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Bacterial htrA genes are typically activated as part of the periplasmic stress response and are dependent on the extracytoplasmic sigma factor rpoE. A putative promoter region, P1, of the sigma(E)-type heat-inducible promoters has previously been identified upstream of the htrA gene of Bartonella henselae. Further analysis of the htrA mRNA by primer extension demonstrated that transcription initiates from P1 and a second region downstream of P1. This second promoter region, termed P2, had no sequence identity to sigma(E)-type heat-inducible promoters. Promoter regions were cloned individually and in tandem into pANT3 upstream of a promoterless version of the green fluorescent protein (GFP) gene (gfpmut3) and transformed into B. henselae by electroporation. The contiguous promoter region containing both P1 and P2 were necessary for the optimal transcriptional activation of the htrA gene. Promoter activity at 37 degrees C was distinctively higher than at 27 degrees C. However, thermal induction at 47 degrees C did not increase expression of gfpmut3. Invasion of human microvascular endothelial cells (HMEC-1) by B. henselae resulted in the formation of well-defined vacuoles containing clusters of bacteria exhibiting marked expression of gfpmut3 transcribed from the P1-P2 region. In addition, a moderate yet significant increase in the ratio of bacterial GFP to DNA was detected for intracellular bacteria compared to extracellular bacteria, indicating upregulation of htrA upon invasion of HMEC-1. The activation of specific genes in the intracellular environment may help us better understand the novel pathogenic mechanisms used by this bacterium.

Infection of BALB/c mice with a sublethal concentration of Legionella pneumophila causes an acute disease that is resolved by innate immune responses. The infection also initiates the development of adaptive Th1 responses that protect the mice from challenge infections. To study the early responses, cytokines induced during the first 24 h after infection were examined. In the serum, interleukin-12 (IL-12) was detectable by 3 h and peaked at 10 h, while gamma interferon was discernible by 5 h and peaked at 8 h. Similar patterns were observed in ex vivo cultures of splenocytes. A transient IL-4 response was also detected by 3 h postinfection in ex vivo cultures. BALB/c IL-4-deficient mice were more susceptible to L. pneumophila infection than were wild-type mice. The infection induced higher serum levels of acute-phase cytokines (tumor necrosis factor alpha [TNF-alpha], IL-1beta, and IL-6), and reducing TNF-alpha levels with antibodies protected the mice from death. Moreover, the addition of IL-4 to L. pneumophila-infected macrophage cultures suppressed the production of these cytokines. Thus, the lack of IL-4 in the deficient mice resulted in unchecked TNF-alpha production, which appeared to cause the mortality. Monocyte chemoattractant protein-1 (MCP-1), a chemokine that is induced by IL-4 during Listeria monocytogenes infection, was detected at between 2 and 30 h after infection. However, MCP-1 did not appear to be induced by IL-4 or to be required for the TNF-alpha regulation by IL-4. The data suggest that the early increase in IL-4 serves to regulate the mobilization of acute phase cytokines and thus controls the potential harmful effects of these cytokines.

A comprehensive peripheral blood immunophenotype analysis of 16 multiple sclerosis (MS) patients was performed by three-color flow cytometric analysis, and the results were compared with those for age-matched healthy controls. The cell subsets quantified included T cells (CD3+), B cells (CD19+), NK cells (CD56+), CD4+ and CD8+ T cells, cytotoxic (CD28+) and suppressor precursor (CD28-) CD8+ T cells, CD45RA+ and CD45RO+ T cells (CD4+ and CD8+), and CD5+ T and B cells. Analysis of MS patients' peripheral blood revealed essentially normal levels of total T, B, and NK cells. In agreement with results obtained by other investigators, it was found that MS patients had an increased CD4/CD8 ratio, primarily due to a decrease in CD8+ T cells. MS patients were found to have a significantly decreased level of suppressor precursor (CD28-) CD8+ T cells compared with that of controls but to have normal levels of cytotoxic (CD28+) CD8+ T cells. These data indicate that MS patients do not have a general decrease in CD8+ T cells but that they have a specific decrease in the suppressor precursor subset only and normal levels of cytotoxic CD8+ T cells. MS patients also had a significant increase in memory (CD45RO+) CD4+ T cells and displayed a trend towards a decrease in naive (CD45RA+) T cells in the peripheral blood.

Legionella pneumophila is a facultative intracellular bacterium which readily grows in cultures of guinea pig and human mononuclear phagocytes. In this report, we demonstrate that the Legionella sp. also grows in thioglycolate-elicited macrophages obtained from A/J mice but not in cells from other mouse strains tested, such as BDF1, DBA/2, C3H/HeN, C57BL/6, and BALB/c. Growth of Listeria monocytogenes and interleukin-1 production in A/J mice were similar to their growth and production in other strains tested, and the growth of Staphylococcus epidermidis was restricted by A/J macrophages. This finding suggests that although A/J macrophages share functional capabilities with cells from other mouse strains, they differ in growth restriction capacity for the Legionella sp. Resident macrophages were less permissive than were thioglycolate-elicited cells in that resident cells from A/J mice failed to support the growth of Legionella pneumophila. Also, resident cells from BDF1 mice rapidly eliminated the bacteria, rather than merely restricting growth. This finding was also observed in in vivo studies in which thioglycolate pretreatment of mice resulted in the enhanced recovery of viable bacteria from the peritoneal cavity of mice infected intraperitoneally. Higher numbers of bacteria were obtained from A/J mice and, in addition, this strain was more susceptible to the lethal effects of Legionella infection. These data suggest that, as with other intracellular bacteria, macrophages may serve a pivotal role in the early stages of Legionella infection and further suggest that the A/J mouse represents a useful animal model for the study of Legionella infection and immunity.

Cultured human monocytes have been shown to be susceptible to lysis by autologous lymphokine-activated killer (LAK) cells. To determine factors that might modulate the sensitivity of monocytes to lysis, we cultured adherent peripheral blood leukocytes (PBL) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) since these cytokines have been reported to affect both functional and physical characteristics of monocytes. Both recombinant human GM-CSF and IL-3 were found to significantly enhance the susceptibility of monocytes to lysis by LAK cells in a dose- dependent manner, with GM-CSF being slightly more effective. In a kinetics study, the lysability of monocytes increased after two days of incubation with either cytokine, with maximal susceptibility occurring after four to six days of culture. The effects of GM-CSF and IL-3 appeared to be specific for monocytes since culture of either nonadherent cells or granulocytes, which are normally resistant to LAK- mediated lysis, did not induce sensitivity. While the effects of GM-CSF and IL-3 have been shown to be synergistic in some cases, they did not act synergistically to induce monocyte susceptibility to LAK lysis. In cold target experiments cytokine-treated monocytes reciprocally blocked lysis, suggesting that similar target structures were modulated with either factor. FACS analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated comparable modulation of surface antigens with either GM-CSF or IL-3. Thus, these cytokines can serve to augment susceptibility of monocytes to LAK cells, emphasizing the complex interactions that occur in the immune system.

Spleen cell cultures from guinea pigs given legionella pneumophila vaccine in complete Freund adjuvant or as a sublethal infection were inhibited in their migration activity in vitro when incubated with specific antigen. Both direct and indirect migration inhibition assays revealed sensitization of the guinea pigs to the bacterium, with demonstrable reactivity 25 to 40 days or more after sensitization. No consistent reactions occurred when the guinea pigs were given the killed Legionella vaccine in incomplete Freund adjuvant in saline. However, spleen cells from guinea pigs injected with sublethal doses of the Legionella vaccine 3 to 4 weeks earlier showed positive migration inhibition factor reactivity. Cutaneous hypersensitivity and lymphocyte blastogenic responsiveness in vitro also developed in guinea pigs sensitized with killed Legionella vaccine in complete adjuvant or given a sublethal infection with the bacterium. These results indicate that in vitro assays for migration inhibitory activity may be utilized to monitor the development of the sensitization of guinea pigs to L. pneumophila, and such reactions correlate with skin reactivity and in vitro lymphocyte blastogenic responses.

Production of T helper 1 and T helper 2 cytokines was investigated in peripheral blood mononuclear cells (PBMCs) from multiple sclerosis (MS) patients by a newly described technique, detection of intracellular cytokines by flow cytometry in conjunction with immunophenotype analysis. T-cell gamma interferon (IFN-gamma) production and interleukin 10 (IL-10) production were examined after PBMC activation with T-cell mitogens at 5 and 24 h, and monocyte spontaneous production of IL-10 and production after PBMC activation with lipopolysaccharide (LPS) for 24 h were also examined. The data indicate that MS patients have decreased percentages of T cells capable of secreting IFN-gama compared with healthy controls, and this change is detectable at 5 and 24 h. the patients displaying decreased T-cell production of IFN-gamma were essentially confined to a group being treated with the newly approved drug Betaseron (berlex Labs, Cedar Knolls, N.J.), a recombinant form of IFN-beta (rIFN-beta 1b). By gating of the entire lymphocyte population, analysis of IFN-gama production in T cells (CD3+ versus that in non-T cells (CD3+) was possible. The percentage of IFN-gamma-producing lymphocytes that was made up of T cells was essentially unchanged between the Betaseron-treated patients, non-Betaseron-treated patients, and controls, indicating that the suppression of IFN-gamma production displayed by betaseron-treated MS patients was a nonspecific suppression of all IFN-gamma-producing lymphocytes as opposed to a suppression of T-cell production only. The data seem to indicate that treatment of MS with Betaseron corresponds to an inhibition of the lymphocyte's ability to produce IFN-gamma. No changes were detected in T-cell production of IL-10 at either time point. We also observed that MS patients in general appear to have small percentages of peripheral blood monocytes spontaneously producing slight but detectable levels of IL-10. No difference was seen regarding monocyte production of IL-10 after PBMC activation with LPS between MS patients and controls. Both populations responded with high percentages of monocytes producing IL-10. The data seem to indicate that treatment of MS with Betaseron, known to decrease the exacerbation rate of relapsing-remitting MS, corresponds to a suppression of peripheral blood lymphocyte production of IFN-gamma. Monocyte production of IL-10 may also play a role in regulating the disease process.

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS). The disease is characterized by inflammatory lesions in the white matter of the CNS, consisting of a specific immune response to the myelin sheath. We investigated peripheral blood mononuclear cell (PBMC) cytokine production by enzyme-linked immunosorbent assays of 21 MS patients and 19 age-matched normal controls in response to the T-cell mitogen phytohemagglutinin (PHA). Peripheral blood mononuclear cells were cultured in medium alone or in medium with 5 micrograms of PHA per ml for 48 h, and culture supernatants were collected for analysis. Cytokines selected for study were interleukin-10 (IL-10), gamma interferon (IFN-gamma), IL-2, and IL-4. All cytokine activities described were expressed as concentrations per 500,000 cells. We found that 48% (10 of 21) of the MS patients produced small but detectable levels of IL-10 in medium alone, compared with 26% (5 of 18) of the controls. We found that the MS patients produced significantly higher quantities of IL-10 protein than the controls in response to PHA (mean supernatant concentrations of IL-10 for patients and controls, 421 and 204 pg/ml, respectively [P < 0.05]). No significant differences were detected in the production of IL-2, IFN-gamma, and IL-4 between patients and controls in response to PHA, although patients appeared to display a trend toward decreased production of IFN-gamma.

Legionella pneumophila is a facultative intracellular bacterium which replicated well in inbred guinea pig strain 2 peritoneal macrophages at a low infectivity ratio. In contrast, the growth of this organism was markedly restricted in mouse (BDF1) peritoneal macrophages, even at a relatively high infectivity ratio. The initial uptake of L. pneumophila organisms by macrophages was similar in both animal species, and both groups of macrophages supported the growth of Listeria monocytogenes. Treatment of L. pneumophila with immune guinea pig serum did not result in restriction of bacterial growth in macrophages, but guinea pig macrophages were readily induced to suppress the growth of L. pneumophila by preincubation with supernatants obtained from mitogen-activated normal guinea pig splenocyte cultures. Thus, lymphokines generated from mitogen-stimulated guinea pig lymphocytes induced a restriction of growth of these organisms similar to that observed naturally with macrophages from mice, which are considered highly resistant to these bacteria. Although guinea pigs are considered highly susceptible to L. pneumophila infections and mice are considered relatively resistant, the mechanism of this difference is not clear. The results of the present study suggest that the restriction of L. pneumophila growth by macrophages relates to host susceptibility to infection and that cell populations permissive for L. pneumophila can be transformed to nonpermissive by products from stimulated lymphocytes but not by opsonization with immune serum.

An agarose microdroplet technique was utilized to assess the cellular immunity of guinea pig lymphoid cells to Legionella pneumophila antigen in vitro. Both direct and indirect migration inhibition procedures were shown to be capable of detecting sensitization of guinea pigs to L. pneumophila antigens. Animals injected with adjuvant alone or unrelated antigens did not yield spleen cells responsive to L. pneumophila, indicating the specificity of the response. Migration inhibition factor induction by Legionella antigen in vitro correlated well with skin test responses in vivo. The positive reaction detected by migration inhibition occurred at times similar to that of skin reactivity but later than that of the earliest serum antibody titers. The assay appears to be useful for monitoring sensitization to Legionella and may be applicable to the study of cell-mediated immunity to this bacterium in infected individuals.

Legionella pneumophilia antigen preparations, either killed whole cell vaccine, a soluble sonic extract, or a purified large-molecular-weight somatic antigen, stimulated blastogenic responses by splenocytes from both normal and Legionella-sensitized mice. Graded amounts of the bacterial preparations, when added to cultures of normal spleen cells, resulted in increased uptake of thymidine into cellular DNA, indicating that the preparations were mitogenic for normal mouse splenocytes. Spleen cells from mice injected with graded numbers of living bacteria showed blastogenic responsiveness to Legionella preparations generally at a higher level than spleen cells from normal animals. The heightened blastogenic response was mainly evident with spleen cells obtained from mice injected with living bacteria 2 to 3 weeks earlier. Splenocytes from mice infected with legionella less than 1 to 2 weeks or for more than 4 to 5 weeks responded generally similar to those obtained from uninjected mice, indicating that sensitization with living organisms had a relatively short duration. Spleen cell suspensions responding to the L. pneumophila antigens appeared to be mainly B-lymphocytes since cell suspensions from athymic nude mice deficient in T-cells responded as well as cells from conventional mice. Furthermore, passage of splenocytes over nylon wool columns to obtain B-cell-enriched preparations resulted in cell populations capable of responding to Legionella antigen. The cell fractions rich in T-cells were much less capable of responding to the Legionella antigens. In addition, treatment of spleen cell populations with antitheta serum plus complement failed to inhibit the blastogenic response, whereas the same spleen cell preparations treated with anti-mouse immunoglobulin serum plus complement markedly diminished blastogenic responsiveness, again consistent with the likelihood that B-lymphocytes were the major cell class responding to the Legionella preparations.

Injection of Legionella pneumophila antigen, either killed vaccine or soluble sonicate thereof, resulted in an enhanced antibody response by mouse spleen cells to sheep erythrocytes as determined by the hemolytic plaque assay. Enhancement was dose dependent and reached a peak response at a concentration of 10(7) bacteria or 50 micrograms of sonicate per animal. Larger doses of antigen were less stimulatory or even depressed the antibody response. Similar enhancement of antibody formation by normal spleen cell cultures to sheep erythrocytes in vitro occurred in the presence of graded amounts of L. pneumophila vaccine or sonicate. In addition, the L. pneumophila antigen stimulated enhanced background antibody formation in vitro in the absence of sheep erythrocytes or specific antigen. It appeared likely that the immunoenhancing activity of the L. pneumophila extract may be unrelated to the presence of lipopolysaccharide since boiling the antigen preparation eliminated much of the antibody-enhancing properties of the extract. A large-molecular-weight surface component from L. pneumophila was also immunomodulatory in vitro. Immunostimulation appeared to be related to effects on macrophages since adherent spleen cell populations rich in macrophages, when derived from spleen cell suspensions incubated with L. pneumophila antigen in vitro, stimulated enhanced antibody formation by normal mouse spleen cells in coculture experiments. Further investigations concerning the mechanism of immunomodulation by L. pneumophila antigen in vivo and in vitro appear to be warranted.

Delta 9-Tetrahydrocannabinol (THC) injection modulates immune cell function, but the significance of this in altering host resistance to infection is not understood. In addition, exposure to THC and other drugs of abuse during infection is associated with an acute mortality syndrome. We examined the effect of THC injection on the survival of mice infected with Legionella pneumophila (Lp). Mice given two injections of THC (8 mg/kg)-one 24 hr before and the second 24 hr after a sublethal Lp infection-experienced acute collapse and death. The drug injection after infection caused death; deaths occurred within 30 min after the injection, and neither one nor two drug injections before infection resulted in death. The THC-induced mortality resembled cytokine-mediated shock in both kinetics and symptoms; therefore, sera from drug-treated animals were measured for the acute-phase cytokines tumor necrosis factor (TNF) and interleukin 6 (IL6). The level of each cytokine was significantly elevated by THC treatment, suggesting a role in the observed mortality. To directly test this role, mice were administered a single injection of either anti-TNF alpha, anti-IL6, or a mixture of anti-IL1 alpha and -IL1 beta antibodies 1 hr before the second THC injection. Results showed that each antibody treatment protected the mice, with anti-IL6 being the most effective. Fluctuations in blood granulocytes levels also supported a role of acute-phase cytokines in THC-induced mortality. These results show that THC injection increases the blood levels of acute-phase cytokines in infected animal and that these elevated levels, at least in part, account for the mortality induced by THC injection.

The lymphocyte blastogenic transformation assay was adapted to study responsiveness of lymphoid cells from animals and humans to Legionella pneumophila antigens in vitro. Spleen cells from guinea pigs after active immunization with Legionella vaccine, but not from normal animals, responded by blast cell transformation when stimulated in vitro with killed Legionella whole-cell vaccine, sonic extracts thereof, or a purified somatic antigen. The response was dose dependent. Similar lymphocyte blastogenesis occurred with spleen cells from mice sensitized to Legionella by sublethal infection with the bacteria. In addition, blastogenesis occurred with peripheral blood leukocytes from human volunteers tested in vitro with whole-cell vaccine, sonic extracts, or purified somatic antigen. Maximum responsiveness generally occurred 4 to 5 days after stimulation of human peripheral blood leukocytes, but a day or two earlier with spleen cells from normal or sensitized mice. Guinea pig spleen cells generally showed peak responses at the same time as human peripheral blood leukocytes after stimulation in vitro. Blastogenic responses with purified antigen were comparable to those with the whole-cell vaccine or sonic extract. Such antigens from Legionella provide a useful material for inducing responses in vitro as a correlate of cellular immunity to these bacteria.