Alternative induction of alpha/beta interferons and gamma interferon by listeria monocytogenes in mouse spleen cell cultures

Production of interferon (IFN) by Listeria monocytogenes (LM) in nonimmunized mouse spleen cell cultures was studied. IFN-gamma defined by virtue of its acid stability and antigenicity was produced in spleen cell cultures obtained from ddY mice, C57BL/6 mice, and BALB/c mice in response to heat-killed (HK) LM within 24 hr. On the other hand, production of IFN-alpha/beta was demonstrated in spleen cell cultures obtained from one of four nude mice (BALB/c, nu/nu). Therefore, it is important to know the reason why the spleen cells of mice other than nude mice did produce only IFN-gamma, but did not produce IFN-alpha/beta in response to HK-LM. Spleen cells obtained from ddY mice were fractionated, and the cellular source for IFN production of either IFN-alpha/beta or IFN-gamma induced by HK-LM was investigated. IFN-gamma was produced only by a mixture of T lymphocytes (nylon wool-nonadherent, Thy-1-positive cells) and macrophages by HK-LM. Neither T lymphocytes nor macrophages alone produced IFN by HK-LM. Macrophage-depleted spleen cells produced neither IFN-gamma nor IFN-alpha/beta, but these cells acquired the ability to produce IFN-alpha/beta, not IFN-gamma, only when they had been treated with IFN-alpha/beta. A possible mechanism of both IFN-gamma and IFN-alpha/beta induction by Listeria in mouse spleen cell cultures is discussed.     

Interferon-gamma induction by lipopolysaccharide: dependence on interleukin 2 and macrophages

Bacterial lipopolysaccharide (LPS) induced fresh murine splenocytes to produce interferon (IFN)-alpha/beta presumably by stimulation of the B lymphocytes and macrophages. However, when the splenocytes were "aged" for 24 to 72 hr in culture before addition of the LPS, the IFN response was significantly increased and was determined to be predominantly IFN-gamma. Because low levels of interleukin 2 (IL 2) were found to be spontaneously produced by the unstimulated splenocytes during the "aging" process, the effect of IL 2 on IFN induction by LPS in fresh splenocytes was examined. The addition of LPS to freshly prepared splenocyte cultures that were treated with human IL 2, either native or recombinant, before exposure to the LPS resulted in the LPS inducing large amounts of IFN-gamma. IL 2 alone induced little if any IFN in the splenocyte cultures. Depletion of T cells and large granular lymphocytes (LGL) from the cultures by anti-Thy-1.2 antibodies plus complement abrogated IFN-gamma production, and the addition of polymyxin B to "aged" splenocyte cultures resulted in loss of IFN production in response to LPS. Cultures that were enriched for T cells and LGL by passage through nylon wool produced significant amounts of IFN-gamma in response to LPS only if first treated with IL 2. Furthermore, the addition of splenic adherent cells to purified nylon wool-non-adherent (NWNA) cells augmented IFN-gamma production, whether or not the NWNA cells were pretreated with IL 2. This enhancement appeared to require direct contact between adherent cells and NWNA cells, because physical separation abrogated IFN production. The addition of recombinant IL 1 or LPS-conditioned supernatants of macrophage cultures did not replace adherent cell activity. These data demonstrate that LPS, which predominantly induces IFN-alpha/beta in fresh murine splenocytes, is able to stimulate T lymphocytes to produce IFN-gamma if the T cells are first exposed to endogenously produced or exogenously applied IL 2. Because IFN-gamma is a potent activator of the bactericidal and cytocidal potential of macrophages, the induction of IFN-gamma by bacterial LPS may play an important role in resistance/recovery mechanisms against bacterial infections.     

Reactivation of latent human cytomegalovirus in CD14(+) monocytes is differentiation dependent.

We have previously demonstrated reactivation of latent human cytomegalovirus (HCMV) in myeloid lineage cells obtained from healthy donors. Virus was obtained from allogenically stimulated monocyte-derived macrophages (Allo-MDM), but not from macrophages differentiated by mitogenic stimulation (ConA-MDM). In the present study, the cellular and cytokine components essential for HCMV replication and reactivation were examined in Allo-MDM. The importance of both CD4(+) and CD8(+) T cells in the generation of HCMV-permissive Allo-MDM was demonstrated by negative selection or blocking experiments using antibodies directed against both HLA class I and HLA class II molecules. Interestingly, contact of monocytes with CD4 or CD8 T cells was not essential for reactivation of HCMV, since virus was observed in macrophages derived from CD14(+) monocytes stimulated by supernatants produced by allogeneic stimulation of peripheral blood mononuclear cells. Examination of the cytokines produced in Allo-MDM and ConA-MDM cultures indicated a significant difference in the kinetics of production and quantity of these factors. Further examination of the cytokines essential for the generation of HCMV-permissive Allo-MDM identified gamma interferon (IFN-gamma) but not interleukin-1 or -2, tumor necrosis factor alpha, or granulocyte-macrophage colony-stimulating factor as critical components in the generation of these macrophages. In addition, although IFN-gamma was crucial for reactivation of latent HCMV, addition of IFN-gamma to unstimulated macrophage cultures was insufficient to reactivate virus. Thus, this study characterizes two distinct monocyte-derived cell types which can be distinguished by their ability to reactivate and support HCMV replication and identifies the critical importance of IFN-gamma in the reactivation of HCMV.     

Participation of macrophages in enhanced in vitro immune interferon (IFN gamma) production with mouse spleen cells

IFN gamma production in cultures of spleen cells obtained from mice sensitized with TH69, a live Streptococcus faecalis preparation, was examined to determine how macrophages participate. It was demonstrated that sensitized spleen macrophages participated in enhanced IFN gamma production by T cells at an early stage (0-6 hr) of incubation, and that this production is mainly dependent on Ia-bearing macrophages In the reconstitution experiments where different combinations of spleen macrophages and T cells obtained from mice sensitized with TH69, OK-432, and BCG were used, T cells required that the identity between the sensitizing organisms in vivo and the stimulating organisms in vitro be the same for enhanced IFN gamma production while macrophages did not. Macrophage-mediated production of IFN gamma appears to be genetically restricted because IFN gamma was only produced in cultures where the H-2 region of macrophages and T cells matched. Further examination revealed that for macrophages to participate in enhanced IFN gamma production, first contact between cycloheximide-treated macrophages and T cells was required. Second, enhanced IFN gamma production occurred when culture supernatants of macrophages obtained from sensitized spleen cells were added to T cells. However, the addition of culture supernatant obtained from sensitized peritoneal macrophages resulted in inhibition of IFN gamma production. These results clearly showed the crucial role of macrophages in enhanced IFN gamma production by spleen T cells in vitro.     

Interleukin 2-mediated immune interferon (IFN-gamma) production by human T cells and T cell subsets

Human interleukin 2 (IL 2, or T cell growth factor), which was free of lectin and interferon activity (IFN), induced human peripheral T lymphocytes to produce immune IFN (IFN-gamma). In contrast, non-T cells and macrophages did not produce IFN-gamma in response to IL 2. IL 2 acted directly on unstimulated T cells to induce IFN-gamma production, and also acted in synergy with a suboptimal dose (2 micrograms/ml) of concanavalin A (Con A) to enhance IFN-gamma production. The IFN-gamma-inducing activity of partially purified IL 2 was absorbed along with the IL 2 activity by murine IL 2-dependent CT-6 cell line cells. This further supports the view that IFN-gamma-inducing activity is identical to IL 2. When T cells were separated further into helper/inducer T4+ and suppressor/cytotoxic T8+ subsets by negative selection with monoclonal antibody and complement, both T4+ and T8+-enriched cells produced significant levels of IFN-gamma in response to IL 2. Complete removal of macrophages from purified T lymphocyte populations by treatment of OKM1 plus complement consistently reduced IFN-gamma production in response to IL 2 to a limited degree; readdition of macrophages restored IFN-gamma production by both T cell subsets. This observation that IL 2 contributes to the production of IFN-gamma by human lymphocytes suggests that a cascade of lymphocyte-cell interactions participates in human immune responses.     

In vitro production of tumor necrosis factor by murine splenic macrophages stimulated with mannoprotein constituents of Candida albicans cell wall.

Mannoprotein components from Candida albicans were investigated for their ability to induce production of tumor necrosis factor (TNF) by cultured splenocytes from naive or Candida-infected mice. Two chromatographically separated mannoproteins preparations, designated F1 and F2, were as able as the heat-inactivated Candida cells to induce the production of TNF from splenocytes of naive animals. In addition, they caused a significant augmentation of basic TNF secretion by splenocytes of Candida-infected animals. Experiments using plastic and/or nylon wool adherence, as well as treatments with antibodies depleting T or NK cells, consistently indicated that most if not all TNF was produced by splenic macrophages. In cultures of splenocytes from Candida-infected mice, mannoprotein addition also stimulated interferon-gamma (IFN-gamma) production by Thy 1.2 positive cells. Depletion of these cells or addition of anti-IFN-gamma antibodies abolished IFN production and reduced TNF secretion by adherent cells to the levels found in the cultures of mannoprotein-stimulated spleen cells from naive mice. These data add further evidence to the immunomodulatory properties possessed by some cell wall constituents of the human commensal microorganism C. albicans and suggest that IFN-gamma is endowed with a regulatory role in TNF production by mouse macrophages in vitro.     

Polarized type 1 cytokine profile in bronchoalveolar lavage T cells of patients with hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is characterized by an inflammatory lymphocytic alveolitis comprised of both CD8+ and CD4+ T cells. Animal models suggest that HP is facilitated by overproduction of IFN-gamma, and that IL-10 ameliorates severity of the disease, indicating a Th1-type response. To determine whether a Th1 phenotype in HP also exists clinically, bronchoalveolar lavage (BAL) and peripheral blood (PB) T cells were obtained from HP individuals and analyzed for Th1 vs Th2 cytokine profiles. It was determined that soluble OKT3-stimulated BAL T cells cocultured with alveolar macrophages produced more IFN-gamma and less IL-10 than PB T cells cocultured with monocytes, but no difference was observed in IL-4 production. The monocytic cells did not account for this difference, as CD80 and CD86 expressions were similar, and coculturing PB T cells with alveolar macrophages resulted in no difference in IFN-gamma production. Similarly, there was no difference in IL-12 production between stimulated BAL or PB T cells; however, addition of rIL-12 significantly increased production of IFN-gamma by BAL T cells, but not by PB T cells. This effect was due to a difference in IL-12R expression. High affinity IL-12R were only present in association with BAL T cells. These studies indicate that clinical HP is characterized by a predominance of IFN-gamma-producing T cells, perhaps resulting from a reduction in IL-10 production and an increase in high affinity IL-12R compared with blood T cells.     

Leishmania sp: comparative study with Toxoplasma gondii and Trypanosoma cruzi in their ability to initialize IL-12 and IFN-gamma synthesis

We compared in vitro and in vivo induction of IL-12 (p40) and IFN-gamma by mouse cells stimulated with Toxoplasma gondii, Trypanosoma cruzi, and different species of Leishmania. Spleen cells cultured in vitro with T. cruzi or T. gondii, but not with Leishmania, produced IL-12 (p40) and IFN-gamma. Accordingly, IL-12 (p40) was produced by macrophages stimulated in vitro with live T. cruzi or T. gondii or membrane glycoconjugates obtained from trypomastigotes or tachyzoites. No IL-12 production was detected when macrophages were stimulated with live parasites or glycoconjugates from Leishmania, regardless of priming with IFN-gamma. In vivo, only T. cruzi and T. gondii induced the synthesis of IL-12 and IFN-gamma by mouse spleen cells after intraperitoneal injection of parasites. When injected subcutaneously, live Leishmania sp. induced IL-12 (p40) and IFN-gamma production by draining lymph node cells, albeit the levels were slightly lower than those induced by infection with T. gondii or T. cruzi using the same route. Together our results indicate that under different conditions, the intracellular protozoa T. gondii and T. cruzi are more potent stimulators of IL-12 and IFN-gamma synthesis by host immune cells than parasites of the genus Leishmania.     

Alterations of interferon production in a mouse model of thermal injury

The effect of thermal injury on the response of interferon (IFN) production in vivo and in vitro after stimulation with eight representative inducers was investigated in a mouse model. The response of mice to immune IFN (IFN-gamma) inducers, staphylococcal enterotoxin A, concanavalin A, and a specific antigen for BCG-sensitized lymphocytes (purified protein derivative) was impaired after a 30% total body surface area third-degree burn. Suppression of IFN-gamma production was observed at day 2 and persisted until day 7 after burn. Decreased IFN-gamma production correlated closely with the percentage of total body surface area burned. When virus type IFN (IFN-alpha/beta) inducers, Newcastle disease virus, polyriboinosinic-polyribocytidylic acid, 10-carboxymethyl-9-acridanone, and E. coli endotoxin, were administered to mice, no change in IFN response was observed after thermal injury. Similar results were obtained when spleen cells obtained from thermally injured mice were stimulated with IFN-gamma inducers in vitro. These studies suggest that although the capacity for IFN-alpha/beta production remains intact in thermally injured mice, IFN-gamma production may be selectively decreased in burned animals and in their spleen cells.     

Endotoxin-induced interferon-gamma production in culture cells derived from BCG-infected C3H/HeJ mice

The cultured cells prepared from the spleens and peritoneal exudate cells of the C3H/HeJ strain of mice produce very little or no interferon (IFN) by stimulation of bacterial lipopolysaccharide (LPS). However, the cells taken from LPS-non-responder C3H/HeJ mice which had been infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) prior to the experiment were capable of producing IFN in culture in the presence of LPS. The peritoneal exudate cells of BCG-primed C3H/HeJ mice were separated into adherent cell and nonadherent cell populations by their adhesiveness to plastic culture dishes. IFN production required the presence of both these cell populations in the same culture, and the IFN activities produced were mainly IFN-gamma. The cultures with nonadherent cells and fixed adherent cells still produced IFN, but the cell cultures reconstituted with the BCG-primed cell population and unprimed cell population produce little if any IFN-gamma. Moreover, when both of the populations were cultured in Marbrook culture vessels separated by a membrane filter, the cultures produced very little or no IFN-gamma. These results indicate that there is a mechanism of IFN-gamma induction by LPS which requires the direct contact between adherent cells and nonadherent cells without the participation of any soluble factor(s) from the adherent cells. The producer cells were mainly in the nonadherent cell population. Previous treatment of nonadherent cells with anti-Thy-1.2 antibody, anti-Lyt-1.1 antibody, anti-L3T4 antibody, or anti-asialo-GM1 antibody and complement diminished the ability of the cells for LPS-induced IFN production with the help of adherent cells. Therefore, it is concluded that both T cells (presumably L3T4+T cells) and asialo-GM1+ natural killer cells in the BCG-primed C3H/HeJ cell cultures produced IFN-gamma in the presence of LPS, and the production was supported by the function of macrophages.     

Mouse CD8+ CD122+ T cells with intermediate TCR increasing with age provide a source of early IFN-gamma production

Although CD8+ IL-2Rbeta (CD122)+ T cells with intermediate TCR reportedly develop extrathymically, their functions still remain largely unknown. In the present study, we characterized the function of CD8+ CD122+ T cells with intermediate TCR of C57BL/6 mice. The proportion of CD8+ CD122+ T cells in splenocytes gradually increased with age, whereas CD8+ IL-2Rbeta-negative or -low (CD122-) T cells conversely decreased. The IFN-gamma production from splenocytes stimulated with immobilized anti-CD3 Ab in vitro increased with age, whereas the IL-4 production decreased. When sorted CD8+ CD122+ T cells were stimulated in vitro by the anti-CD3 Ab, they promptly produced a much larger amount of IFN-gamma than did CD8+ CD122- T cells or CD4+ T cells, whereas only CD4+ T cells produced IL-4. The depletion of CD8+ CD122+ T cells from whole splenocytes greatly decreased the CD3-stimulated IFN-gamma production and increased the IL-4 production, whereas the addition of sorted CD8+ CD122+ T cells to CD8+ CD122+ T cell-depleted splenocytes restored the IFN-gamma production and partially decreased IL-4 production. It is of interest that CD8+ CD122+ T cells stimulated CD4+ T cells to produce IFN-gamma. The CD3-stimulated IFN-gamma production from each T cell subset was augmented by macrophages. Furthermore, CD3-stimulated CD8+ CD122+ T cells produced an even greater amount of IFN-gamma than did liver NK1.1+ T cells and also showed antitumor cytotoxicity. These results show that CD8+ CD122+ T cells may thus be an important source of early IFN-gamma production and are suggested to be involved in the immunological changes with aging.     

Interferon production and tumor cell killing by human lymphocytes stimulated in mixed-lymphocyte culture

The in vitro synthesis of interferon (IFN) by human lymphocytes stimulated in mixed-lymphocyte culture (MLC) was examined. The production of IFN in MLC was restricted to T lymphocytes and maximum levels of IFN were detected in supernatants from cells incubated for 5 to 7 days. The IFN produced was identified as IFN-gamma by antibody neutralization. To identify the T cell responsible for IFN production, purified T lymphocytes were separated into subpopulations after incubation in 5 mM theophylline. Theophylline-resistant (T-res) T cells retain the ability to form sheep erythrocyte (SRBC) rosettes and are depleted in IgG Fc receptor-positive T cells (T gamma cells). Theophylline-sensitive (T-sens) T cells fail to form rosettes after theophylline treatment and are enriched in T gamma cells. In addition, analyses using monoclonal antibodies showed that T-sens cells were enriched in OKM1-, HNK-1-, and 7.2-positive cells and T-res cells contained increased numbers of 9.6- and OKT4-positive cells. Following MLC stimulation, equivalent levels of IFN-gamma were produced by T-res and T-sens cells and both subpopulations maintained natural killer (NK)-like cytotoxicity against K562 target cells. Addition of partially purified IFN-gamma to unstimulated T-res and T-sens cells resulted in the maintenance of NK-like cytotoxicity in a manner analogous to that observed after MLC. Additional experiments indicated that peripheral blood lymphocytes depleted of 9.6- or OKM1-positive cells by complement-mediated lysis were devoid of cytotoxicity against K562 cells. Furthermore, MLC stimulation of 9.6- or OKM1-depleted cells failed to restore cytotoxic activity. In summary, these experiments demonstrate that the maintenance of NK-like cytotoxicity by MLC-stimulated T cells is associated with the synthesis of IFN-gamma, that MLC stimulated T-res and T-sens T-cell subsets produce equivalent amounts of IFN, and that 9.6- or OKM1-positive cells are required for the maintenance of NK-like cytotoxicity in MLC.     

Antigen presentation to Th1 but not Th2 cells by macrophages results in nitric oxide production and inhibition of T cell proliferation: interferon-gamma is essential but insufficient

The induction and role of nitric oxide (NO) during antigen presentation by macrophages to T helper (Th) cell subsets was examined. When cultured with Th1 clones, macrophage APC produced NO only in the presence of cognate Ag, which in turn suppressed T cell proliferation. IFN-gamma production by the activated Th1 cells was essential for the induction of NO. Th2 cells presented with the same cognate Ag did not induce NO production and proliferated uninhibited. Coactivation of Th1 and Th2 cells specific for the same Ag indicated that Th2 cells did not inhibit NO production, but were sensitive to NO induced by stimulated Th1 cells. Antigenic activation of Th2 cells in the presence of rIFN-gamma resulted in NO-mediated inhibition of proliferation. Th2 cells provided only a cell-associated cofactor, whereas Th1 cells secreted a soluble cofactor for IFN-gamma as well, i.e., TNF-alpha. Finally, a role for IFN-gamma and NO during immune responses was studied in spleen cells obtained from immunized IFN-gamma(-/-) mice. NO production and subsequent inhibition of Ag-specific proliferation ex vivo was observed only after the addition of rIFN-gamma. These studies suggest an IFN-gamma-dependent regulatory role for NO during Ag-specific Th cell activation involving macrophages, with obvious implications for Th subset-dependent immune responses in general.     

Impaired production of gamma-interferon by newborn cells in vitro is due to a functionally immature macrophage

The decreased production of gamma-(PHA-induced) interferon (IFN) by leukocytes of normal newborns could be due to functionally immature T cells, macrophages, or both. We studied gamma-IFN production by macrophages and T cells, alone and in combination, obtained from 50 cord blood samples and 14 adult blood samples in a series of experiments. Adherent macrophages were cultivated for 7 days before the addition of T cells. After 48 hr, PHA-stimulated macrophage-T cell supernatants were harvested and assayed for IFN by a microassay. Macrophage-T cell cultures of autologous and nonautologous cells in 14 adults showed enhanced IFN production (GMT 121 +/- 5 IU) as compared with Ficoll-Hypaque mononuclear cells (GMT 42 +/- 5 IU). No IFN was detected in supernatants from PHA-stimulated Ficoll-Hypaque cord cells alone or macrophage-T cord combined cultures. Combined cord macrophages and adult T cells produced minimal IFN (GMT 13 +/- 3 IU); however, cord T cells combined with adult macrophages showed enhanced IFN production (GMT 195 +/- 47 IU). This cord macrophage dysfunction was not due to an inhibitor and improved with the time of in vitro cultivation. These results indicate that the neonatal macrophage is primarily responsible for the impaired gamma-IFN response by the newborn cells.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

Spontaneous production of gamma-interferon in cultures of T lymphocytes obtained from patients with Beh?et's disease

The spontaneous production of interferon (IFN) in the cultures of peripheral blood mononuclear leukocytes (PBML) obtained from 30 patients with Beh?et's disease was investigated. PBML obtained from each of 26 patients in the convalescent stage and four patients in the exacerbation stage were cultured at least twice without stimulation, and IFN activity in the culture fluid 2 to 7 days after the cultivation was assayed. Twenty PBML of normal healthy donors were also cultured simultaneously. PBML of all patients in the convalescent stage spontaneously produced high-titered IFN (60.0 +/- 9.5 IU/ml), but IFN activity produced in PBML cultures of four patients in the exacerbation stage was very low or was undetectable. Similarly, IFN was always detectable in the circulation of the patients whose PBML spontaneously produced IFN. All IFN activity detected both in the circulation and in the fluid of PBML culture from these patients was gamma-IFN, defined by virtue of its acid lability and antigenicity neutralized with antiserum for gamma-IFN and not with antisera for alpha- and beta-IFN. The cellular source of this gamma-IFN in the patients' PMBL was T lymphocytes and not non-T cells or macrophages. T lymphocytes did not require the help of macrophages. It is suggested that T lymphocytes of these patients may be stimulated by unknown causative agents in vivo and may produce gamma-IFN in vivo as well as in in vitro culture of T lymphocytes.     

T cell regulation of interferon-alpha/beta (IFN-alpha/beta) production by alloantigen-stimulated bone marrow cells

We have previously reported that mouse bone marrow cells produce high levels of interferon-alpha/beta (IFN-alpha/beta) after 5 to 6 days of in vitro culture with irradiated allogenic spleen cells. The current study was initiated to determine whether or not T cells are important for alloantigen-induced IFN-alpha/beta production by mouse bone marrow cells. Bone marrow cells and spleen cells were obtained from C57BL/6 mice. These cells were treated with different monoclonal antisera and complement, and then were cultured 5 to 6 days with irradiated DBA spleen cells. The results from these experiments indicated that optimal IFN-alpha/beta production by alloantigen-stimulated bone marrow cells required Lyt-1+2+ T cells. In addition, when bone marrow cells obtained from nu/nu B10 mice were cultured with alloantigen, only low levels of IFN were produced when compared with IFN production by bone marrow cells obtained from normal littermate B10 mice. The addition of nylon wool-enriched splenic T cells to cultures containing bone marrow cells and alloantigen resulted in an augmentation of IFN-alpha/beta production by three-fold to fivefold. Furthermore, bone marrow cells obtained from alloantigen-immunized mice produced much higher levels of IFN-alpha/beta and in a shorter period of time (2 to 3 days) when compared with bone marrow cells obtained from control or non-immunized mice. Cyclosporin A (CsA) has been shown to inhibit predominantly T cell-dependent responses. The effect of CsA on IFN production by alloantigen-stimulated bone marrow and spleen cells was investigated. The addition of CsA at concentrations as low as 0.1 micrograms/ml inhibited not only IFN-gamma production by alloantigen-stimulated spleen cells, but also IFN-alpha/beta production by alloantigen-stimulated bone marrow cells. In contrast, IFN-alpha/beta production by Newcastle disease virus-infected spleen cells, bone marrow cells, or L cells was not inhibited by the addition of CsA (1 microgram/ml). Thus, the ability of bone marrow cells to produce high levels of IFN-alpha/beta after in vitro culture with alloantigen is dependent upon T cells resident in the bone marrow. IFN-alpha/beta production by alloantigen-stimulated bone marrow cells may play a major role in the pathogenesis associated with graft-vs-host disease and in T cell regulation of hematopoiesis.     

Regulation of IFN-gamma induction in human peripheral blood cells by exogenous and endogenously produced interleukin 2

Interferon (IFN)-gamma production, stimulated by the addition of exogenous interleukin (IL) 2, T cell mitogens, or tuberculin purified protein derivative (PPD) was studied in cultures of separated human mononuclear cells or unseparated peripheral blood leukocytes (PBL). IFN-gamma was induced by the addition of IL 2 to cultures of otherwise unstimulated cells. The minimal concentration of exogenous IL 2 required to cause a reproducible stimulation of IFN-gamma was about 10 U/ml, i.e., approximately 50 times the minimal concentration required to stimulate proliferation in an IL 2-dependent murine cytotoxic T cell line. Approximately 500 to 1000 IL 2 U/ml were required to produce maximal stimulation of IFN-gamma production in otherwise unstimulated cultures. Monoclonal antibody anti-Tac, specific for an epitope associated with the IL 2 receptor (IL 2 R), inhibited IFN-gamma induction by exogenous IL 2 less strongly than induction by phytohemagglutinin (PHA) or concanavalin A (Con A). The highest degree of inhibition was exerted by anti-Tac on IFN-gamma production stimulated with PPD. Stimulation of IFN-gamma induction by exogenous IL 2 and the inhibitory action of anti-Tac on IFN-gamma production were also seen in cultures of irradiated (2000 R) cells. Treatment of cells with subinducing doses of Con A or phorbol myristate acetate increased IFN-gamma induction by exogenous IL 2. Taken together, the data suggest that endogenously generated IL 2 is a major mediator of IFN-gamma induction in PBL cultures stimulated with antigens or T cell mitogens.     

GM-CSF-mediated T-cell activation by macrophages infected with recombinant BCG that secretes major membrane protein-II of Mycobacterium leprae

The potential of Mycobacterium bovis Bacillus Calmette–Guerin (BCG) needs to be augmented to efficiently activate CD4⁺ T cells through macrophages. Mycobacterium leprae -derived recombinant major membrane protein (MMP)-II induced GM-CSF production from macrophages. A recombinant BCG-SM that secretes MMP-II more efficiently produced GM-CSF and activated interferon (IFN)-γ-producing CD4⁺ T cells than did vector control BCG when infected with macrophages. The T-cell activation by BCG-SM was dependent on the GM-CSF production by macrophages. Interleukin (IL)-10 production by macrophages stimulated with M. leprae was inhibited in a GM-CSF-dependent manner when the precursor monocytes were infected with BCG-SM. BCG inducing GM-CSF production was effective in macrophage-mediated T-cell activation partially through IL-10 inhibition.     

Interferon-induced transfer of viral resistance by human B and T lymphocytes

Enriched human B lymphocytes cocultivated with mouse L cells produced human leukocyte interferon (IFN-alpha) and shortly thereafter transferred antiviral activity to the recipient cells (99% inhibition of expected virus yield). In contrast, cocultivation of enriched T-cell populations with mouse L cells resulted in no IFN production or transfer of antiviral activity. In addition, both T and B lymphocytes pretreated with exogenous IFN or stimulated in vitro by mitogens could transfer antiviral activity to human WISH cells. The transfer of antiviral activity was not blocked by antibodies to IFN. The data indicate that both T and B cells can be recruited by IFN to transfer antiviral activity. Thus, once cells are recruited by IFN they can transfer antiviral activity in the absence of IFN and protect cells locally or distally from the site of infection.