Effect of Listeria monocytogenes infection on serum levels of colony-stimulating factor and number of progenitor cells in immune and nonimmune mice.

Studies were performed to determine changes in serum macrophage colony-stimulating factor (M-CSF) levels and the number of macrophage progenitor cells in bone marrow and spleens of nonimmune and immune mice infected with Listeria monocytogenes. Immunity in mice was established by infecting mice 6 weeks before use with a sublethal dose of L. monocytogenes. When challenged with 10(4) L. monocytogenes organisms, immune mice had an early (12 h) peak in M-CSF serum concentrations. Levels remained elevated for 24 h but fell towards normal by 48 h. By contrast, M-CSF levels in nonimmune mice did not rise until 24 h after challenge, remained elevated for 7 days, and returned to normal by 14 days. The number of macrophage progenitor cells in the bone marrow of immune mice rose slightly during infection, whereas the number in nonimmune mice fell significantly by days 4 and 7. Progenitor cells in spleens of immune mice more than doubled during infection; in nonimmune mice, a sixfold increase was noted. These results indicate that important parameters of monocyte production differ in immune and nonimmune mice during listeria infection and suggest a possible mechanism for differences in resistance to infection.     

Cross-linking of immune complexes by human mononuclear phagocytes

Incubation of immune complexes (IC) bound to plastic surfaces with human blood monocytes for 48 hours resulted in the cross-linking of a proportion of antibody molecules. This process was largely inhibited by the addition of sodium azide to the cultures. Cross-linking was defined as the inability of strong chaotropic solutions (3 M MgCl₂ or 5 M guanidine) or acid pH (0.1 N HCl) to solubilize¹²⁵J-labeled rabbit anti-human serum albumin attached to plastic-bound antigen. Addition to the cultures of a suitable hydrogen donor such as catechol (0.5 mM) resulted in a large increase in cross-linking of IC. This process was shown to depend on the presence of viable phagocytic cells because incubation with dead monocytes or with viable T lymphocytes failed to induce cross-linking. Quantitation of rabbit immunoglobulin remaining in the wells by enzyme-linked immunoassy techniques excluded the possibility that the increase in¹²⁵I bound was merely due to a transiodination reaction. Experiments using various oxygen metabolite inhibitors and scavengers indicated that catechol-dependent protein cross-linking depended on the action of hydrogen peroxide and enzyme systems inhibitable by sodium azide, probably monocyte-peroxidase. Superoxide dismutase,¹O₂, and OH · radical scavengers failed to inhibit cross-linking, whereas addition of catalase resulted in almost complete abolition of the process. These observations suggest that catechol-dependent cross-linking of IC may be due to oxidation of catechol to orthoquinone and that this strong oxidant is responsible for nonenzymic chemical action on proteins leading to intermolecular covalent bond formation. Cell-mediated protein cross-Sinking by oxidative mechanisms may be a prominent feature of drug-related reactions and of acute and chronic inflammatory processes in general. The possible mechanisms involved in catechol-dependent and -independent cross-linking of IC by human mononuclear phagocytes are discussed.     

Protection of mice against the intracellular bacterium Listeria monocytogenes by recombinant immune interferon

Immune interferon, available at high specific activity through recombinant DNA technology, is known to activate macrophages to intra- and extracellular cytotoxicity. We now report that murine recombinant IFN-gamma activates macrophages to cytotoxicity also when applied in vivo. Furthermore, recombinant IFN-gamma can protect mice in vivo against the intracellular bacterial pathogen Listeria monocytogenes in a local as well as in a systemic infection model. The role of T lymphocyte-produced lymphokines in acquired resistance to facultative intracellular pathogens and their possible involvement in novel immunotherapy are discussed.     

Ingestion and killing of Listeria monocytogenes by blood and milk phagocytes from mastitic and normal cattle.

Human listeriosis resulting from consumption of listeria-contaminated dairy products is emerging as a significant public health concern. There is a need to understand better the processes involved in the pathogenesis of Listeria monocytogenes-induced bovine mastitis. In the present report, we describe the results of the in vitro interaction of L. monocytogenes with bovine blood and milk leukocytes. Induction of an experimental L. monocytogenes mastitis resulted in a rapid and dramatic increase in neutrophils in the milk of infected cows. Blood neutrophils and mononuclear cells and milk leukocytes from listeria-infected and uninfected cows readily ingested L. monocytogenes in the presence of serum opsonins. These leukocytes also killed a portion of the ingested listeriae. Ingestion of listeriae evoked a vigorous chemiluminescence response by blood neutrophils and a relatively weak response by blood mononuclear cells. Ingestion, killing, and chemiluminescence by milk leukocytes were directly related to the percentage of neutrophils that were present. Blood neutrophils from healthy donor cattle ingested and killed L. monocytogenes when leukocyte-depleted milk and whey from mastitic cows were the sole sources of opsonins, although fewer listeriae were ingested than when normal bovine serum was present. These results indicate that bovine blood and milk phagocytes, like blood and inflammatory phagocytes from other mammalian species, can ingest and kill L. monocytogenes in vitro.     

Impaired function of immune reactivity to Listeria monocytogenes in diet-fed mice.

Mice fed a diet high in cholesterol, lard, and sucrose were shown to exhibit an impairment of specific immunity to Listeria monocytogenes. Whereas titers of L. monocytogenes in livers of normal mice decreased rapidly after 6 days of infection, L. monocytogenes persisted in livers of diet-fed mice. Adoptive transfer experiments indicated that L. monocytogenes-immune spleen cells are generated in diet-fed mice. However, the function of immune spleen cells from donors of either nutritional status was impaired in diet-fed recipients. The results indicate that the site(s) of impairment of specific immunity to L. monocytogenes in diet-fed mice occurs at a stage beyond the generation of immune T-cells.     

Concomitant induction of an inflammatory response and immunosuppression by an extract from Listeria monocytogenes

An immunosuppressive agent (ISA) present in an aqueous extract from Listeria monocytogenes diminished the immune response in vivo to subsequently injected heterologous antigen. Intraperitoneal injection of ISA induced an inflammatory response and activation of the reticuloendothelial system, both of which coincided with the period of immune hyporesponsiveness. Mice treated with ISA exhibited increased accumulation of labelled antigen to phagocytic peritoneal cells but decreased delivery of labelled antigen to the spleen. Both delivery of antigen to spleen and the immune response could be improved by injecting either ISA or antigen or both intravenously, or by increasing the dose of antigen. The response of ISA-treated animals could also be improved by intraperitoneal injection of latex beads, colloidal carbon, or carrageenan shortly (60 min) before immunization. Spleen cells from ISA-treated mice adoptively transferred to irradiated syngeneic recipients were able to mount a normal immune response. These results suggest that ingestion of antigen by the enlarged population of phagocytes in peritoneal cavities of ISA-treated mice prevented antigen delivery to the spleen and was partly responsible for the observed immunosuppression.     

Chronic inflammatory bowel disease--increased plasminogen activator secretion by mononuclear phagocytes.

The secretion of the neutral protease plasminogen activator (PA) by cultured macrophages (M phi) was studied in hospitalized patients suffering from chronic inflammatory bowel disease (IBD). There was markedly enhanced secretion of PA by M phi derived from circulating monocytes of the IBD population (18) compared to an age-matched population (16) which was not afflicted by intestinal disease (P less than 0 . 001). Mean M phi PA activity was greater in the population of 11 Crohn's disease patients (P less than 0 . 01) than in a group of seven ulcerative colitis sufferers (P less than 0 . 05) when compared to the control population. While both the treated and untreated hospitalized IBD populations showed increased M phi PA specific activity, results for the nine untreated patients (5 . 56 +/- 1.14 units/micrograms M phi DNA) were substantially higher than those found in the treated IBD population (2 . 91 +/- 0 . 62 units/micrograms M phi DNA) (P less than 0 . 01). These findings reflect the activity of M phi in IBD and suggest a means by which tissue injury is mediated in these conditions.     

Immunomodulation of GvH reaction in mice by Listeria monocytogenes

In the first part of experiments the GvHR activity of the spleen cell suspensions from normal parental donors (Balb/c) or infected with Listeria monocytogenes was compared. GvHR was examined in (Balb/c X AKR) F1 mice. Full suspensions from these donors or depleted of adherent cells or with addition of macrophages were studied. It has been proved that adherent cells population plays a significant role in the GvHR development. The addition of syngeneic macrophages from F1 hybrids results in a greater augmentation of GvH reactivity of parental lymphocytes. However, the addition of macrophages harvested from F1 hybrids immunized with L. monocytogenes 7 days before, brings about a weaker GvH reaction. Spleen cells of parental donors injected with L. monocytogenes 5-6 days earlier induced stronger GvHR as compared with the splenocytes of normal donors. Similarly, F1 hosts treated with L. monocytogenes 3-5 days before injection of normal donor's spleen cells showed increased GvHR, but those infected 7-9 days before injection of spleen cells, developed only very weak GvHR.     

An ultrastructural study of peritoneal mononuclear phagocytes from Corynebacterium parvum-injected mice.

Peritoneal mononuclear phagocytes harvested from mice 24 h after i.p. injection of C. parvum displayed hypertrophy of the Golgi and smooth endoplasmic reticulum complex with attendant increase in lysosome production. The ingested bacilli were identified within phagolysosomes, within which large myelin figures accumulated. In addition to heterophagic vacuoles, autophagosomes and lipid droplets were observed. These latter two inclusions may reflect a direct or indirect cytopathic effect of C. parvum on the phagocytes.     

Effect of benzydamine on exocytosis and respiratory burst in human neutrophils and mononuclear phagocytes

The effect of benzydamine on stimulus-dependent respiratory burst activity and enzyme release was tested in human neutrophils, monocytes and monocyte-derived macrophages. Establihsed anti-inflammatory compounds, indomethacin, phenylbutazone and bufexamac, were tested for comparison. Care was taken to avoid cytotoxic or cytolytic concentrations of the test compounds, and their effect on release of lactate dehydrogenase was also tested.Release of specific and azurophil granules contents were induced in human neutrophils by A23187, PMA and fMLP with and without cytochalasin B pretreatment. Benzydamine inhibited stimulus-dependent release of vitamin B₁₂-binding proteins, a marker for the specific granules, in a concentration-dependent fashion. By contrast, phenylbutazone and bufexamac were practically inactive. The effect of benzydamine on exocytosis of azurophil granules was tested in cytochalasin B-pretreated neutrophils. Benzydamine, again in contrast to the two reference anti-inflammatory compounds, inhibited release concentration-dependently also under these conditions. The concentration of the compound which inhibited exocytosis by 50% was 30–100 M in normal and 3–10 M in cytochalasin B-treated neutrophils.The effect of benzydamine and reference compounds on the respiratory burst was tested by assaying for superoxide formation in neutrophils and H₂O₂ formation in mononuclear phagocytes. Benzydamine was inactive on neutrophils and inhibited slightly the burst response of monocytes and macrophages. Two reference compounds, bufexamac and phenylbutazone, were generally more active. The strongest inhibitory effect was that of phenylbutazone on fMLP-stimulated cells. Benzydamine lacked activity receptor of formylated chemotactic peptides.The profile of activity of benzydamine shown in these experiments on human phagocytes suggest that this compound may act therapeutically by decreasing the release of enzymes and other granule constitutents from stimulated neutrophils.     

Effect of two feeding formulas on immune responses and mortality in mice challenged with Listeria monocytogenes

Cell-mediated immunity and natural killer cells play an important role against facultative intracellular organisms. The effect of two commercially available tube feeding formulas used for patients with acute or chronic debilitating and life-threatening illnesses was studied in mice challenged with Listeria monocytogenes. C57BL/6 X DBA/2 F1 hybrid mice were given ad libitum access to one of two formulas or to chow. Sixty mice in each of the feeding groups were challenged with 4.8 X 10(3) organisms intraperitoneally. Mortality was significantly less in animals fed Impact, a formula enriched with arginine, RNA and selected fatty acids. This was associated with reduced number of viable organisms in the spleen on day 7 after challenge. There was no difference in the spleen/body weight index between the different groups. Delayed cutaneous hypersensitivity was slightly higher in the Impact group but this was not statistically significant. Natural killer cell activity was significantly higher in the Impact group compared with the other two feeding regimens. These observations suggest that selective manipulation of the composition of tube feeding formulas may have a significant impact on immune responses and on morbidity and mortality following infectious challenge.     

Evidence for involvement of peptidoglycan in the triggering of an oxidative burst by Listeria monocytogenes in phagocytes

We have shown previously that in listeric encephalitis of cattle and rats, nitrotyrosine was produced in microabscesses, implying that both superoxide anion (O(2) (-)) and nitric oxide (NO) are present and react with each other. Evidence of local synthesis of NO by macrophages was provided, but the source of O(2) (-) remained unknown. Here we have examined whether phagocytes exposed to viable and heat-killed Listeria monocytogenes (LMDelta) produce O(2) (-) and, if so, whether this results from direct interaction of phagocytes with the bacterial surface of L. monocytogenes or whether prior opsonization is required. Using lucigenin-enhanced chemiluminescence (LCL) for the measurement of O(2) (-), we show that LMDelta induces an oxidative burst in human neutrophils, monocytes and monocyte-derived macrophages (Mphi). Viability is not required, and opsonization by antibodies and/or complement does not enhance the LCL signal. As Toll-like receptors (TLR) were shown recently to mediate an oxidative burst, TLR agonists representative for pathogen-associated molecular patterns (PAMPs) were tested for their ability to elicit an oxidative burst. These included lipoteichoic acid (LTA), bacterial peptidoglycan (PGN), recombinant flagellin, CpG-containing DNA and double-stranded RNA. Only PGN and flagellin consistently elicited an LCL signal resembling that induced by LMDelta with regard to the kinetics and cell spectrum stimulated. However, flagellin was unlikely to be responsible for the LMDelta-mediated burst, as a flagellin-deficient mutant showed no decrease in LCL. We therefore assume that in LMDelta, core PGN acts as a PAMP and directly induces an oxidative burst in all phagocyte populations. We conclude that in cerebral lesions superoxide anion is generated locally by phagocytes recognizing bacterial PGN.     

Differences in the Rate of Intracellular Killing of Catalase-Negative and Catalase-Positive Listeria monocytogenes by Normal and Interferon-Gamma-Activated Macrophages

Intracellular killing of catalase-positive bacteria by murine resident macrophages requires the presence of extracellular serum, whereas killing of catalase-negative bacteria can occur in the absence of serum. To find out whether the intracellular killing of bacteria by rIFN-γ-activated macrophages also requires serum stimulation, we investigated the handling of ingested catalase-negative and -positive Listeria monocytogenes by peritoneal macrophages of normal Swiss mice and mice injected i. p. with I × 10⁴U rIFN-γ 18 h earlier. In the absence of extracellular serum. rlFN-γ-activated macrophages killed ingested catalase-negative Listeria more efficiently (P<0.01) than normal resident macrophages. Maximal killing of catalase-negative bacteria by rlFN-γ-activated macrophages required an extracellular serum concentration of only 1.0 to 2.5% compared with the 10% needed by normal macrophages. No differences were observed in the rates of intracellular killing of catalase-positive Listeria by rlFN-γ-activated and normal resident macrophages: both populations of macrophages required 10% extracellular serum for maximal killing of these bacteria, and killing was minimal in the absence of scrum. The rIFN-γ-activated macrophages displayed enhanced O₂-consumption after stimulation with phorbol myristate acetate and heat-killed Listeria compared with macrophages from normal mice. These findings indicate that, under suboptimal stimulation by extracellular serum. rlFN-γ enhances the intracellular killing of catalase-negative Listeria which lack endogenous catalase acting as a scavenger of reactive oxygen intermediates. The mechanism underlying the enhancement is probably the amplification of the respiratory burst by IFN-γ.     

Morphological changes induced by dextran sulfate 500 in mononuclear phagocytes of listeria-infected mice

Morphological changes involving mononuclear phagocytes in Listeria-infected mice after treatment with dextran sulfate 500 were investigated. Mononuclear phagocytes in livers and spleens, both circulating monocytes and fixed macrophages, showed uptake of electron-dense material. Mononuclear phagocyte changes were most pronounced within granulomatous lesions, where many phagocytes showed large membrane-bound inclusions and extensive cellular damage. It is concluded that dextran sulfate 500 selectively damages mononuclear phagocytes and that, in listerial infection, dextran sulfate 500 renders mononuclear phagocytes unable to express cellular resistance.     

Administration of superantigens protects mice from lethal Listeria monocytogenes infection by enhancing cytotoxic T cells

Superantigens stimulate T-cell-receptor Vbeta-selective T-cell proliferation accompanying the release of cytokines, which may eventually protect the host from microbial infections. We investigated here whether superantigens can rescue the host from lethal bacterial infection. Mice were pretreated with Staphylococcus aureus enterotoxin B (SEB) 1 and 2 days before bacterial infection, and the mortality of infected mice was assessed. SEB pretreatment protected mice from lethal infection with Listeria monocytogenes but not from lethal infection with Streptococcus pyogenes. This enhanced protection was also observed upon pretreatment with recombinant streptococcal pyrogenic exotoxin A. Furthermore, L. monocytogenes-specific delayed-type hypersensitivity (DTH) due to type 1 helper T (Th1) cells and the cytotoxicity of CD8(+) T cells were significantly enhanced after SEB administration and bacterial infection. Depletion of either CD4(+) T cells or CD8(+) T cells in SEB-pretreated mice completely abolished this protection. This phenomenon was ascribed to the elimination of L. monocytogenes-specific CD8(+) cytotoxic T lymphocytes (CTL). It was found that CD4(+) T cells contributed to the induction of the CTL populations. Furthermore, SEB pretreatment of heat-killed L. monocytogenes-immunized mice enhanced the protection from challenge of L. monocytogenes. Taken together, these results indicated that administrations of superantigens protected mice from infection with L. monocytogenes, which was dependent on the enhanced L. monocytogenes-specific CTL activity in the presence of CD4(+) T cells, and superantigens exhibited adjuvant activity in the immunization against intracellular pathogens.     

Listeria monocytogenes in laboratory mice: a model of short-term infectious and pathogenic processes controllable by regulated protective immune responses

Summary: The mammalian immune system is an integrated network of tissues, leukocytes and effector and regulatory molecules. All these components operate i) to maintain the proper structure of and processes expressed by each tissue, and ii) to protect the hosts from those microorganisms that generally invade them as part of their life cycle. Among the invading microorganisms, Listeria monocytogenes (L. monocytogenes) can persist as a live organism independent of the host, and is, thus, able to drive short-term infectious and pathogenic processes that are controlled by integrated innate and adaptive protective immune responses driven by CD8 and CD4 type 1 T lymphocytes acting on non-T non-B leukocytes. Although the effector functions and the fine specificity of T lymphocytes have been more and more characterized, an understanding of the precise regulation of both leukocyte traffic and T-lymphocyte migration depends on knowledge of the early tissue distribution of L. monocytogenes, points that are addressed in this review.     

Purified human and recombinant murine interleukin-1 alpha induced accumulation of inflammatory peritoneal neutrophils and mononuclear phagocytes: possible contributions to antibacterial resistance

Interleukin-1 (IL-1) mediates a number of proinflammatory biological responses that are thought to contribute to antibacterial resistance. In the present study we examined the ability of IL-1 to recruit inflammatory neutrophils and mononuclear phagocytes in vivo; a function that has been reported to be closely related to antibacterial resistance. Intraperitoneal injection of small amounts (1-10 LAF units) of purified human or recombinant murine IL-1 alpha (rIL-alpha) resulted in an increased influx of inflammatory neutrophils into the peritoneal cavity that peaked at 4-14 h after IL-1 injection. A small but consistent increase in peritoneal macrophages also was observed at 72 h after rIL-1 alpha injection. The ability of rIL-1 alpha to induce neutrophil accumulation was uninfluenced by polymyxin B, was sensitive to heat treatment (100 degrees C for 1 h), and was observed after i.p. injection into LPS-nonresponsive C3H/HeJ mice. These three lines of evidence suggested that contaminating LPS did not contribute substantially to rIL-1 alpha induced accumulation of neutrophils. Treatment of mice with indomethacin or nordihydroguaiaretic acid did not abrogate rIL-1 alpha induced neutrophil accumulation. Mice injected i.p. with increasing amounts of rIL-1 alpha demonstrated a corresponding enhancement of their resistance to an i.p. L. monocytogenes challenge 4 h later, thus suggesting that IL-1 mediated inflammatory phagocyte accumulation may contribute in part to nonspecific antibacterial resistance.     

Characteristics of resistance to Listeria monocytogenes enhanced by Corynebacterium parvum in mice

In mice pre-treated with Corynebacterium parvum, Listeria monocytogenes was cleared rapidly from the blood and bacterial growth in the liver and spleen was inhibited effectively during the early phase of infection. This enhanced resistance could be transferred with peritoneal exudate cells (PEC) but not with non-adherent spleen cells. In spite of earlier elimination of bacteria, pre-treated mice developed lower levels of delayed-type hypersensitivity (DTH) to bacteria than untreated immune control mice, but the control levels of DTH could be reached by increasing the challenge dose of bacteria in C. parvum-pre-treated mice. Additionally, C. parvum did not inhibit the expression of antibacterial immunity when immune mice were rechallenged. It appeared that the active suppression of the T-cell mediated immune response by C. parvum-activated macrophages was not seen during the course of L. monocytogenes infection, and that the lower levels of DTH seen in mice pre-treated with C. parvum were attributable to an insufficient antigenic stimulus following the accelerated elimination of bacteria by non-specifically activated macrophages during the early phase of infection.