Phenotype and frequency of cells secreting IL-2, IL-4, IL-6, IL-10, IFN and TNF-α in human peripheral blood

The phenotype and frequency of cells in normal human peripheral blood spontaneously secreting IL-2, IL-4, IL-6, IL-10, IFN and TNF-α ex vivo was determined using ELIspot assays. CD4⁺T cells were the dominant source of IL-2 and IL-4 while multiple cell types (primarily CD8⁺lymphocytes) produced IFN. Fewer than 0.05% of mononuclear cells were spontaneously secreting these T cell derived factors. By comparison, IL-6, IL-10 and TNF-α were produced by 0.7–20% of PBMC. The primary sources of the latter cytokines were CD14⁺macrophages/monocytes. A significant positive correlation was found in the frequency of cells secreting IL-6, IL-10 and TNF-α ex vivo, suggesting that the release of such factors was coordinately regulated. No such correlation was found among IL-2, IL-4 and IFN secreting cells, indicating that the production of predominantly T cell derived cytokines was regulated independently.     

Possible regulation of autologous mixed lymphocyte reaction by OKM1+ NK cells

T cells are stimulated by autologous non-T cells and interleukin 2 (IL-2) is produced in the conventional autologous mixed lymphocyte reaction (AMLR) in young healthy controls. The role of cells with natural killer (NK) cell markers (OKM1+ cells or Leu 7+ cells) in the AMLR was studied. There were significant inverse correlations between the percentage of input OKM1+ cells minus monocyte (OKM1+ NK cells) and either AMLR proliferation (gamma = -0.9, P less than 0.001) or IL-2 production (gamma = -0.75, P less than 0.01) in the AMLR cultures after 7 days measured at 7 days. A statistically significant correlation was observed between the percentage of input Leu 7+ cells and AMLR proliferation (gamma = -0.64, P less than 0.05), but not IL-2 production. These results suggest that the AMLR is controlled by OKM1+ NK, perhaps acting through IL-2 regulation.     

Generation of nondividing high rate Ig-secreting plasma cells in cultures of human B cells stimulated with anti-CD3-activated T cells.

The capacity of human B cells to differentiate into high rate nondividing antibody-secreting plasma cells was investigated. Highly purified human peripheral blood B cells were stimulated with polyclonal B cell activators in the presence of a variety of recombinant cytokines (IL-2, IL-4, IL-6). Maximal production of Ig of all isotypes was observed when B cells were stimulated with intact T cells that were activated with mAb to the CD3 molecular complex. In these cultures, Ig production continued for more than 16 days. Moreover, differentiation to nondividing high rate Ig-producing cells was induced, as evidenced by a ninefold increase in the amount of Ig produced per Ig-secreting cell and the acquisition of resistance of ongoing Ig secretion to the inhibitor of DNA synthesis, hydroxyurea. To determine whether intact T cells were required for the entire culture period to achieve maximal Ig production, B cells were cultured with activated T cells for various lengths of time, reisolated and cultured with fresh activated T cells or various cytokines, then analyzed for Ig secretion. B cells preactivated for 6 days with anti-CD3-stimulated T cells required contact with intact T cells for continued Ig secretion. However, after 9 days of preactivation, dividing B cells responded maximally to anti-CD3-stimulated T cells, whereas cytokines were able to drive continued IgG secretion by nondividing B cells in the absence of intact T cells. IL-6 alone, or in combination with either IL-2 or IL-4, was the major cytokine driving ongoing Ig secreting by nondividing preactivated B cells. These results suggest that continued clonal expansion of Ig-secreting B cell blasts requires intact anti-CD3-activated T cells, whereas terminal differentiation of B cells into plasma cells after extensive clonal expansion is driven by cytokines, most notably IL-6.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

The human liver contains multiple populations of NK cells, T cells, and CD3+CD56+ natural T cells with distinct cytotoxic activities and Th1, Th2, and Th0 cytokine secretion patterns.

The human liver contains significant numbers of T cells, NK cells, and lymphocytes that coexpress T and NK cell receptors. To evaluate their functional activities, we have compared the cytotoxic activities and cytokines produced by normal adult hepatic CD3+CD56- (T) cells, CD3-CD56+ (NK) cells, and CD3+CD56+ (natural T (NT)) cells. In cytotoxicity assays using immunomagnetic bead-purified NK cell, T cell, and NT cell subpopulations as effectors, fresh hepatic NK cells lysed K562 targets, while NT cells could be induced to do so by culturing with IL-2. Both NT and T cells were capable of redirected cytolysis of P815 cells using Abs to CD3. Flow cytometric analysis of cytokine production by fresh hepatic lymphocyte subsets activated by CD3 cross-linking or PMA and ionomycin stimulation indicated that NT cells and T cells could produce IFN-gamma, TNF-alpha, IL-2, and/or IL-4, but little or no IL-5, while NK cells produced IFN-gamma and/or TNF-alpha only. The majority of NT cells produced inflammatory (Th1) cytokines only; however, approximately 6% of all hepatic T cells, which included 5% of Valpha24 TCR-bearing NT cells and 2% of gammadeltaTCR+ cells, simultaneously produced IFN-gamma and IL-4. The existence of such large numbers of cytotoxic lymphocytes with multiple effector functions suggests that the liver is an important site of innate immune responses, early regulation of adaptive immunity, and possibly peripheral deletion of autologous cells.     

High-dose leptin activates human leukocytes via receptor expression on monocytes

Leptin is capable of modulating the immune response. Proinflammatory cytokines induce leptin production, and we now demonstrate that leptin can directly activate the inflammatory response. RNA expression for the leptin receptor (Ob-R) was detectable in human PBMCs. Ob-R expression was examined at the protein level by whole blood flow cytometry using an anti-human Ob-R mAb 9F8. The percentage of cells expressing leptin receptor was 25 +/- 5% for monocytes, 12 +/- 4% for neutrophils, and 5 +/- 1% for lymphocytes (only B lymphocytes). Incubation of resting PBMCs with leptin induced rapid expression of TNF-alpha and IL-6 mRNA and a dose-dependent production of TNF-alpha and IL-6 by monocytes. Incubation of resting PBMCs with high-dose leptin (250 ng/ml, 3-5 days) induced proliferation of resting cultured PBMCs and their secretion of TNF-alpha (5-fold), IL-6 (19-fold), and IFN-gamma (2.5-fold), but had no effect on IL-4 secretion. The effect of leptin was distinct from, and additive to, that seen after exposure to endotoxin or activation by the mixed lymphocyte reaction. In conclusion, Ob-R is expressed on human circulating leukocytes, predominantly on monocytes. At high doses, leptin induces proinflammatory cytokine production by resting human PBMCs and augments the release of these cytokines from activated PBMCs in a pattern compatible with the induction of Th1 cytokines. These results demonstrate that leptin has a direct effect on the generation of an inflammatory response. This is of relevance when considering leptin therapy and may partly explain the relationship among leptin, proinflammatory cytokines, insulin resistance, and obesity.     

Interleukin 10 production in patients undergoing cardiopulmonary bypass: evidence of inhibition of Th-1-type responses

The cardiopulmonary bypass (CPB) procedure has long been associated with a generalized immunosuppression. To understand further the cytokine-mediated regulation of the complex physiological and immunological changes induced by CPB, the authors decided to investigate whether CPB affects the release of interleukin (IL)-10, as well as other cytokines, in correlation to the inhibition of T cell responses. Using phytohaemagglutinin (PHA) as mitogen and peripheral blood mononuclear cells (PBMC) isolated from patients undergoing CPB, we investigated whether this procedure has an effect on the secretion of different patterns of cytokines (Th1- and Th2-type) and PBMC proliferation. In all patients, CPB significantly enhances IL-10 and IL-6 production in resting and PHA-stimulated PBMC. On the other hand, IL-2 production, in response to PHA, was significantly diminished. Reduced IL-2 and enhanced IL-10 production were associated with a significant decrease in PBMC proliferation. Immunosuppression was also associated to lymphopenia, while neutrophil counts were significantly enhanced. These results show that after CPB there is a transient but clear unbalanced immune response demonstrated by a differentiated production of Th1- and Th2-type cytokines. The release of different patterns of cytokines observed after CPB may be helpful in understanding and preventing the development of infectious and immune complications in surgical procedure employing CPB.     

Effect of hydrogenated and saturated, relative to polyunsaturated, fat on immune and inflammatory responses of adults with moderate hypercholesterolemia

Consumption of diets high in hydrogenated fat/trans fatty acids has been shown to have an adverse affect on lipoprotein profiles with respect to cardiovascular disease risk. Dietary fat and cholesterol play an important role in the regulation of immune and inflammatory responses shown to be involved in atherogenesis. We investigated the effects of diets containing hydrogenated fat on cellular immune response and production of inflammatory cytokines in human subjects with moderately elevated cholesterol levels (LDL cholesterol >130 mg/dl). In a double blind cross-over study, 19 subjects consumed three diets, 30% of calories as fat, of which two thirds were provided as soybean oil, soybean oil-based stick margarine, or butter for 32 days, each in a randomized order. Production of proinflammatory mediators, prostaglandin (PG)E(2), interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha); delayed type hypersensitivity (DTH) response, in vitro lymphocyte proliferation, and production of IL-2 were determined. Production of IL-6 and TNF-alpha was significantly higher after consumption of stick margarine diet compared with soybean oil diet. IL-1beta and TNF-alpha production correlated positively with ratios of total cholesterol to HDL cholesterol (r = 0.499, P < 0.001 and r = 0.291, P = 0.04, respectively). There was no significant difference in DTH response, lymphocyte proliferation, or levels of IL-2 and PGE(2) produced among three groups. Our results indicate that consumption of a diet high in hydrogenated fat does not adversely affect cellular immunity but increases production of inflammatory cytokines that have been associated with the pathophysiology of atherosclerosis.     

Rotavirus 2/6 virus-like particles administered intranasally in mice, with or without the mucosal adjuvants cholera toxin and Escherichia coli heat-labile toxin, induce a Th1/Th2-like immune response

We investigated the rotavirus-specific lymphocyte responses induced by intranasal immunization of adult BALB/c mice with rotavirus 2/6 virus-like particles (2/6-VLPs) of the bovine RF strain, by assessing the profile of cytokines produced after in vitro restimulation and serum and fecal antibody responses. The cytokines produced by splenic cells were first evaluated. Intranasal immunization with 50 microg of 2/6-VLPs induced a high serum antibody response, including immunoglobulin G1 (IgG1) and IgG2a, a weak fecal antibody response, and a mixed Th1/Th2-like profile of cytokines characterized by gamma interferon and interleukin 10 (IL-10) production and very low levels of IL-2, IL-4, and IL-5. Intranasal immunization with 10 microg of 2/6-VLPs coadministered with the mucosal adjuvants cholera toxin and Escherichia coli heat-labile toxin (LT) considerably enhanced the Th1/Th2-like response; notably, significant levels of IL-2, IL-4, and IL-5 were observed. Since rotavirus is an enteric pathogen, we next investigated the production of IL-2 and IL-5, as being representative of Th1 and Th2 responses, by Peyer's patch and mesenteric lymph node cells from mice immunized intranasally with 2/6-VLPs and LT. The results were compared to those obtained from splenic and cervical lymph node cells. We found that both cytokines were produced by cells from each of these lymphoid tissues. These results confirm the Th1/Th2-like response observed at the systemic level and show, on the assumption that T cells are the primary cells producing the cytokines after in vitro restimulation, that rotavirus-specific T lymphocytes are present in the intestine after intranasal immunization with 2/6-VLPs and LT.     

Contribution of astrocyte-derived IL-15 to CD8 T cell effector functions in multiple sclerosis

The contribution of local factors to the activation of immune cells infiltrating the CNS of patients with multiple sclerosis (MS) remains to be defined. The cytokine IL-15 is pivotal in the maintenance and activation of CD8 T lymphocytes, a prominent lymphocyte population found in MS lesions. We investigated whether astrocytes are a functional source of IL-15 sufficient to enhance CD8 T lymphocyte responses and whether they provide IL-15 in the inflamed CNS of patients with MS. We observed that human astrocytes in primary cultures increased surface IL-15 levels upon activation with combinations of proinflammatory cytokines. Expanded human myelin autoreactive CD8 T lymphocytes cultured with such activated astrocytes displayed elevated lytic enzyme content, NKG2D expression, and Ag-specific cytotoxicity. These functional enhancements were abrogated by anti-IL-15-blocking Abs. Immunohistochemical analysis of brain tissue sections obtained from patients with MS demonstrated colocalization for IL-15 and the astrocyte marker glial fibrillary acidic protein within white matter lesions. The majority of astrocytes (80-90%) present in demyelinating MS lesions expressed IL-15, whereas few astrocytes in normal control brain sections had detectable IL-15. IL-15 could be detected in the majority of Iba-1-expressing microglia in the control sections, albeit in lower numbers when compared with microglia/macrophages in MS lesions. Furthermore, infiltrating CD8 T lymphocytes in MS lesions were in close proximity to IL-15-expressing cells. Astrocyte production of IL-15 resulting in the activation of CD8 T lymphocytes ascribes a role for these cells as contributors to the exacerbation of tissue damage during MS pathogenesis.     

Exogenous glutamine requirement is confined to late events of T cell activation

Glutamine is required for the proliferation of lymphocytes, but quantitative effects on discrete steps of activation remain unknown to date. Therefore the influence of glutamine (range: 0 mM–1 mM) on the in vitro response of human peripheral blood mononuclear cells (PBMC) to a mitogenic anti-CD3 monoclonal antibody (mAb) was investigated. Expression of surface activation markers by flow cytometry, presence of mRNA of cytokine genes by polymerase chain reaction, release of cytokines by ELISA, and entering into the cell cycle by flow cytometry were sequentially analyzed. Proliferation was measured by a ³H-thymidine incorporation assay. mRNA coding for IL-2, IL-2 receptor, IL-4, IL-5, GM-CSF, and IFN-γ was detectable independently from exogenous glutamine provision; expression of the cell surface activation marker CD69 was also glutamine independent. In contrast, later activation events including the expression of the surface activation markers CD25, CD45RO, and CD71 as well as the production of IFN-γ were found to require exogenous glutamine supply. In contrast, production of TNF-α could be observed in the absence of glutamine and was increased to a limited extent by exogenous glutamine. The overall lymphocyte response as reflected by entering into the cell cycle and proliferation was directly correlated with the glutamine concentration of the culture medium. Efficient progression through the cell cycle was found to require at least 0.5 mM glutamine and an increase in glutamine concentration from 0.1 mM to 1 mM enhanced proliferation by 50%. These results were supported by data obtained following anti-CD3 stimulation of a CD4⁺ T cell clone. Altogether, these data underline that a complete cellular immune response depends on an exogenous glutamine supply. Regarding glutamine requirements, they define early, glutamine-independent and late, glutamine-dependent lymphocyte activation stages.     

Induction of cytokines in human peripheral blood mononuclear cells by mycoplasmas.

Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated with Mycoplasma pneumoniae, M. hyorhinis, M. arginini, M. salivarium, M. orale, M. gallisepticum or A. laidlawii for 48 hr, and the activities of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN) in the supernatants were determined by ELISA or bioassay. All mycoplasma species induced IL-1 beta, IL-6 and TNF-alpha, although IL-2 was induced only by M. pneumoniae. IFN was induced by 5 of the 7 species, and the IFN produced was antigenically confirmed to be mainly IFN-alpha. On the other hand, mycoplasma-stimulated cultures did not contain detectable amounts of IFN-beta and IL-4 activities. Furthermore, the cytokines were induced by mycoplasmal contaminating cells in human PBMC as well as by mycoplasma alone. These results suggest that many kinds of cytokines induced by mycoplasma contamination in cell culture affect immunological experiments in vitro.     

Presence of CTAK/CCL27, MCP-3/CCL7 and LIF in human colostrum and breast milk

Human colostrum and breast milk are known to contain high levels of cytokines and chemokines, which are thought to contribute to the development of the newborn. The aim of this study was to investigate the difference in the presence and levels of 21 soluble cytokines and chemokines in paired samples of human colostrum (day 2 after delivery) and breast milk (day 4–5 after delivery) by using the multiplex technology. Of the 21 cytokine investigated in 10 pairs of samples, only β-NGF was absent in both colostrum and milk, while INF-α2, SCF and TNF-β were present in colostrum but not in human milk. As a general rule, colostrum contained higher concentrations of cytokines and chemokines with respect to breast milk. The majority of cytokines, detected in colostrum alone or in colostrum and human milk (IL-1α, IL-2Rα, IL-3, IL-16, IL-18, GRO-α, HGF, IFN-α2, M-CSF, MIF, MIG, TNF-β, SDF-1α, TRAIL) have been described in previous studies, while for the first time we describe the presence of additional cytokines either in colostrum alone (SCF) or in both colostrum and breast milk (CTAK/CCL27, MCP-3/CCL7, LIF). Our data confirm and expand previous studies showing that some cytokines/chemokines, which might contribute to the development of the gastro-intestinal and nervous systems, are overexpressed in human colostrum and breast milk, and might contribute to the development of these systems.     

Interleukin-15 modulates interferon-gamma and beta-chemokine production in patients with HIV infection: implications for immune-based therapy

Interleukin (IL)-15 is a cytokine that has lymphocyte stimulatory activity similar to that of IL-2, and plays important immunoregulatory functions during HIV disease. To evaluate the role of IL-15 in HIV infection the following patients were studied: 18 antiretroviral-naive patients with advanced disease; 19 patients with continuous viral suppression and immunological response after 48-120 weeks of highly active antiretroviral therapy (HAART); and 12 patients with evidence of virological and immunological HAART treatment failure. Nineteen healthy blood donors were included as controls. The production of IL-15 by human peripheral blood monocytes stimulated with lipopolysaccharide and Mycobacterium avium complex, the priming effect of IL-15 on IFN-gamma production from purified CD4(+) and CD8(+) T cells, and the ability of IL-15 to stimulate the beta-chemokine release from purified CD4(+) and CD8(+) T cells were analyzed. In the present work IL-15 production by human peripheral blood monocytes was significantly increased in HIV-infected patients with long-term virological and immunological response to HAART. IL-15 enhanced the in vitro priming of CD4(+) and CD8(+) T cells for IFN-gamma production, also in patients receiving HAART. Finally, IL-15 had positive effects on RANTES, MIP-1alpha, and MIP-1beta release by CD4(+) and CD8(+) T cells. In conclusion IL-15 could affect the immune response of HIV-infected patients by augmenting and/or modulating IFN-gamma production and beta-chemokine release. These data about functional properties of IL-15 could provide new implications for immune-based therapies in HIV infection.     

Haptoglobin and the Th1/Th2 balance: hints from in vitro and in vivo studies

A variety of immunomodulatory effects have previously been attributed to haptoglobin (Hp). These are supposed to be partly mediated through binding of Hp to CD11b. In the present study, we assessed its effects on T-helper (Th) cytokine production following both in vitro and in vivo stimulation of T-cells. Hp exhibits a dose-dependent inhibitory effect on human T lymphocyte release of the Th2 cytokines (IL-4, IL-5, IL-10 and IL-13) in vitro, whereas it has no clear effect on Th1 cytokine (IL-2 and IFN-gamma) release. When administered an anti-CD3 monoclonal antibody, Hp knockout mice produced more IL-4 and less IFN-gamma than did their wild-type litter-mates. Our findings imply that Hp may be regarded as a regulator of the Th1/Th2 balance in both human and murine immune systems.     

The schistosome oligosaccharide lacto-N-neotetraose expands Gr1(+) cells that secrete anti-inflammatory cytokines and inhibit proliferation of naive CD4(+) cells: a potential mechanism for immune polarization in helminth infections.

Immunomodulatory oligosaccharides found on helminths also are found in human milk, and both helminths and milk have been shown to be immunosuppressive. We have been examining the immunomodulatory capabilities of two oligosaccharides expressed in milk and on helminth parasites, lacto-N-fucopentaose III and lacto-N-neotetraose (LNnT). In an attempt to dissect mechanisms that lead to Th2 polarization and immune suppression, we examined the early response in mice to the glycoconjugate LNnT-Dextran (LNnT-Dex). We found that injection of LNnT-Dex expanded a cell population, phenotypically defined as Gr1(+)/CD11b(+)/F4/80(+), as early as 2 h after injection. Examination of spontaneous cytokine production showed that this Gr1(+)/F4/80(+) population of cells spontaneously produced low levels of proinflammatory cytokines, but higher levels of IL-10 and TGF-beta ex vivo, compared to peritoneal cells from mice injected with Dex. Gr1(+) cells adoptively suppressed naive CD4(+) T cell proliferation in vitro in response to anti-CD3/CD28 Ab stimulation. Suppression of naive CD4(+) cells involved cell contact and was dependent on IFN-gamma and NO, with a discrete role played by IL-10. Coculture of naive CD4(+)T cells with Gr1(+) suppressor cells did not lead to CD4(+) T cell apoptosis, although it did imprint on naive CD4(+) T cells a response characterized by lower levels of IFN-gamma, coincident with increased IL-13 production. Our results suggest that both human milk and helminth parasites may share a ligand-specific mechanism involved in the generation of anti-inflammatory mediators that suppress Th1-type and inflammatory responses.     

Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production.

Trypanosoma cruzi, which causes Chagas' disease, has been shown to cause polyclonal proliferation of lymphocytes after infection in vivo. This paper demonstrates that coculture of human PBMC with T. cruzi CL strain leads to proliferation of lymphocytes, which peaks on days 5 to 7 after infection. Approximately 15% of lymphocytes in culture undergo blast transformation. The proliferation of lymphoblasts can be measured by [3H]TdR incorporation, because the parasites incorporate little TdR. Parasites derived from autologous PBMC cultures or xenogeneic rat fibroblasts stimulate lymphocyte transformation similarly. By immunofluorescent cytometry, lymphoblasts from these cultures are 23 to 46% B cells (CD19+) and 39 to 64% T cells (CD3+), and approximately half of the T cells are CD4+ and half CD8+. A high percentage of lymphoblasts express MHC class II and IL-2R p55, suggesting both B and T lymphoblasts express these molecules. Anti-MHC class II and anti-IL-2R p55 mAb significantly inhibit the proliferative response of PBMC to T. cruzi. The mRNA for cytokines IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TNF-alpha are detected after T. cruzi coculture with PBMC, peaking on day 3. No IL-4 or IL-10 mRNA are detected. Large quantities of bioactive IL-1 and IL-6 are found in the supernatants of these PBMC. Monocytes, infected in the apparent absence of lymphocytes, assume activated morphology and accumulate mRNA for IL-1 beta, TNF-alpha, and IL-6. T cells require accessory cells to proliferate and produce cytokine mRNA. A trypsin-sensitive activity in lysates of T. cruzi stimulates lymphocyte proliferation. The data presented demonstrate that T. cruzi coculture with PBMC leads to lymphocyte proliferation, monocyte activation, and cytokine production.     

Cytokine production by peripheral lymphocytes in melanoma

BACKGROUND: The differentiation of T cells towards a T helper 1 (Th1) or Th2 phenotype based on their profile of cytokine production, is of great relevance in the regulation of immune responses. We have determined by flow cytometry, the expression of selected Th1 and Th2 cytokines by activated T cells in whole blood samples (WB) from normal donors and from patients with different clinical stages of melanoma in different clinical stages. METHODS: WB samples from 6 normal donors and 19 patients with melanoma were activated over 4 hours with PMA + ionomycin in presence or absence of a protein secretion inhibitor. Following surface staining (CD3-Cy5+CD8-FITC), fixation and permeabilization, cells were stained with PE-labelled antibodies against Th1 cytokines (IL-2, IFN-gamma, TNF-alpha) and Th2 cytokines (IL-4, IL-10). RESULTS: The most relevant results were related to IFN-gamma and IL-10 production. The percentage of IFN-gamma producer cells was significantly lower in melanoma patients, independent of the stage, than in controls. IL-10 production was significantly increased in melanoma patients with respect to normal donors. CONCLUSIONS: Our data support the notion that the pattern of cytokines produced by lymphocytes from melanoma patients may help to explain the impairment in their T cell immune response. More extensive studies regarding the pattern of cytokines, not only in peripheral blood, but also in tumour tissue and sentinel lymph nodes, are needed to confirm these data.     

Ixodes scapularis: effects of repeated infestations with pathogen-free nymphs on macrophage and T lymphocyte cytokine responses of BALB/c and C3H/HeN mice.

Schoeler, G. B., Manweiler, S. A., and Wikel, S. K. 1999. Ixodes scapularis: Effects of repeated infestations with pathogen-free nymphs on macrophage and T lymphocyte cytokine responses of BALB/c and C3H/HeN mice. Experimental Parasitology 92, 239-248. Ixodes scapularis is the principal vector in the United States of Borrelia burgdorferi, the causative agent of Lyme borreliosis, the human granulocytic ehrichiosis agent, and Babesia microti. Infestation with I. scapularis nymphs has previously been shown to modulate host T lymphocyte cytokine production. Tick-induced host immunomodulation is increasingly recognized as a contributing factor in successful transmission and/or establishment of tick-borne pathogens. This study was conducted to determine the effects of repeated infestations with pathogen-free I. scapularis nymphs on the production of the macrophage cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha and the T lymphocyte cytokines IL-2, IL-4, IL-10, and interferon-gamma in both BALB/c and C3H/HeN mice. The pattern of T lymphocyte cytokine production was evaluated to determine if repeated tick infestation polarizes the immune response toward a Th-1 or Th-2 cytokine profile. Female BALB/c and C3H/HeN mice were infested one to four times with pathogen-free I. scapularis nymphs, with a 14-day tick-free period between each exposure. After each infestation, tick biology parameters were measured and macrophage and T lymphocyte cytokine production was assessed. Elaboration of T lymphocyte and macrophage cytokines was quantitated by antigen capture enzyme-linked immunosorbent assay. Acquired resistance to I. scapularis feeding was not developed by either mouse strain. Significant differences in cytokine production were observed between infested and noninfested mice, as well as between the two mouse strains, following tick infestation. Infestation of both strains with pathogen-free I. scapularis results in a polarization of the host immune response toward a Th-2, anti-inflammatory pattern, with a corresponding suppression of Th-1 responses.     

IL-6 production by human T lymphocytes. Expression in HTLV-1-infected but not in normal T cells

IL-6 is an important regulator of humoral and cellular immunity. Although this cytokine is produced by diverse cell types, it is not known whether it is produced by T lymphocytes under physiologic conditions or which agents can induce T cell expression of IL-6. We analyzed the production of IL-6 by human peripheral blood T cells, human thymocytes, and human T cell lines. In pure populations of these cells, stimulated with different combinations of various mitogens and cytokines, IL-6 activity could not be detected. Analysis of purified T-alpha beta and T-gamma delta cells showed that neither T cell subset produced IL-6. Similarly, IL-6 mRNA was not detected in T cell or thymocyte populations for up to 48 h after stimulation. With the use of a PCR assay, IL-6 mRNA in T cells was found to be virtually negligible, and did not change after T cell activation. By in situ hybridization it was shown that the cells expressing IL-6 mRNA after mitogen activation of PBMC do not belong to the T cell lineage. To analyze whether human T cells express IL-6 in vivo, we examined lymphoid tissues by in situ hybridization. In normal human thymus there was no detectable signal for IL-6. Tonsils showed only few positive cells within the parenchyma, but strong expression of IL-6 by epithelial cells in crypts. In contrast to normal lymph node, which contained only rare cells positive for IL-6, a lymph node from a patient with Castleman's disease showed IL-6 expression in cells occupying the marginal sinus and interfollicular areas. Screening of various human T cell lines showed that all cell lines infected with HTLV-1 secrete IL-6 activity and express IL-6 mRNA. In addition, in vitro infection of peripheral blood T cells with HTLV-1 induced de novo synthesis and secretion of IL-6. Furthermore, IL-6 expression in HTLV-1-infected cells was enhanced by stimulation with IL-1 beta or TNF-alpha. In contrast, IL-6 was not detectable in non-infected T cell lines. These studies indicate that IL-6 may not be a physiologic product of human T lymphocytes and that infection of T cells with HTLV-1 results in aberrant expression of this cytokine.