Deficient tumor necrosis factor secretion by cord blood mononuclear cells upon in vitro stimulation with Listeria monocytogenes.

We examined the production of tumor necrosis factor (TNF) by mononuclear cells (MNC) after incubating adult or cord blood MNC with Listeria monocytogenes in vitro. With adult MNC cultures, we found that TNF activity reached a peak at 6 h (606 +/- 120 x 10(3) units/liter) and declined to the baseline by day 3. In contrast, using cord blood MNC, we found that TNF activity increased gradually reaching a peak at 24 h. In addition, the peak TNF activity using newborn MNC (189 +/- 26 x 10(3) U/liter) at 24 h was still lower than the peak using adult MNC at 6 h (p < 0.0002). In seeking an explanation for the decreased TNF secretion from newborn MNC, we examined the possibility that newborn cells produce TNF but failed to secrete it. However, lysates of newborn cells contained functionally and antigenically less TNF than adult cells. Based on these observations, we conclude that the overall TNF production by newborn cells incubated with L monocytogenes is decreased compared with similarly stimulated adult cells.

Interferon-gamma detection in cultures of newborn cells exposed to Listeria monocytogenes.

Clearance of Listeria monocytogenes in experimental models of infection has underscored the importance of interferon-gamma (IFN-gamma) in host resistance to intracellular pathogens. Because L. monocytogenes infections are more severe in newborns than adults, we compared IFN-gamma accumulation in the supernatants of mononuclear cells infected in vitro from newborns with those from adults. Supernatants were assayed for IFN-gamma using an enzyme-linked immunosorbent assay. Uninfected newborn and adult mononuclear cells had less than 50 pg/ml of IFN-gamma at all times tested. IFN-gamma levels in supernatants from infected adult mononuclear cells at 24 h of culture (1.15 x 10(3) +/- 0.92 pg/ml) were greater than supernatants from infected newborn mononuclear cells (0.19 x 10(3) +/- 0.33 pg/ml). IFN-gamma concentrations in newborn cell cultures plateaued on day 3 of culture (1.6 x 10(3) +/- 1.1 pg/ml) and were not significantly less than concentrations from adult cells. However, adult cell IFN-gamma was further increased by day 5 (18.7 x 10(3) +/- 21.8 pg/ml). Because IFN-gamma plays a critical role in the host defense against L. monocytogenes, this delay in the release of IFN-gamma may be a factor in the increased susceptibility and severity of infection in the neonate.

Production of interleukin 8 (IL-8) by cord blood mononuclear cells induced by Listeria monocytogenes.

The defective ability of human newborns to mobilize phagocytes to the site of infection led us to examine the ability of cord blood mononuclear cells to secrete interleukin-8, a major neutrophil chemotactic factor, in response to stimulation with Listeria monocytogenes. Adult or cord blood mononuclear cells were incubated with L. monocytogenes for varying lengths of time, and IL-8 was measured in the culture supernatants by the enzyme-linked immunosorbent assay (ELISA). Spontaneous IL-8 secretion by unstimulated cells was undetectable or at the minimal detection limit of the assay. By 24 h of cell incubation with L. monocytogenes, newborn cells produced as much IL-8 as adult cells did (300 +/- 113 versus 269 +/- 189 ng/ml, respectively). Over the next 2-4 days, IL-8 output by adult cells was slightly higher than that by newborn cells, but the difference was not statistically significant. The in vitro results suggested that newborns are as able as adults to produce IL-8, although they are defective in mobilizing neutrophils, the IL-8 target cells, to the site of infection.

Role of tumor necrosis factor-alpha and interferon-gamma in newborn host defense against Listeria monocytogenes infection.

The fetus and newborn are particularly susceptible to Listeria monocytogenes infection. We used a newborn rat animal model to investigate neonatal host defense against Listeria. In this animal model, newborn (3-d-old) rats are more susceptible to L. monocytogenes than older animals. Juvenile (23-d-old) L. monocytogenes-infected rats pretreated with lipopolysaccharide (LPS) had a lower bacterial load in blood than control animals, whereas LPS pretreated newborn rats had a higher bacterial load. Because LPS is a potent inducer of tumor necrosis factor (TNF) and TNF enhances host defense against this organism in adult animals, we assessed TNF content in splenic homogenates for animals of different ages. The age at which TNF was detectable in L. monocytogenes-Infected rats corresponded to the age at which LPS became active in preventing severe bacteremia. TNF was less than 1 unit/mL in splenic homogenates taken from rats less than 8 d of age, whereas 16-d-old rats infected with L. monocytogenes 1 d earlier had greater than 80 units/mL (p less than 0.0001 for 3-d-old versus 16-d-old rats). We also assessed the responsiveness of rats to exogenous TNF-alpha. Juvenile rats pretreated with TNF-alpha before L. monocytogenes infection had decreased bacterial load in spleen (p less than 0.02 versus controls) and better survival at 7 d (p less than 0.05 versus controls), whereas newborn rats did not improve with TNF-alpha pretreatment (p greater than 0.05 treated versus controls for splenic bacterial load and 7-d survival).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of recombinant human granulocyte colony-stimulating factor (rh G-CSF) on murine resistance against Listeria monocytogenes.

Recombinant human granulocyte colony-stimulating factor (rh G-CSF) enhanced resistance of mice against Listeria monocytogenes (LM) as determined by survival and bacterial growth. Mice pretreated with rh G-CSF twice daily for 5 days survived better than untreated animals to the challenge with LM. Number of bacteria in peritoneal cavity (PC) and spleen was lower in treated mice than that in the control group. Rh G-CSF increased mainly polymorphonuclear cells (PMN) in blood and spleen. After LM inoculation, a larger number of PMN and monocyte-macrophages accumulated in PC and spleen of tested mice. In addition, PMN primed in vivo with rh G-CSF released more superoxide anions when stimulated with phorbol myristate acetate. The inhibition of bacterial growth in PC and spleen could be ascribed to the accumulation of phagocytic cells at the infection sites and the increased oxidative metabolism. The results provided further evidence of the important contribution of G-CSF and neutrophils, as target cells, to the host defence against the intracellular bacteria.

Difference in the functional maturation of T cells against Listeria monocytogenes in lymph nodes and spleen.

After subcutaneous immunization of mice with viable Listeria monocytogenes (LM), we evaluated the function of T cells induced in draining lymph nodes (LN) and spleen as determined by the local transfer of delayed-type hypersensitivity (DTH), acquired cellular resistance (ACR) and in vitro lymphokine production. LN cells could transfer specifically DTH but not ACR. In contrast, spleen cells from the same donor mice evoked the DTH response as well as bacterial clearance at the reaction site. Comparison of bacterial counts in spleen and in LN upon subcutaneous inoculation of mice with LM suggested that the lack of bacterial proliferation in LN underlay the failure to induce protective T cells in this lymphoid tissue. Spleen and LN T cells expressed CD4 and CD8 surface antigens equally and DTH response was solely dependent on CD4+ cells. Another major difference between LN and spleen cells was the profile of lymphokines produced in vitro. Upon the in vitro restimulation with killed Listeria, immune spleen cells produced macrophage chemotactic factor (MCF), interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). In contrast, LN cells could produce all of the measured lymphokines but not IFN-gamma. The results provided strong evidence for the dissociation of DTH and ACR. Listerial growth appeared to be the requirement for full maturation of anti-listerial immunity that may coincide with the generation of IFN-gamma-producing T cells.

A dissociated induction of MCF-producing and MAF-producing T cells specific for Listeria monocytogenes in the in vitro primary culture system.

Using an in vitro primary culture system which we had previously established, the induction phase of Listeria monocytogenes-specific effector cells was analysed with respect to their abilities to produce effector lymphokines, macrophage chemotactic factor (MCF) and macrophage-activating factor (MAF). Listeria-specific effector cells generated after in vitro culture of normal spleen cells with viable L. monocytogenes for 5 days conveyed L3T4+, Lyt-2-, Thy-1+ surface antigens and produced MCF and MAF in response to the secondary stimulation with heat-killed L. monocytogenes. The cells required for the induction of Listeria-specific effector cells, which produce effector lymphokines, MCF and MAF, were L3T4+, Lyt-2-, Thy-1+ cells. The kinetic analysis revealed that the ability of these effector cells to produce MCF was generated earlier than that to produce MAF. Furthermore, using passive transfer of cells, the effector cells producing only MCF, which were generated early in culture, conferred delayed-type hypersensitivity (DTH) alone, but MCF- and MAF-producing effector cells generated late in culture conferred sufficient levels of DTH and acquired cellular resistance (ACR). These results indicate a dissociated production of MCF and MAF by L. monocytogenes-specific T cells generated in the primary in vitro culture system.

Role of antibodies against outer-membrane proteins in murine resistance to infection with encapsulated Klebsiella pneumoniae.

In the assessment of immunity to the encapsulated virulent strain of Klebsiella pneumoniae and its avirulent mutant defective for capsular polysaccharide (CPS), killed bacterial vaccine of both strains could protect mice equally against challenge with 100 x LD50 of encapsulated wild strain. Antisera to each strain conferred the same level of protection on naive mice upon transfer; the protective anti-mutant serum was highly capable of opsonizing the encapsulated bacteria. In addition to the common antigenic components shared by both strains, the wild strain had antigen(s) unrelated to the mutant since the protective capacity of the anti-wild serum was not affected by preabsorption with the mutant strain; the protection conferred by the anti-mutant serum was mediated by antibodies against non-capsular antigens since the antiserum did not contain antibodies against purified CPS detectable by ELISA. As possible candidates among the non-capsular antigens, outer-membrane proteins (OMPs) extracted from the mutant strain were examined for their immunogenicity. Immunoblotting of the protein-containing fraction and ELISA using LPS-free OMP suggested that a number of proteins were involved in the immune response evoked by K. pneumoniae. Furthermore, mice immunized with OMP or anti-OMP serum could overcome a lethal challenge with the wild strain. These results indicated that OMPs of K. pneumoniae are implicated as the protective antigens and may pave the way for the development of non-capsular, proteinaceous vaccines.

A low degree of requirement for Ia-positive macrophages and IL 2 in the induction phase of Listeria monocytogenes-specific T cells in vitro.

We have analyzed the priming process of Listeria-specific T cells using an in vitro primary culture system. Listeria-specific T cells mediating delayed footpad reaction (DFR) and acquired cellular resistance (ACR) upon passive local transfer into naive recipients were generated from non-immune mouse spleen cells when stimulated with viable Listeria monocytogenes primarily in vitro. The effectors were detected on the third day of culture, and culturing for 5 days was sufficient for the generation of effectors mediating the peak level of DFR and ACR. The requirement of T cell subsets, Ia antigen and interleukin 2 (IL 2) for inducing effectors was studied. Presence of macrophages (M phi) and their contact to T cells were required for priming of Listeria-specific T cells in vitro. The presence of Ia antigens on macrophages was absolutely required for priming, but this requirement was lower than that in secondary immune response of Listeria-specific T cells. Effectors could not be generated when L3T4+ cells were depleted, but effectors capable of conferring a full level of DFR and ACR were induced even after the depletion of Lyt2+ cells. Contribution of IL 2 to the generation of effectors during early phase of priming was not observed. IL 2 was not produced in the supernatant of the in vitro primary culture. Precursor cells of the effectors did not respond to exogenously added recombinant IL 2 (rIL 2). Some mechanisms operating in the induction phase of Listeria-specific T cells were clarified in this study.

Co-operative effect of MCF and MAF(IFN-gamma) in the protection of mice against Listeria monocytogenes.

The effects of macrophage chemotactic factor (MCF) and macrophage-activating factor (MAF) on protection to Listeria monocytogenes were analysed using 'MCF-rich fraction' and murine recombinant interferon-gamma (rIFN-gamma). Recombinant IFN-gamma showed no macrophage chemotactic activity in the assays performed in vitro and in vivo. Although a single injection of either MCF-rich fraction or rIFN-gamma into the footpads of mice led to a significant degree of anti-listerial activity, the highest degree of protection was observed when injected together. The i.v. administration of 20,000 U rIFN-gamma did not raise significant protective activity against Listeria; however, injection with rIFN-gamma prior to that with the MCF-rich fraction into the footpad produced a higher level of protective activity than the group treated with MCF alone. In addition to MAF(IFN-gamma), MCF seems to play another important role in the full expression of protection against L. monocytogenes.

A new T cell subset expressing B220 and CD4 in lpr mice: defects in the response to mitogens and in the production of IL-2.

Autoimmune-prone mice homozygous for the lpr gene develop prominent lymphadenopathy composed mainly of Thy-1+ CD8- CD4- B220+ cells. Expression patterns of B220 vs CD4 on lymph node cells from lpr mice were analysed using two-colour flow microfluorometry. B220+CD4+ cells, which were hardly seen in lymph nodes of B6-+/+ mice, increased significantly in B6-lpr mice with ageing. Functional analysis of purified B220+ CD4+ cells from lpr mice revealed that these cells scarcely responded to T cell mitogens with or without rIL-2. Furthermore, B220+ CD4+ cells were defective in IL-2 production when cultured with Con A. On the other hand, B220-CD4+ cells from B6-lpr mice showed an ability to respond to T cell mitogens similar to that of B220- CD4+ cells from B6-+/+ mice. These results indicate that an unusual T cell subset expressing both B220 and CD4 in lpr mice is functionally defective, but the intrinsic ability of B220-CD4+ cells is almost intact as compared with the counterpart in normal mice.

Positive effects of indomethacin on restoration of splenic nucleated cell populations in mice given sublethal irradiation.

Splenic cellularity during the recovery phase after 400-R irradiation was evaluated in mice, in which the level of prostaglandin was regulated by indomethacin and exogenous prostaglandin E2, following sublethal irradiation. Two weeks after irradiation, administration to these mice of indomethacin, an inhibitor of prostaglandin synthesis, augmented the recovery of all nucleated spleen cell populations, whereas the thymus was drastically depopulated. This treatment had little effect upon the total number of bone marrow cells but inversed the ratio of PNA+/PNA- cells. Cell transfer experiments using heavily irradiated mice as recipients showed that the stem cell proliferation was positively affected by indomethacin treatment in the bone marrow rather than in the spleen. These results suggest that cell migration from primary lymphoid organs, particularly from the bone marrow to the spleen, is regulated by a prostaglandin-mediated system and that a prostaglandin E2 synthesis inhibition would have a positive effect on the restoration of peripheral nucleated cells following irradiation.

Indomethacin-induced sialic acid-mediated changes in surface markers from "cortical type" to "medullary type" in murine thymoma line EL-4.

The effect of indomethacin (INDO), a specific inhibitor of prostaglandin (PG) synthesis, on the expression of markers during thymocyte differentiation such as Thy-1, H-2K, and peanut agglutinin (PNA) receptor was examined by immunofluorescence, using poorly differentiated thymoma, EL-4, as the indicator cells. EL-4 cells grown in the culture medium in the presence of INDO exhibited lower levels of Thy-1 and PNA, and a higher level of H-2K, compared with the EL-4 cells grown in medium without INDO. The decrease in PNA level by INDO was attributed to an increased density of sialic acid bound to PNA receptors on the cell surface, because treatment with neuraminidase (Nase) released more sialic acid from such cells, as compared with control cells, and markedly increased the detectable amounts of PNA receptors. On the other hand, a decrease of Thy-1 or an increase of H-2K may be ascribed to the decrease of Nase-resistant sialic acid on the cell surface, determined by analyses with FITC-LPA, sialic acid-specific lectin, and by metabolic labeling of surface sialic acid. These results suggest that the PG-system modulates the metabolism of sialic acid located on the thymocyte surface and alters the expression of surface markers of thymocytes.