Change of mouse CD5(+) B1 cells to a macrophage-like morphology induced by gamma interferon and inhibited by interleukin-4

The in vitro effects of gamma interferon (IFN-gamma) on the mouse CD5(+) B1-cell line, TH2.52, a hybridoma between mouse B lymphoma and mouse splenic B cells that expresses a series of B1 markers, were investigated. A significant number of macrophage-like cells appeared in the cultures of TH2.52 cells exposed to IFN-gamma, these adhering to plastic dishes and exhibiting phagocytic activity. Positive for esterase staining, the macrophage-like cells returned to the original TH2.52 morphology upon removal of IFN-gamma. The change was prevented by treatment with SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase and by transfection of a p38 MAP kinase dominant-negative mutant. Further, interleukin-4 (IL-4) inhibited IFN-gamma-induced phosphorylation of p38 MAP kinase and the appearance of macrophage-like cells. IFN-gamma and IL-4 exhibited contradictory actions on morphological change of CD5(+) B1 cells into macrophage-like cells. Differential regulation of CD5(+) B1 cells by IFN-gamma, a Th1 cytokine, and IL-4, a Th2 cytokine, may have clear immunological significance.     

Interleukin-4-promoted T helper 2 responses enhance Nippostrongylus brasiliensis-induced pulmonary pathology

The role of CD4⁺ T-cell interleukin-4 (IL-4) receptor alpha (IL-4Rα) expression in T helper 2 (TH2) immune responses has not been defined. To examine this role, we infected CD4⁺ T-cell IL-4Rα knockout (KO) mice with the parasitic nematode Nippostrongylus brasiliensis, which induces strong host TH2 responses. Although N. brasiliensis expulsion was not affected in CD4⁺ T-cell IL-4Rα KO mice, the associated lung pathology was reduced. Infected CD4⁺ T-cell IL-4Rα KO mice showed abrogation of airway mucus production. Furthermore, CD4⁺ T-cell IL-4Rα KO mouse lungs contained reduced numbers of lymphocytes and eosinophils. Restimulation of pulmonary region-associated T-cell populations showed that TH2 cytokine responses were disrupted. Secretion of IL-4, but not secretion of IL-13 or IL-5, from mediastinal lymph node CD4⁺ T cells was reduced in infected CD4⁺ T-cell IL-4Rα KO mice. Restimulation of tissue-derived CD4⁺ T cells resulted in equivalent levels of IL-4 and IL-13 on day 7 postinfection (p.i.) in control and CD4⁺ T-cell IL-4Rα KO mice. By day 10 p.i. the TH2 cytokine levels had significantly declined in CD4⁺ T-cell IL-4Rα KO mice. Restimulation with N. brasiliensis antigen of total lung cell populations and populations with CD4⁺ T cells depleted showed that CD4⁺ T cells were a key TH2 cytokine source. These data demonstrated that CD4⁺ T-cell IL-4 responsiveness facilitates eosinophil and lymphocyte recruitment, lymphocyte localization, and TH2 cytokine production in the allergic pathology associated with N. brasiliensis infections.     

Temporal regulation of interleukin-12p70 (IL-12p70) and IL-12-related cytokines in splenic dendritic cell subsets during Leishmania donovani infection

Dendritic cells (DC) play an essential role in initiating and directing T-cell responses, in part by production of interleukin-12p70 (IL-12p70), IL-23, and IL-27. However, comparative studies on the capacity for cytokine production of DC subsets are rare. Here, we compare splenic CD8α⁺, CD4⁺, and double-negative (DN) DC, isolated 5 h to 28 days after Leishmania donovani infection, for (i) production of IL-12p70, (ii) accumulation of IL-12/23p40, IL-12p35, IL-23p19, and IL-27p28 mRNAs, and (iii) their capacity to direct CD4⁺ T-cell differentiation. At 5 h, conventional DC (cDC) accumulated mRNA for IL-12/23p40 (CD8α>CD4>DN), IL-23p19 (CD4>CD8α>DN), and IL-27p28 (CD8α>CD4>DN), in an infection dose-dependent manner. IL-12p70 was restricted to CD8α⁺ cDC, reflecting the subset-specific accumulation of IL-12p35 mRNA. In contrast, cDC from mice infected for 14 to 28 days accumulated little mRNA for IL-12p40 and IL-12p19, though IL-27p28 mRNA remained detectable (CD8α>DN>CD4). IL-12p70 secretion by CD8α⁺ cDC was also absent, reflecting deficient IL-12/23p40, rather than IL-12p35, mRNA accumulation. The capacity of CD8α⁺ cDC isolated early after infection to direct Th1 cell differentiation was mediated through IL-12/23p40, whereas this ability in CD4⁺ and DN cDC was independent of IL-12/23p40 and did not result from overexpression of Delta 4 Notch-like ligand. However, DN cDC produced gamma interferon (IFN-γ) and also contained a rare population of CD11c^hi DX5⁺ IFN-γ-producing cells. Our data illustrate the extensive diversity in, and temporal regulation of, splenic cDC subsets during infection and suggest caution in interpreting data obtained with unfractionated or minimally purified DC.     

Reciprocal Regulation of Th1- and Th2-Cytokine-Producing T Cells during Clearance of Parasitemia in Plasmodium falciparum Malaria

Flow cytometry for the intracellular detection of T-cell cytokines was performed for 15 Gabonese patients during acute uncomplicated Plasmodium falciparum malaria. A striking expansion of CD4⁺ and CD8⁺ T cells producing gamma interferon (IFN-γ) was found during drug-induced clearance of parasitemia, paralleled by a decrease of interleukin-2 (IL-2) production. The frequency of IL-4- and IL-13-producing CD4⁺ cells gradually decreased, whereas the frequency of T cells producing IL-2⁺–IFN-γ⁺, IL-4⁻–IL-5⁺, and IL-4⁺–IL-5⁺ cytokines as well as IL-4⁺–IFN-γ⁺ and IL-13⁺–IFN-γ⁺ cytokines was not significantly altered. The capacity for IL-10 production within the CD4⁺ subset increased due to an expansion of both IL-10⁺–IFN-γ⁻ and IL-10⁺–IFN-γ⁺ cytokine-expressing cells. Thus, a more pronounced Th2-driven immune response during acute untreated P. falciparum infection with a shift towards Th1 responsiveness induced by parasite clearance is suggested.     

Assessment by Flow Cytometry of Cytokine Production in Malnourished Children

Malnutrition in children is associated with an increased risk of infection and death. Multiple abnormalities in the immune response, including cytokine production, in protein energy-malnourished children have been described and could account for the increased severity and frequency of infections. In this study, we used flow cytometry to investigate the effects of malnutrition on the production of cytokines (interleukin-2 [IL-2], gamma interferon [IFN-γ], IL-4, and IL-10) in CD4⁺ and CD8⁺ cells and the activation capability (as indicated by CD69⁺ and CD25⁺ cells). CD4⁺ and CD8⁺ cells from malnourished children showed increased production of IL-4 and IL-10 cytokines and decreased production of IL-2 and IFN-γ cytokines compared to that in cells from well-nourished, uninfected and well-nourished, infected children. In addition, malnourished children showed impaired activation capability, since the fluorescence intensity of CD69⁺ and CD25⁺ cells was lower than that in cells from well-nourished, uninfected and well-nourished, infected children. These results indicate that malnutrition alters the capacity of CD4⁺ and CD8⁺ cells to produce IL-2, IFN-γ, IL-4, and IL-10 in response to stimulus. We concluded that both cytokine production and activation capacity were impaired in malnourished children. This functional impairment may be involved in the failure to develop a specific immune response and the predisposition to infection in these children.     

Interleukin-15 May Be Responsible for Early Activation of Intestinal Intraepithelial Lymphocytes after Oral Infection with Listeria monocytogenes in Rats

Exogenous interleukin-15 (IL-15) stimulates intestinal intraepithelial lymphocytes (i-IEL) from mice to proliferate and produce gamma interferon (IFN-γ) in vitro. To determine whether endogenous IL-15 is involved in activation of i-IEL during intestinal infection, we examined IL-15 synthesis by intestinal epithelial cells (i-EC) after infection with Listeria monocytogenes in rats. In in vitro experiments, invasion of L. monocytogenes into IEC-6 cells, a rat small intestine epithelial cell line, evidently induced IL-15 mRNA expression coincident with nuclear factor κB (NF-κB) activation, which is essential for IL-15 gene expression. IL-15 synthesis was detected in rat i-EC on day 1 after an oral inoculation of L. monocytogenes in vivo. The numbers of T-cell receptor (TCR) γδ⁺ T cells, NKR.P1⁺ cells, and CD3⁺ CD8⁺ αα cells in i-IEL were significantly increased on day 1 after oral infection. The i-IEL from infected rats produced larger amounts of IFN-γ upon stimulation with immobilized anti-TCR γδ or anti-NKR.P1 monoclonal antibodies. These results suggest that IL-15 produced by i-EC may stimulate significant fractions of i-IEL to produce IFN-γ at an early phase of oral infection with L. monocytogenes.     

Spontaneous Cytokine Production and Its Effect on Induced Production

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Multiparameter flow cytometry is often used to examine cell-specific cytokine production after in vitro phorbol 12-myristate 13-acetate and ionomycin induction, with brefeldin A or other agents added to inhibit protein secretion. Spontaneous ex vivo production reportedly rarely occurs. We examined the spontaneous production of interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) by peripheral-blood B lymphocytes, T cells, CD8⁻ T cells, CD8⁺ T cells, CD3⁻ CD16/56⁺ lymphocytes (natural killer [NK] cells), CD3⁺ CD16/56⁺ lymphocytes (natural T [NT] cells), and/or monocytes of 316 acutely ill hospitalized persons and 62 healthy adults in Malawi, Africa. We also evaluated the relationship between spontaneous and induced cytokine production. In patients, spontaneous TNF-α production occurred most frequently, followed in descending order by IFN-γ, IL-8, IL-4, IL-10, IL-6, and IL-2. Various cells of 60 patients spontaneously produced TNF-α; for 12 of these patients, TNF-α was the only cytokine produced spontaneously. Spontaneous cytokine production was most frequent in the immunoregulatory cells, NK and NT. For IL-2, IL-4, IL-6, IL-8, and IL-10, spontaneous cytokine production was associated with greater induced production. For TNF-α and IFN-γ, the relationships varied by cell type. For healthy adults, IL-6 was the cytokine most often produced spontaneously. Spontaneous cytokine production was not unusual in these acutely ill and healthy persons living in an area where human immunodeficiency virus, mycobacterial, malaria, and assorted parasitic infections are endemic. In such populations, spontaneous, as well as induced, cell-specific cytokine production should be measured and evaluated in relation to various disease states.     

Dysregulated Synthesis of Intracellular Type 1 and Type 2 Cytokines by T Cells of Patients with Cutaneous T-Cell Lymphoma

Mycosis fungoides (MF) and Sezary syndrome (SS) are the two main clinical entities of cutaneous T-cell lymphoma (CTCL). As the disease progresses from MF to SS, a switch from a type 1 (interleukin [IL]-2 and gamma interferon [IFN-γ]) to a type 2 (IL-4) cytokine production profile occurs. Although roles for type 1 and type 2 cytokines in the pathogenesis of CTCL have been proposed, the cellular origins of these cytokines are unclear. Using flow cytometry to identify individual T-cell subsets, we studied cytokine synthesis by the T cells of 13 patients with SS and 12 with MF and 9 hematologically healthy donors. Upon activation with phorbol 12-myristate 13-acetate (PMA), the numbers of T cells synthesizing IL-2 were similar for all study groups. Whereas the predominant T-cell producing IL-2 in healthy donors and in those with MF was CD7⁺, in patients with SS, it was CD7⁻. Although the number of IL-4⁺ CD4⁺ T cells was low for all study groups, there was a significantly higher number of IL-4⁺ CD8⁺ T cells in patients with MF than in those with SS or healthy donors. There was a decline in the number of IFN-γ-producing T cells in CTCL donors compared to that in healthy donors. More importantly, there was a significant decrease in the number of IFN-γ-producing T cells with disease progression from MF to SS. The inability of these T cells to synthesize IFN-γ may have prognostic value in CTCL, since it may be responsible for the progression of the disease from MF to SS.     

Depressed Type 1 Cytokine Synthesis by Superantigen-Activated CD4+ T Cells of Women with Human Papillomavirus-Related High-Grade Squamous Intraepithelial Lesions

Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4⁺ T and CD8⁺ T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-γ], and tumor necrosis factor alpha [TNF-α]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4⁺ T cells that produced IL-2 (P = 0.045), IFN-γ (P = 0.040), and TNF-α (P = 0.015) and a significantly lower percentage of activated CD8⁺ T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4⁺ T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8⁺ T cells.     

Effects of CXCL10 on dendritic cell and CD4+ T-cell functions during Leishmania amazonensis infection

Leishmania amazonensis can cause progressive disease in most inbred strains of mice. We have previously reported that treatment with CXCL10 activates macrophage (MΦ) effector function(s) in parasite killing and significantly delays lesion development in susceptible C57BL/6 mice via enhanced gamma interferon (IFN-γ) and interleukin 12 (IL-12) secretion; however, the mechanism underlying this enhanced immunity against L. amazonensis infection remains largely unresolved. In this study, we utilized stationary promastigotes to infect bone marrow-derived dendritic cells (DCs) of C57BL/6 mice and assessed the activation of DC subsets and the capacity of these DC subsets to prime CD4⁺ T cells in vitro. We found that CXCL10 induced IL-12 p40 production but reduced IL-10 production in uninfected DCs. Yet L. amazonensis-infected DCs produced elevated levels of IL-10 despite CXCL10 treatment. Elimination of endogenous IL-10 led to increased IL-12 p40 production in DCs as well as increased proliferation and IFN-γ production by in vitro-primed CD4⁺ T cells. In addition, CXCL10-treated CD4⁺ T cells became more responsive to IL-12 via increased expression of the IL-12 receptor β₂ chain and produced elevated levels of IFN-γ. This report indicates the utility of CXCL10 in generating a Th1-favored, proinflammatory response, which is a prerequisite for controlling Leishmania infection.     

An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible to Mycobacterium tuberculosis

I/St mice, previously characterized as susceptible to Mycobacterium tuberculosis H37Rv, were given 10³ or 10⁵ CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44⁻ CD45RB⁺ cells disappeared within 2 weeks postinfection, while the number of CD44⁺ CD45RB^−/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3⁺ CD4⁺ CD8⁻ T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ⁺ IL-4⁻) clone and one Th0-like (IFN-γ⁺ IL-4⁺ IL-10⁺) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.     

Dendritic Cell and NK Cell Reciprocal Cross Talk Promotes Gamma Interferon-Dependent Immunity to Blood-Stage Plasmodium chabaudi AS Infection in Mice

Dendritic cells (DCs) are important accessory cells for promoting NK cell gamma interferon (IFN-γ) production in vitro in response to Plasmodium falciparum-infected red blood cells (iRBC). We investigated the requirements for reciprocal activation of DCs and NK cells leading to Th1-type innate and adaptive immunity to P. chabaudi AS infection. During the first week of infection, the uptake of iRBC by splenic CD11c⁺ DCs in resistant wild-type (WT) C57BL/6 mice was similar to that in interleukin 15^−/− (IL-15^−/−) and IL-12p40^−/− mice, which differ in the severity of P. chabaudi AS infection. DCs from infected IL-15^−/− mice expressed costimulatory molecules, produced IL-12, and promoted IFN-γ secretion by WT NK cells in vitro as efficiently as WT DCs. In contrast, DCs from infected IL-12p40^−/− mice exhibited alterations in maturation and cytokine production and were unable to induce NK cell IFN-γ production. Coculture of DCs and NK cells demonstrated that DC-mediated NK cell activation required IL-12 and, to a lesser extent, IL-2, as well as cell-cell contact. In turn, NK cells from infected WT mice enhanced DC maturation, IL-12 production, and priming of CD4⁺ T-cell proliferation and IFN-γ secretion. Infected WT mice depleted of NK cells, which exhibit increased parasitemia, had impaired DC maturation and DC-induced CD4⁺ Th1 cell priming. These findings indicate that DC-NK cell reciprocal cross talk is critical for control and rapid resolution of P. chabaudi AS infection and provide in vivo evidence for the importance of this interaction in IFN-γ-dependent immunity to malaria.     

Exposure of cord blood to Mycobacterium bovis BCG induces an innate response but not a T-cell cytokine response

Despite routine vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) soon after birth, tuberculosis in babies and adults remains epidemic in South Africa. The immune responses of the naïve newborn child and how they are affected by vaccination with BCG are as yet not fully understood. Immunity during pregnancy and in healthy human newborns may be skewed toward type 2 cytokine production; however, it is type 1 cytokines that are required for protection against M. tuberculosis infection. To better understand neonatal cytokine responses prior to and following exposure to mycobacteria, we have collected cord blood and peripheral blood samples and evaluated the cytokine response following ex vivo incubation with BCG. Gamma interferon (IFN-γ), interleukin 10 (IL-10), IL-12, and low levels of IL-13 and IL-5 but no IL-4 were secreted into the culture supernatant of cord blood mononuclear cells. Intracellular staining showed that IL-10 and IL-12 were produced by monocytes and that IFN-γ was produced by natural killer (NK) cells but not by CD4⁺ or CD8⁺ T cells. In contrast, in the peripheral blood samples collected from babies 13 weeks post-BCG vaccination, IFN-γ was detected within CD4⁺ and CD8⁺ cells. Taken together, the data suggest a central role for Th1 cytokines in naïve as well as BCG-vaccinated neonates in the protective immune response to tuberculosis. NK cell-derived IFN-γ produced in naïve neonates likely plays a key protective role via monocyte activation and the priming of a subsequent adaptive Th1 response.     

Ambiguous Role of Interleukin-12 in Yersinia enterocolitica Infection in Susceptible and Resistant Mouse Strains

Endogenous interleukin-12 (IL-12) mediates protection against Yersinia enterocolitica in C57BL/6 mice by triggering gamma interferon (IFN-γ) production in NK and CD4⁺ T cells. Administration of exogenous IL-12 confers protection against yersiniae in Yersinia-susceptible BALB/c mice but exacerbates yersiniosis in resistant C57BL/6 mice. Therefore, we wanted to dissect the different mechanisms exerted by IL-12 during Yersinia infections by using different models of Yersinia-resistant and -susceptible mice, including resistant C57BL/6 mice, susceptible BALB/c mice, intermediate-susceptible wild-type 129/Sv mice, 129/Sv IFN-γ-receptor-deficient (IFN-γR^−/−) mice and C57BL/6 tumor necrosis factor (TNF) receptor p55 chain-deficient (TNFR p55^−/−) mice. IFN-γR^−/− mice turned out to be highly susceptible to infection by Y. enterocolitica compared with IFN-γR^+/+ mice. Administration of IL-12 was protective in IFN-γR^+/+ mice but not in IFN-γR^−/− mice, suggesting that IFN-γR-induced mechanisms are essential for IL-12-induced resistance against yersiniae. BALB/c mice could be rendered Yersinia resistant by administration of anti-CD4 antibodies or by administration of IL-12. In contrast, C57BL/6 mice could be rendered more resistant by administration of transforming growth factor β (TGF-β). Furthermore, IL-12-triggered toxic effects in C57BL/6 mice were abrogated by coadministration of TGF-β. While administration of IL-12 alone increased TNF-α levels, administration of TGF-β or TGF-β plus IL-12 decreased both TNF-α and IFN-γ levels in Yersinia-infected C57BL/6 mice. Moreover, IL-12 did not induce toxicity in Yersinia-infected TNFR p55^−/− mice, suggesting that TNF-α accounts for IL-12-induced toxicity. Taken together, IL-12 may induce different effector mechanisms in BALB/c and C57BL/6 mice resulting either in protection or exacerbation. These results are important for understanding the critical balance of proinflammatory and regulatory cytokines in bacterial infections which is decisive for beneficial effects of cytokine therapy.     

T-Cell-Mediated Immune Responses in Patients with Cutaneous or Mucosal Leishmaniasis: Long-Term Evaluation after Therapy

T-cell immune responses in patients with cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) were studied during the active disease, at the end of therapy, and 1 to 17 years posttherapy (long-term follow-up). Lymphocyte proliferative responses, phenotypic characterization of CD4⁺ and CD8⁺ Leishmania-reactive T cells, and cytokine production were assayed. Patients with active ML and CL showed higher proportions of CD4⁺ than CD8⁺ T cells. In CL, the healing process was associated with a decrease of CD4⁺ and an increase of CD8⁺, leading to similar CD4⁺ and CD8⁺ proportions. This pattern was only seen in ML after long-term therapy. Long-term follow-up of patients with CL showed a positive CD4⁺/CD8⁺ ratio as observed during the active disease, although the percentages of these T cell subsets were significantly lower. Patients with CL did not show significant differences between gamma interferon (IFN-γ) and interleukin-5 (IL-5) production during the period of study. Patients with active ML presented higher IFN-γ and IL-5 levels compared to patients with active CL. IL-4 was only detected during active disease. Patients long term after cure from ML showed increasing production of IFN-γ, significant decrease of IL-5, and no IL-4 production. Two apparently beneficial immunological parameters were detected in tegumentary leishmaniasis: (i) decreasing proportions of CD4⁺ Leishmania-reactive T cells in the absence of IL-4 production associated with cure of CL and ML and (ii) decreasing levels of IL-5 long after cure, better detected in patients with ML. The observed T-cell responses maintained for a long period in healed patients could be relevant for immunoprotection against reinfection and used as a parameter for determining the prognosis of patients and selecting future vaccine preparations.     

Activation of Human NK Cells by Staphylococci and Lactobacilli Requires Cell Contact-Dependent Costimulation by Autologous Monocytes

NK cells are instrumental in innate immune responses, in particular for the early production of gamma interferon (IFN-γ) and other cytokines necessary to control certain bacterial, parasitic, and viral infections. NK cell-mediated effector functions are controlled by a fine balance between distinct receptors mediating activating and inhibitory signals; however, little is known about activating receptors on NK cells and their corresponding ligands. Several studies have shown that commensal lactobacilli isolated from the human gastrointestinal tract activate human mononuclear cells and are potent inducers of IFN-γ and monocyte-derived interleukin 12 (IL-12). NK cell activation was shown for Lactobacillus johnsonii La1. In this study the cellular mechanisms of in vitro NK cell activation by gram-positive bacteria were analyzed. Staphylococcus aureus- and L. johnsonii La1-mediated activation of CD3⁻ CD16⁺ CD56⁺ human peripheral blood NK cells, including expression of the activation antigen CD69 and secretion of IFN-γ, required cell contact-dependent costimulation by autologous monocytes. S. aureus- and L. johnsonii-preactivated monocytes retained their capacity to induce NK cell activation. In contrast, cytokine-primed monocytes completely failed to induce NK cell activation unless bacteria were present. This suggests that phagocytosis of bacteria provided additional coactivation signals on accessory cells that may differ from those induced by tumor necrosis factor and IFN-γ. Blocking of costimulatory molecules by B7.1, B7.2, and IL-12 but not CD14 monoclonal antibodies inhibited S. aureus- and L. johnsonii-induced effector function of NK cells. Our data suggest an important role for accessory cell-derived signals in the process of NK cell activation by gram-positive bacteria.     

Association of CD4+ CD25+ T Cells with Prevention of Severe Destructive Arthritis in Borrelia burgdorferi-Vaccinated and Challenged Gamma Interferon-Deficient Mice Treated with Anti-Interleukin-17 Antibody

CD4⁺ CD25⁺ T cells are a population of regulatory T cells responsible for active suppression of autoimmunity. Specifically, CD4⁺ CD25⁺ T cells have been shown to prevent insulin-dependent diabetes mellitus, inflammatory bowel disease, and pancreatitis. Here, we present evidence that CD4⁺ CD25⁺ T cells also play a major role in controlling the severity of arthritis detected in Borrelia burgdorferi-vaccinated gamma interferon-deficient (IFN-γ°) C57BL/6 mice challenged with the Lyme spirochete. When B. burgdorferi-vaccinated and challenged IFN-γ° mice were treated with anti-interleukin-17 (IL-17) antibody, the number of CD4⁺ CD25⁺ T cells increased in the local lymph nodes. Furthermore, histopathologic examination showed the mice to be free of destructive arthritis. When these anti-IL-17-treated B. burgdorferi-vaccinated and challenged mice were also administered anti-CD25 antibody, the number of CD4⁺ CD25⁺ T cells in the local lymph nodes decreased. More importantly, severe destructive arthropathy was induced. In addition, delayed administration of anti-CD25 antibody decreased the severity of the arthritis. These results suggest that CD4⁺ CD25⁺ T cells are involved in regulation of a severe destructive arthritis induced with an experimental model of vaccination and challenge with B. burgdorferi.     

Sulfasalazine prevents T-helper 1 immune response by suppressing interleukin-12 production in macrophages

Interleukin-12 (IL-12) plays a pivotal role in the development of T-helper 1 (Th1) immune response, which may be involved in the pathogenesis of chronic inflammatory autoimmune disorders. In this study we investigated the effects of sulfasalazine, a drug for treating inflammatory bowel disease and rheumatoid arthritis, on the production of IL-12 from mouse macrophages stimulated with lipopolysaccharide (LPS). Sulfasalazine potently inhibited the production of IL-12 in a dose-dependent manner, in part through the down-regulation of nuclear factor κB (NFκB) activation in IL-12 p40 gene. Activation of macrophages by LPS resulted in markedly enhanced binding activities to the κB site, which significantly decreased upon addition of sulfasalazine as demonstrated by an electrophoretic gel shift assay. Importantly, macrophages pretreated with sulfasalazine either in vitro or in vivo reduced their ability to induce interferon-γ (IFN-γ) and increased the ability to induce IL-4 in antigen-primed CD4⁺ T cells. From these results, sulfasalazine may induce the Th2 cytokine profile in CD4⁺ T cells by suppressing IL-12 production in macrophages, and sulfasalazine-induced inhibition of IL-12 production in macrophages may explain some of the known biological effects of sulfasalazine.     

Activated Pulmonary Macrophages Are Insufficient for Resistance against Pneumocystis carinii

CD4⁺ T cells are pivotal for elimination of Pneumocystis carinii from infected lungs, and alveolar macrophages are considered the main effector cells clearing the infected host of P. carinii organisms. To investigate this issue, several mutant mouse strains were used in a previously established experimental setup which facilitates natural acquisition of disease through inhalation of airborne fungal organisms. Mutant mice deficient in major histocompatibility complex class II molecules (Aβ^−/−), T-cell receptor αβ cells (TCRβ^−/−), or all mature T and B lymphocytes (RAG-1^−/−) were naturally susceptible to P. carinii, whereas mouse mutants lacking the gamma interferon (IFN-γ) receptor (IFN-γ-R^−/−) or tumor necrosis factor alpha (TNF-α) type I receptor (p55) (TNF-α-RI^−/−) resisted disease acquisition. Analysis of pulmonary cytokine patterns and free radical expression revealed the presence of superoxide, nitric oxide, and interleukin-1 (IL-1) mRNA and elevated levels of IFN-γ, TNF-α, and IL-12 in diseased TCRβ^−/− and RAG-1^−/− mice. Pulmonary macrophages of all diseased mouse mutants expressed scavenger and mannose receptors. Morbid Aβ^−/− mutants displayed significant NO levels and IL-1 mRNA only, whereas heterozygous controls did not exhibit any signs of disease. Interestingly, neither IFN-γ nor TNF-α appeared to be essential for resisting natural infection with P. carinii, nor were these cytokines sufficient for mediating resistance during established disease in the absence of CD4⁺ T lymphocytes. Taken together, the results indicated that an activated phagocyte system, as evidenced by cytokine and NO secretion, in diseased mutants was apparently operative but did not suffice for parasite clearance in the absence of CD4⁺ TCRαβ cells. Therefore, additional pathways, possibly involving interactions of inflammatory cytokines with CD4⁺ T lymphocytes, must contribute to successful resistance against P. carinii.Immunocompromised patients, especially those suffering from AIDS, are at elevated risk of acquiring Pneumocystis carinii pneumonia (PCP), a major cause of premature mortality among AIDS patients (8, 35, 53). Various studies have emphasized that CD4⁺ T lymphocytes play a pivotal role in the orchestration of resistance to P. carinii (22, 43, 45), an opportunistic fungus, but the mechanisms underlying protection remain a conundrum. Pulmonary macrophages are considered the main effector cells in clearing the immunocompetent host from invading P. carinii organisms (25). It seems conceivable, therefore, that macrophage-activating functions mediated by CD4⁺ T cells are central to resistance. Impaired gamma interferon (IFN-γ) production by T cells from AIDS patients is thought to enhance susceptibility to P. carinii (34, 41). This notion is supported by reports that application of exogenous IFN-γ ameliorates disease in experimental animal models (2, 45). In contrast, in vivo neutralization of IFN-γ in spleen cell-reconstituted severe combined immunodeficiency (SCID) mice by a specific monoclonal antibody (MAb) does not affect parasite clearance (5). Further studies point to a critical role of tumor necrosis factor alpha (TNF-α) (5) and interleukin-1 (IL-1) (6) in maintaining an immunocompetent state. Both cytokines are mainly produced by macrophages and induce inflammatory responses (4, 10, 26). Overall, these findings support involvement of macrophage-derived cytokines in successful host resistance against P. carinii.To analyze in more depth the role of inflammatory and Th1/Th2-related pulmonary defense mechanisms in control of aerogenically acquired PCP, we took advantage of naturally susceptible gene disruption mutant mice lacking major histocompatibility complex (MHC) class II molecules (and therefore conventional CD4⁺ T cells) (Aβ^−/−), T-cell receptor (TCR) αβ cells (TCRβ^−/−), or all mature T and B lymphocytes (RAG-1^−/−) (19). We further exploited mice deficient in the IFN-γ receptor (IFN-γ-R^−/−) or the TNF-α type I receptor (p55) (TNF-α-RI^−/−) to analyze their capacity to cope with aerogenic P. carinii organisms.Bronchoalveolar lavage (BAL) cells of healthy and diseased mice were investigated for expression of the proinflammatory cytokines IL-1, TNF-α, IFN-γ, and IL-12, as well as IL-4, IL-5, and IL-10. The latter three cytokines counteract IFN-γ- and IL-12-mediated responses but participate in protection against certain extracellular pathogens (9). Moreover, production of superoxide (SO) and nitric oxide (NO), putative effector molecules of antimicrobial defense, was taken as a further indicator of macrophage activation. Contact with foreign material induces a rapid respiratory burst in professional phagocytes which results in SO production as a first line of defense. SO has been implicated in destruction of P. carinii (31), whereas NO produced by IFN-γ-stimulated macrophages encountering pathogens (4, 18, 30) does not appear to participate in control of P. carinii infection (47). Of further interest was the role of macrophage-expressed mannose receptors (MR) and scavenger receptors (SR). MR were previously found crucial for mediating P. carinii internalization (11, 37). The relevance of SR with respect to PCP has not been evaluated, but they are mainly expressed by tissue macrophages (36) and nonspecifically bind a large array of molecules, including surface molecules of microorganisms (39). Receptors with such broad pattern reactivity may be involved in direct differentiation of self from non-self, and recent data suggest that not only MR but also SR aid pattern recognition by macrophages and subsequent internalization of invading pathogens (27).We found that BAL cells from P. carinii-diseased RAG-1^−/− and TCRβ^−/− mutants secreted elevated IFN-γ, TNF-α, IL-12, NO, and SO levels and expressed IL-1 mRNA. In contrast, cells from morbid Aβ^−/− mice produced IL-1 mRNA and high levels of NO only, whereas all other parameters were low to absent in these mutants. SR were expressed on pulmonary macrophages of all diseased RAG-1^−/−, TCRβ^−/−, and Aβ^−/− mutants, whereas MR were also expressed by macrophages of healthy animals. Yet, the apparently activated phagocyte system in the lung, most pronounced in morbid TCRβ^−/− and RAG-1^−/− mutant mice, was insufficient for protection against natural P. carinii infection. Elevated levels of IFN-γ and TNF-α in morbid mutants (not in Aβ^−/− mice) and the naturally resistant status of IFN-γ-R^−/− and TNF-α-RI^−/− mice further argue not only for independence from IFN-γ and TNF-α. Our findings indicate that CD4⁺ αβ T lymphocytes prevent and clear infection with P. carinii by mechanisms distinct from, or in addition to, pulmonary macrophage activation.(This study is part of the Ph.D. thesis of R. Hanano.)     

Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway

Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach-allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P < 0·05), while enhanced soluble CD40 level in the culture supernatant (P < 0·05) compared to the heat-inactivated Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)-12 and interferon (IFN)-γ secretion by DCs (P < 0·05) compared to heat-inactivated Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Active Per a 10 stimulation caused low nuclear factor-kappa B (NF-κB) activation in DCs compared to heat-inactivated Per a 10. Inhibition of the NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs, further indicating that NF-κB is required for CD40 up-regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38 mitogen-activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion of IL-4, IL-6, IL-12 and TNF-α by CD4⁺ T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels, thereby modulating DC-mediated immune responses.