro-C4, extending from the alpha-chain through the gamma-chain; four nucleotide substitutions are evident in comparing 316 bp of the sequence of clone pBmS10 to that of a previously described mouse C4 clone, pMLC4/w7-2 (23). By using these probes, Northern blot analysis of total cellular RNA revealed similar C4 mRNA levels in resident peritoneal macrophages from high-C4 (B10.A) and low-C4 (C3HeB) strains. Pulse and pulse-chase studies of C4 and factor B synthesis were performed on resident, starch-elicited, and thioglycollate-elicited peritoneal macrophages at two culture time periods, 0 to 9 and 24 to 33 hr, and total cellular RNA was isolated from each population at 4.5 and 28.5 hr of culture for Northern blot analysis of C4 and factor B mRNA content. The data demonstrate that as previously reported, C4 production decreases in elicited compared with resident macrophages and decreases with time in culture; however, factor B synthesis does not differ among resident and elicited cells and it increases with time in culture. The variations in C4 and factor B production by mouse peritoneal macrophages are not associated with alterations in C4 and factor B protein processing, catabolism, or secretion; rather, they are a function of differences in net amounts of C4 and factor B mRNA. These data provide direct evidence that the regulation of expression of these class III MHC genes in mouse peritoneal macrophages is a pretranslational event.     

Priming and triggering of tumoricidal and schistosomulicidal macrophages by two sequential lymphokine signals: interferon-gamma and macrophage cytotoxicity inducing factor 2

We describe a new lymphokine activity, macrophage cytotoxicity inducing factor 2 (MCIF2), in the T cell mitogen-induced supernatant of a murine T cell clone in long-term culture. MCIF2 has the following properties: it elutes from a Sephadex G-100 column in three m.w. forms (10, 34, and 100 KD); it is acid labile (pH 2 to 4) and heat sensitive (80 min at 56 degrees C); it is not constitutively secreted, coexists in the same supernatant with immune interferon (IFN-gamma), and synergizes with IFN-gamma for induction of tumoricidal and schistosomulicidal resident peritoneal mouse macrophages. We uncoupled this synergy and show that IFN-gamma serves as the first ("priming") and MCIF2 as the second ("triggering") signal for macrophage activation. Application of the lymphokines in the reverse order was ineffective. These data demonstrate a two-step mechanism of macrophage activation.     

Regulation of lymphocyte motility by macrophages: characterization of a lymphocyte migration inhibitory factor derived from a macrophage-like cell line

An inhibitory factor on lymphocyte migration was detected using a capillary random migration assay in the culture supernatant of peritoneal exudate macrophages cultured at concentrations greater than 8 x 10(6) cells/ml. After examining different macrophage-like cell lines, J774A.1 cells were found to produce this inhibitory factor, which was termed lymphocyte migration inhibitory factor (LMIF). The inhibitory effect of LMIF on the migration of spleen lymphocytes, thymocytes, and bone marrow cells was determined. The migration of thymocytes was more sensitive to LMIF than was the migration of spleen lymphocytes and bone marrow cells. Interestingly, when the effect of LMIF was tested on the migration of spleen T cells and B cells, T cells were more sensitive than B cells. When the thymocytes were separated by peanut agglutinin into mature and immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes to the effect of LMIF, suggesting that the greatest effect of LMIF was on the migration of mature T cells. Partial purification of LMIF by ion-exchange and gel-filtration chromatography revealed that it is approximately 14,000 in molecular weight and could exist in either monomeric or dimeric forms. The possible role of this factor in an immune response is discussed.     

Models for the study of the toxic action of Legionella pneumophila

L. pneumophila virulent culture and the filtrate of this culture disintegrated with ultrasound were shown to be toxic for guinea-pig peritoneal macrophages in vitro and for AKR mice. The virulent culture of this infective agent and the filtrate of the supernatant fluid obtained from the washings of its virulent culture had no toxic effect on a macrophage monolayer culture and on mice. The use of these models for characterizing Legionella intracellular toxic activity, as well as for characterizing Legionella strains isolated from patients and the environment, is proposed.     

Mastocytoma cell migration in vitro: inhibition by MIF-containing supernatants.

Supernatants with macrophage migration inhibition factor (MIF) activity were obtained from cultures of antigen-stimulated guinea pig and human lymphocytes, and from SV40-infected monkey kidney cells. The monkey and human but not guinea pig preparations were effective in inhibiting migration of mastocytoma cells as well as macrophages. This inhibition of migration was not associated with cytotoxicity and was reversible.     

Plasmacytoma growth factor activity of murine granulocyte-macrophage colony-stimulating factor

Mouse plasmacytoma FLOPC21 was adapted to culture in the presence of a mouse Th cell supernatant. A stable factor-dependent cell line was derived from this culture and the factor responsible for its growth was identified as granulocyte-macrophage colony-stimulating factor.     

Soluble factors produced by macrophages/monocytes inhibit lymphokine-activated killer activity in rat splenocyte cultures

In the present study we investigated the inhibition of interleukin-2(IL-2)-induced lymphokine-activated killer (LAK) activity in rat splenocyte cultures in relation to the presence of 2-mercaptoethanol and macrophages/monocytes. The presence of 2-mercaptoethanol is necessary for induction of LAK activity in rat splenocyte cultures. Removal of macrophages/monocytes from rat splenocytes by plastic or nylon-wool adherence, or iron ingestion resulted in LAK induction by IL-2 in the absence of 2-mercaptoethanol. The effect of macrophages/monocytes on LAK activity was also studied in transwell co-cultures. In the absence of 2-mercaptoethanol, the induction of LAK activity was very low in macrophage/monocyte-depleted splenocytes with macrophages/monocytes in the upper compartment of a transwell culture. In contrast, in the presence of 2-mercaptoethanol a high level of LAK activity was induced in these transwell cultures, showing that 2-mercaptoethanol abolished the LAK-inhibiting capacity of macrophages/monocytes. In addition, established LAK activity was strongly inhibited when, after LAK induction, splenocytes were cultured with supernatant of unfractionated splenocytes, which were cultured with IL-2 but in the absence of 2-mercaptoethanol. Addition of 2-mercaptoethanol abrogated the inhibiting effect of the supernatant completely. These experiments demonstrate that rat macrophages/monocytes produce 2-mercaptoethanolsensitive soluble LAK-inhibiting factors. Ultrafiltration of conditioned culture medium of macrophages/monocytes revealed the presence of LAK-inhibiting factors larger than 10 kDa. We concluded that 2-mercaptoethanol-sensitive soluble factors produced by macrophages/monocytes determine the level of LAK induction in rat splenocyte cultures.