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A Zlotnik

Publications and source records attributed to A Zlotnik.

At least 109 records · Page 6Linked to original sources
PubMed1991

Cytokine production by mature and immature thymocytes.

We have studied the ability of subpopulations of activated thymocytes to produce four cytokines (IL-2, IL-4, IFN-gamma and TNF-alpha) which are believed to play roles in T cell development. Supernatants from various thymocyte subsets activated with calcium ionophore and PMA were tested for these cytokines. All CD3hi thymocyte subsets (CD4+8-, CD4-8- and CD4-8+) produced high titers of these four cytokines except CD3+4-8+ thymocytes, which did not produce IL-4. In contrast, CD4+8+ thymocytes did not produce any detectable cytokines. CD3-4-8- thymocytes produced IL-2, IFN-gamma, and TNF-alpha (but not IL-4) when activated by calcium ionophore + PMA and IL-1. We then separated CD3-4-8- thymocytes into IL-2R+ and IL-2R-. CD3-4-8-IL-2R+ thymocytes only produced small amounts of IL-2 when activated with calcium ionophore + PMA + IL-1, whereas CD3-4-8-IL-2R- thymocytes did not require IL-1 to produce IL-2, IFN-gamma, and TNF-alpha. Finally, CD4-8+3- thymocytes (an immature population believed to be an intermediate between CD3-4-8- and CD4+8+ thymocytes) only produced marginally detectable levels of IL-2 upon stimulation with calcium ionophore, PMA, and the addition of IL-1 did not result in increased levels of cytokine production. These observations indicate discrete patterns of cytokine production by the subsets studied and suggest specific controls of cytokine gene expression during T cell development.

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PubMed1991

IL-7 maintains the T cell precursor potential of CD3-CD4-CD8- thymocytes.

We and other investigators have reported that IL-4 (in the presence of PMA) or IL-7 (used alone) induce proliferation of both adult and fetal (gestation day 15) CD4-CD8- thymocytes. These results suggested that these cytokines may be growth factors for pre-T cells. However, we recently observed that among adult CD4-CD8- thymocytes, only the CD3+ subset proliferates in response to IL-7, whereas IL-4 + PMA induces proliferative responses in both CD3- and CD3+ subsets. Thus, we concluded that IL-7 used alone is not a potent growth stimulus for adult thymic CD3-CD4-CD8- triple negative (TN) T cell precursors. Interestingly, the viability of adult TN thymocytes in culture was improved by IL-7 for up to 1 wk, in spite of the inability of IL-7 to induce significant [3H]TdR incorporation in these cells. After culture in IL-7 for 4 days, the viable cells remained CD4-CD8-, but 25 to 35% expressed CD3 whereas the rest remained CD3-. In contrast, most of the cells cultured with IL-4 + PMA for 4 days remained TN. To investigate whether adult TN thymocytes that survive in vitro in the presence of IL-4 + PMA or IL-7 retain T cell progenitor potential, we tested whether they could reconstitute lymphoid cell-depleted (2-deoxyguanosine-treated) fetal thymus organ cultures. Our results demonstrate that TN cells cultured in IL-7 retain T cell progenitor potential.

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PubMed1991

Antigen presenting ability of thymic macrophages and epithelial cells: evidence for defects in the antigen processing function of thymic epithelial cells.

We compared the antigen presenting ability of cloned thymic macrophage and epithelial cell lines using T cell hybridomas with well-characterized activation requirements. A cloned thymic epithelial cell line (3D.1), preinduced with interferon-gamma (IFN-gamma) activated the T cell hybridoma 3DO-18.3 but not the T cell hybridoma DO-11.10. Analyses using preprocessed antigen suggest that the failure of 3D.1 to activate DO-11.10 is due to its inability to process chicken ovalbumin to produce a peptide recognized by the Ag:MHC T cell receptor of DO-11.10. The epithelial cell line 3D.1 was able to activate DO-11.10 if the superantigen staphylococcal enterotoxin B was used for activation instead of ovalbumin. These observations indicate that IFN-gamma-induced 3D.1 expresses sufficient I-Ad molecules to activate DO-11.10 but is unable to produce the peptide of ovalbumin recognized by DO-11.10. Furthermore, 3D.1 appears to be representative of nonmacrophage thymic stromal cells cultured in vitro, since heterogeneous cultures containing epithelial cells exhibited the same selective T cell activation characteristics. In contrast, thymic macrophage cell lines activated all T cells studied. These results suggest that there is a functional difference between the capacity of thymic epithelial cells and macrophages to process and present antigen to T cells.

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PubMed1991

Lymphokine requirements for the development of specific cytotoxic T cells from single precursors.

A high cloning efficiency, filler cell-free culture system was developed for the growth of single murine cytotoxic T lymphocyte precursors (CTLp) and their differentiation into cytotoxic T lymphocytes (CTL). The system used nonspecific stimulation with phorbol ester and calcium ionophore in the presence of recombinant lymphokines. The optimal lymphokine combination was interleukin 2 throughout, together with interferon-gamma during the first 6 days and interleukin 6 during the last 2 days of culture. Under these conditions half of all CD4-CD8+ T cells became CTL clones. The CTL were CD4-CD8+CD3+ TcR alpha/beta+ and were derived from CD4-CD8+Pgp-1- precursors.

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PubMed1991

Regulation of thymocyte proliferation by alpha beta TcR+ CD3+ CD4- CD8- cloned natural suppressor (NS) cells.

Cloned alpha beta TcR+ CD3+ CD4- CD8- T cells, with natural suppressor (NS) activity, were cocultured with thymocytes in the presence or absence of mitogen or cytokines. Whereas thymocytes show a minimal response to phytohemagglutinin (PHA), IL-2, or IL-4 alone, they proliferate vigorously when cocultured with irradiated cloned NS cells in the presence of PHA or IL-2 or IL-4, but not with IL-1, IL-3, IL-6, IL-7, IFN-gamma, or GM-CSF. Among a total of 11 NS cell clones, derived from the spleen or thymus, only one clone (NR-1) did not induce thymocyte activation in synergy with PHA. This costimulation is most likely mediated by soluble factor(s), since supernatants, obtained from NS cells activated with phorbol ester (PMA) and calcymicin (A23187) or with solid phase anti-CD3 mAb, enhance thymocyte DNA synthesis in the presence of a mixture of PHA, IL-2, and IL-4. The latter factor does not appear to be a previously described lymphokine, since PMA- and A23187-activated NS cells secrete IL-3, TGF-beta, IFN-gamma, GM-CSF, and TNF-alpha, but not IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, nor IL-10. None of the factors, identified in the NS cell supernatants, was able to stimulate thymocyte DNA synthesis. This study shows that, in addition to their previously reported suppressor function, cloned NS cells can exert immunostimulatory activities by virtue of a soluble mediator.

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PubMed1991

Interleukin 10.

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PubMed1990

IL-10, a novel growth cofactor for mature and immature T cells.

We identified a new cytokine, B cell-derived T cell growth factor (B-TCGF), that is produced by a murine B cell lymphoma and induces proliferation of mature and immature thymocytes in the presence of IL-2 and IL-4. Both adult and day 15 fetal thymocytes (CD4-8-, CD4+8-, CD4-8+) proliferate strongly in the presence of IL-2, IL-4, and B-TCGF. B-TCGF alone does not stimulate thymocyte proliferation. B-TCGF appears to be identical to a novel cytokine whose cDNA was recently isolated at our institution, cytokine synthesis-inhibitory factor (CSIF; IL-10). rIL-10 has B-TCGF activity, and mAb specific for IL-10 inhibit the B-TCGF activity present in CH12 supernatants. Further studies have shown that day 15 fetal thymocytes cultured in the presence of IL-10, IL-2, and IL-4 remain CD4- and CD8- but exhibit increased CD3 expression. Adult CD4- CD8- thymocytes cultured under the same conditions proliferate whether they are CD3+ or CD3-. The CD3- population becomes enriched in CD3+ cells after 4 days of culture. IL-10 is secreted by day 15 fetal thymocytes, adult thymocytes, and adult splenocytes when stimulated via their TCR. IL-10 is strongly homologous to the EBV gene BCRFI, and BCRFI has CSIF activity. In contrast to IL-10, BCRFI does not exhibit detectable thymocyte-stimulating activity, suggesting the existence of at least two functional epitopes on the IL-10 molecule.

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PubMed1990

Growth-promoting activity of IL-1 alpha, IL-6, and tumor necrosis factor-alpha in combination with IL-2, IL-4, or IL-7 on murine thymocytes. Differential effects on CD4/CD8 subsets and on CD3+/CD3- double-negative thymocytes.

Many cytokines (including IL-1, IL-2, IL-4, IL-6, and TNF-alpha) have been shown to induce thymocyte proliferation in the presence of PHA. In this report, we demonstrate that certain cytokine combinations induce thymocyte proliferation in the absence of artificial comitogens. IL-1 alpha, IL-6, and TNF-alpha enhanced the proliferation of whole unseparated thymocytes in the presence of IL-2, whereas none of them induced thymocyte proliferation alone. In contrast, of these three enhancing cytokines, only IL-6 enhanced IL-4-induced proliferation. We also separated thymocytes into four groups based on their expression of CD4 and CD8, and investigated their responses to various cytokines. The results indicate that each cytokine combination affects different thymocyte subsets; thus, IL-1 alpha enhanced the proliferation of CD4-CD8- double negative (DN) thymocytes more efficiently than IL-6 in the presence of IL-2, whereas IL-6 enhanced the responses of CD4+CD8- and CD4-CD8+ single positive (SP) thymocytes to IL-2 or IL-4 better than IL-1 alpha. TNF-alpha enhanced the proliferation of both DN and both SP subsets in the presence of IL-2 and/or IL-7. None of these combinations induced the proliferation of CD4+CD8+ double positive thymocytes. Finally, DN were separated into CD3+ and CD3- populations and their responsiveness was investigated, because recent reports strongly suggest that CD3+ DN thymocytes are a mature subset of different lineage rather than precursors of SP thymocytes. CD3+ DN proliferated in response to IL-7, TNF-alpha + IL-2, and IL-1 + IL-2. CD3- DN did not respond to IL-7 or to IL-1 + IL-2, but did respond to TNF-alpha + IL-2. Finally, we detected TNF-alpha production by a cloned line of thymic macrophages, as well as by DN adult thymocytes. These results suggest that cytokines alone are capable of potent growth stimuli for thymocytes, and indicate that different combinations of these molecules act selectively on thymocytes at different developmental stages.

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PubMed1990

Tumor necrosis factor-alpha and P40 induce day 15 murine fetal thymocyte proliferation in combination with IL-2.

Cytokines are known to play a key role in the development of several hemopoietic lineages including lymphocytes. Two cytokines: IL-4 (in the presence of PMA) and IL-7 have been shown to induce immature fetal thymocyte proliferation. It has also been suggested that IL-2 plays an important role in fetal T cell development. In this report, we investigated the effects of several cytokines (known to be growth factors for T-lineage cells) on fetal thymocyte proliferation. Our results indicate that: 1) TNF-alpha and a newly described cytokine, P40, enhance fetal thymocyte proliferation stimulated by IL-2 (but not IL-4 or IL-7). 2) The enhancement induced by P40 is not mediated by TNF-alpha because blocking antibodies against this cytokine failed to inhibit this response. 3) IL-4 inhibits fetal thymocyte proliferation in response to TNF-alpha + IL-2 or to IL-7 but not to P40 + IL-2. Finally, 4) the proliferating cells to all cytokine combinations used were Thy-1+. These observations suggest that these cytokine combinations induce independent pathways of T cell proliferation in the developing thymus.

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PubMed1990

Identification of a novel thymocyte growth-promoting factor derived from B cell lymphomas.

We found a unique thymocyte growth-promoting activity in supernatants (SN) from subclones of the B cell lymphoma CH12.LX. We have tentatively named this activity B-TCGF (for B cell-derived T cell growth factor) and characterized the activity produced by the CH12.LX.4866 subclone. This SN did not induce thymocyte proliferation alone, however, it enhanced both adult and fetal (Day 15 of gestation) murine thymocyte proliferation in the presence of IL-2, IL-4, or IL-7. Other known cytokines were screened for a B-TCGF-like activity using both adult and fetal thymocytes. IL-6 was found to be active only on adult thymocytes, while TNF alpha and GM-CSF were found to be active only on fetal thymocytes. However, neutralizing antibodies against these cytokines did not block the B-TCGF activity present in CH12.LX.4866 using either adult or fetal thymocytes. These observations suggest that the B-TCGF activity is mediated by a novel factor(s). The apparent molecular weight of this novel molecule(s) was 27-50 kDa determined by sizing HPLC.

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PubMed1990

IL-2, IL-6, and IFN-gamma have distinct effects on the IL-4 plus PMA-induced proliferation of thymocyte subpopulations.

We have previously reported complex effects of cytokine-containing T cell supernatants on the interleukin (IL)4 plus phorbol 12-myristate 13-acetate (PMA)-induced proliferative response of murine thymocytes. Here we show that recombinant murine IL-2, IL-6, and IFN-gamma each differentially regulate the IL-4/PMA-driven growth of thymocyte subpopulations. Thymocytes fractionated into four subpopulations on the basis of CD4 and CD8 expression were stimulated to proliferate by IL-4/PMA. Interferon-gamma (IFN-gamma) caused almost complete inhibition of the CD4+/CD8- response but had no measurable effect on the growth of CD4-/CD8+ or CD4-/CD8- populations. This inhibitory effect was also observed on splenic CD4+/CD8- T cells. In contrast, IL-6 strongly enhanced the proliferative response of CD4+/CD8- thymocytes, but showed no effect on peripheral CD4+/CD8- T cells, suggesting that IL-6 may be an important regulator of growth in the thymus. IL-2 also enhanced the proliferation of both CD4-/CD8+ and CD4-/CD8- thymocytes to IL-4 and PMA. To test whether the IL-4/PMA stimulus provided all the signals required to initiate growth in each subpopulation, we titrated cell number and examined the relationship between cell dose and cell response. Growth of CD8+/CD4- cells was cell density independent, indicating that IL-4/PMA is sufficient stimulus to induce growth of these cells. In contrast, growth of CD4-/CD8- and CD4+/CD8- cells is cell density dependent, suggesting a requirement for another signal provided by the cells themselves. These observations suggest that more signals remain to be identified in this thymocyte growth system.

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PubMed1989

Distinct patterns of lymphokine requirement for the proliferation of various subpopulations of activated thymocytes in a single cell assay.

The frequency and capacity for clonal expansion of several murine thymocyte subpopulations responsive to various IL (fetal day 15, and adult CD4-8-, CD4+8- and CD4-8+) were investigated using a single-cell limiting-dilution cell culture system without filler cells. This assay requires the presence of PMA and ionomycin. The main conclusions of these studies are the following: 1) IL-4 is a better growth factor than IL-2 for immature thymocytes (fetal day 15 or adult CD4-8-). 2) IL-2 is a better growth factor than IL-4 for mature phenotype thymocytes (CD4+8- and CD4-8+). 3) IL-4 is a relatively poor growth factor for adult CD4-CD8- thymocytes and CD4+CD8- thymocytes, while it induced strong responses in fetal day 15 and CD4-8+ thymocytes. 4) IL-6 enhanced the response of CD4+8- thymocytes to either IL-2 or IL-4. 5) Cortisone-resistant thymocytes grown initially with IL-4 and then switched to IL-2 showed a significant decrease in cloning efficiency. No inhibitory effect was observed when cells were cultured first with IL-2 and then switched to IL-4. 6) Finally, supernatant from Con-A stimulated rat spleen cells induced maximal growth of all adult thymocyte populations tested, suggesting that unidentified thymocyte growth factor(s) remain to be characterized. These results indicate that the maturational stage of thymocytes determines their requirements for activation and proliferation.

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PubMed1989

Biological activity of recombinant murine interleukin-6 in interleukin-1 T cell assays.

Interleukin-6 (also called B cell stimulatory factor 2, hepatocyte activating factor, interferon-beta 2) has been shown to have effects on various lineages of hemopoietic cells. Some of its activities appear to overlap those of interleukin-1. In particular, recombinant murine IL-6 induced proliferation of phytohemagglutinin-activated thymocytes, an assay widely used to detect IL-1. In this report, we compared several features of IL-1 and IL-6 dependent thymocyte proliferation. The results indicate that IL-2 is the major second mediator of both IL-1 and IL-6 dependent proliferation. Finally, we tested whether IL-6 would also have activity in other T cell-based IL-1 assays using the T cell lymphoma LBRM33 1A5 and the T cell clone D10-G4.1. IL-6 had no activity in the latter two assays. These results indicate that IL-1 assays using LBRM33 1A5 and D10-G4.1 selectively detect Il-1, and are more specific assays for the detection of IL-1 in samples that may also contain IL-6.

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PubMed1989

Role of interleukin-4 in T-cell ontogeny: changes in cell surface phenotype and lymphokine production of immature thymocytes after culture with interleukin-4 and phorbol ester.

We have analyzed the phenotype and functional capabilities of adult and fetal CD4 8 thymocytes after 4 d of culture in IL-4 and PMA. Both adult and day 14 fetal CD4 8 thymocytes failed to acquire CD4 or CD8 antigens following culture. However, changes in expression of other antigens typical of immature thymocytes were observed. For example, the frequency with which cells expressed high levels of J11d or IL-2-R was greatly decreased following culture, whereas the frequency with which high levels of MEL-14, the lymph node homing receptor were expressed were greatly increased. This phenomenon may be due to direct induction by IL-4 and PMA of MEL-14 expression on purified MEL-14lo CD4-8- thymocytes. The frequency of cells expressing CD3, Ly-1 and Pgp-1 changed only slightly. Functionally, the cultured cells produced large amounts of interferon gamma but very little IL-2 or IL-4, although freshly isolated CD4-8- thymocytes produced all three lymphokines. These results suggest that in addition to a proliferative stimulus, culture in IL-4/PMA alters the expression of several early thymocyte antigens, the functional capabilities of CD4-8- progenitor thymocytes, and may act as a selective differentiation stimulus to MEL-14lo CD4-8- thymocytes.

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PubMed1989

IL-7 is a growth and maintenance factor for mature and immature thymocyte subsets.

The specific signals inducing the growth and maturation of thymocytes remain undefined. We show here that recombinant IL-7 induces growth of fetal and adult mouse CD4-8- thymocytes. IL-7 also induces a lower but significant response in CD4+8- and CD4-8+ thymocytes. Day 14 fetal thymic lobes cultured in IL-7 for 6 days show a 2-fold increase in cell number when compared to control cultures. The thymocyte subsets that proliferate in response to IL-7 can be maintained in culture for extended periods of time. CD4-8- thymocytes maintained in IL-7 did not change their phenotype with respect to CD4 and CD8 expression. In addition, we show that the combination of IL-7 plus IL-6 provides a potent growth stimulus for CD4+8- and CD4-8+ thymocytes. A cloned thymic epithelial cell, that can be induced to express MHC class II molecules, transcribes both IL-7 and IL-6 mRNA. A cloned thymic macrophage cell line produces IL-6 but no detectable IL-7 mRNA. The pattern of biological activities present in the supernatants of these cell lines is also presented. These observations suggest that the thymic epithelial and macrophage cell types may be an in vivo source of signals which mediate thymocyte development.

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PubMed1989

Effects of IL-4 on macrophage functions: increased uptake and killing of a protozoan parasite (Trypanosoma cruzi).

We studied whether interleukin-4 (IL-4) could modulate two macrophage functions relevant to their microbicidal activity (uptake and killing), using non-invasive [amastigote (AMA)] forms of the protozoan parasite Trypanosoma cruzi. Treatment of cultures of mouse resident peritoneal macrophages (MPM) with the supernatant of cultures of cells transfected with IL-4 cDNA increased both the capacity of the MPM to take up the organisms and the rate of intracellular killing with respect to MPM mock-treated with medium alone. The presence in the medium of a monoclonal antibody specific for IL-4 during MPM treatment inhibited both effects, pointing to recombinant IL-4 (rIL-4) as the active principle in the supernatant. Kinetic studies revealed that at least a 24-hr pretreatment of the MPM with the rIL-4-containing supernatant was required for these effects to be produced. The rate of intracellular parasite killing was also significantly increased when the rIL-4 treatment was applied after AMA ingestion by MPM. This result confirmed that MPM could be activated by rIL-4 for greater intracellular killing and showed that this enhancement was not necessarily dependent on the initial rIL-4-mediated increase in parasite load. The use of scavengers of reactive oxygen reduction intermediates indicated that hydrogen peroxide, superoxide anion and singlet oxygen, but apparently not hydroxyl radicals, were involved in parasite killing modulated by rIL-4. These results document for the first time the capacity of IL-4 to enhance the microbicidal activity of macrophages and suggest that this lymphokine might play a role in host defence against T. cruzi infection.

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