Enhanced production of IL-6 in tumor-bearing mice and determination of cells responsible for its augmented production

IL-6 is a cytokine secreted in normal individuals by monocytes, fibroblasts, and endothelial cells. We have found increased levels of IL-6 in the sera from MH134 hepatoma- and CSA1M fibrosarcoma-bearing mice. Concerning the capacity of these tumor cells themselves to produce IL-6 in vitro, they exhibited the distinct contrast, i.e., the MH134 tumor cells produced high levels of IL-6 whereas the CSA1M generated a marginal level of IL-6. It was, however, demonstrated that appreciably enhanced IL-6 production was observed in spleen cell culture supernatants from both types of tumor-bearing mice when compared to those obtained from normal mice. More importantly, in contrast to the production of IL-6 by non-T cell compartment of normal spleen cells, enhanced IL-6 production of spleen cells from tumor-bearing mice was ascribed to T cell compartment. Analysis of T cell phenotype has revealed that enhanced IL-6 production was mediated predominantly by Lyt-2+ but not by L3T4+ T cell subset. Thus, these results indicate that increased circulating IL-6 is elicited in the tumor-bearing state and that irrespective of the potential of tumor cells themselves to produce IL-6, T cells, especially Lyt-2+ T cells from tumor-bearing mice are responsible for such a high level of IL-6 production.     

Myelosuppressive effects in vivo of purified recombinant murine macrophage inflammatory protein-1 alpha.

Purified recombinant murine macrophage inflammatory protein-1 alpha (rmuMIP-1 alpha), a cytokine with myelopoietic activity in vitro, was assessed in vivo by injection into C3H/HeJ mice for effects on proliferation (percentage of cells in S phase DNA synthesis of the cell cycle) and absolute numbers of granulocyte-macrophage, erythroid, and multipotential progenitor cells in the femur and spleen, and on nucleated cellularity in the bone marrow, spleen, and blood. rmuMIP-1 alpha rapidly decreased cycling rates (at 2 to 10 micrograms/mouse i.v.) and absolute numbers (at 5 to 10 micrograms/mouse i.v.) of myeloid progenitor cells in the marrow and spleen. These effects were dose- and time-dependent and reversible. Suppressive effects were noted within 3 to 24 h for cell cycling and absolute numbers of progenitor cells in the marrow and spleen, and by 48 h for circulating neutrophils. A study comparing the effects of i.v. injection of rmuMIP-1 alpha versus rmuMIP-1 beta, a biochemically similar molecule but with no myelosuppressive effects in vitro, demonstrated myelosuppression in vivo by rmuMIP-1 alpha, but not by rmuMIP-1 beta. The results suggest that rmuMIP-1 alpha has myelosuppressive activity in vivo and offers the possibility that it may be a useful adjunct to treatments involving cytotoxic drugs because of its reversible suppressive effects on normal progenitor cell cycling.     

Production of BSF-1 during an in vivo, T-dependent immune response

BSF-1, a cytokine produced by some T lymphocyte tumors, has been shown to act with anti-Ig antibodies to stimulate B lymphocyte proliferation, to independently induce resting B lymphocytes to increase their expression of surface Ia antigen, and to induce some activated B lymphocytes to differentiate into IgG1- or IgE-secreting cells. To determine whether BSF-1 might be secreted by normal lymphoid cells in the course of a physiologic immune response, BALB/c mice were injected with an affinity-purified goat antibody to mouse IgD (GaM delta), which induces the generation of a large, polyclonal T-dependent IgG1 response; 4-hr culture supernatants of spleen cells from these mice were prepared, and these supernatants were assayed for BSF-1 activity by analyzing their ability to induce BALB/c nu/nu spleen cells to increase their expression of cell surface Ia in vitro. Culture supernatants of unfractionated spleen cells removed from mice 4 to 8 days after GaM delta antibody injection induced substantial increases in B lymphocyte surface Ia expression; these increases were blocked by a monoclonal anti-BSF-1 antibody. Culture supernatants of spleen cells from untreated BALB/c mice or from untreated or GaM delta antibody-treated BALB/c nu/nu mice induced small to moderate increases in B cell surface Ia expression, and GaM delta antibody itself induced large increases in B cell surface Ia expression; however, these increases were not significantly blocked by a monoclonal anti-BSF-1 antibody. A culture supernatant of T cell-enriched spleen cells from untreated mice induced small increases in B cell surface Ia expression that were inhibited by anti-BSF-1 antibody, as was the larger increase in B cell Ia expression induced by a culture supernatant of T cell-enriched spleen cells from mice sacrificed 3 days after GaM delta injection. On the other hand, T cell-depleted spleen cells from BALB/c mice injected with GaM delta antibody 7 days before sacrifice failed to generate culture supernatants with BSF-1 activity. Supernatants prepared from spleen cells taken from untreated mice or mice treated with GaM delta antibody 1 to 3 days before sacrifice did not block the ability of purified BSF-1 to induce an increase in B cell surface Ia expression, and thus did not contain inhibitors of BSF-1 activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rat macrophage activation after treatment with the bleomycin group of antitumour antibiotics in vivo

Summary We have previously reported that bleomycin and its derivative peplomycin enhance the release of cytokines by rat spleen cells during mitogen-stimulated cell culture in vitro, but liblomycin, another derivative of bleomycin, decreases cytokine release to below untreated control levels. Cytokine release correlated well with the inhibition of subcutaneous tumour growth after treatment with equivalent doses of the three analogues. In contrast, ascites tumour growth is completely inhibited by liblomycin and appears to be at least partly macrophage-mediated because the antitumour effect can be significantly inhibited by carageenan. This study shows that bleomycin and its analogues activate rat peritoneal macrophages and increase interleukin-6 release, O₂ ^– production, cell spreading, phagocytosis and random migration of macrophages, but only bleomycin enhances peritoneal macrophage invasion into a monolayer of rat lung endothelial cells in vitro. This study also shows that although liblomycin decreases spleen cell cytokine production and is less effective than bleomycin against subcutaneous tumour, as we have previously reported, the antitumour drug activates peritoneal macrophages and, compared to bleomycin, has a remarkable therapeutic effect on rat ascites tumour.     

Interleukin-13 and IgE production in rat experimental schistosomiasis

We have previously demonstrated in rat experimental schistosomiasis an upregulation of IL-4 expression at the mRNA and protein levels which could explain, at least in part, the increased IgE production observed during infection. Using this model, we have investigated the expression of IL-13 which is also involved in the induction of the IgE response. In the present study, we have shown a significant increase in IL-13 mRNA expression in spleen, liver and lungs following primary and secondary infection. IL-13 protein was detected by intracellular staining in spleen cells from infected rats, and in the supernatants of antigen-stimulated spleen cells. Furthermore, circulating levels of IL-13 were increased in sera from infected rats as compared to those from non-infected control animals. These findings show that, similarly to IL-4, IL-13 is upregulated and secreted during rat schistosomiasis, suggesting an involvement of both cytokines in IgE induction. In the in vivo experiments, only rats cotreated with neutralizing anti-IL-4 and anti-IL-13 antibodies showed significant decrease in the IgE levels. Moreover, administration of IL-13 enhanced total IgE levels. These results demonstrate the implication of IL-4 and IL-13 in vivo in IgE production, and provide a relevant animal model for a better understanding of the role of IL-4 and IL-13 in humans.     

Prevention of the in vitro myelosuppressive effects of glucocorticosteroids by interleukin 1 (IL 1)

We investigated the effects of dexamethasone on the formation of granulocyte/macrophage colonies by murine bone marrow cells cultured with colony-stimulatory factors (CSF) in semisolid agar. Dexamethasone (10(-7) M) completely inhibited the formation of colonies in response to L929 CSF but had no effect on the response to CSF in the culture supernatants of the murine macrophage cell line, PU5-1.8. We postulated that a cofactor, interleukin 1, present in the PU5-1.8 supernatants was responsible for protecting colony formation against steroid suppression. Interleukin 1, isolated from culture supernatants of PU5-1.8 and from culture supernatants of human acute monocytic leukemia cells, blocked the inhabitory effects of dexamethasone on colony formation in response to L929 CSF. Moreover, dexamethasone inhibited colony formation in response to PU5-1.8 culture supernatants when interleukin 1 was absent. We also examined interleukin 2 for possible protective effects. Although crude interleukin 2 preparations (supernatants of spleen cells cultured with concanavalin A) blocked dexamethasone inhibition, purified interleukin 2 had no protective effects. These data indicate that interleukin 1 protects colony formation by a pathway that is independent of interleukin 2 and that supernatants of spleen cells activated with concanavalin A probably contain significant amount of interleukin 1.     

Oxidative stress by visible light irradiation suppresses immunoglobulin production in mouse spleen lymphocytes

In this study, we attempted to induce the oxidative stress in mouse spleen lymphocytes with visible light irradiation and examined the effects of lipid peroxidation on immunoglobulin (Ig) production. The spleen lymphocytes were isolated from 8-week-old male balb/c mice and irradiated with 300 W visible light. When the cells were cultured for 72 hr, Ig contents in culture supernatants were decreased gradually by irradiation for over 30 min. The cell viability was also lowered by the irradiation. Intracellular phosphatidylcholine hydroperoxide (PCOOH) levels and thiobarbituric acid-reactive substances (TBARS) values in culture supernatants were measured as indices of lipid peroxidation and we found that Ig production by mouse spleen lymphocytes was suppressed accompanied with the progress of peroxidation of intracellular phospholipids. Cell membrane fluidity was also significantly decreased, but the intracellular Ig level was not changed in the irradiated cells. These results suggest that the peroxidation of intracellular lipids is a cause of the suppression of Ig production by mouse spleen lymphocytes via lowering cell viability and suppressing Ig synthesis and secretion.     

A study of cytokine production in acute graft-vs-host disease

The role of cytokines in the development of acute graft-vs-host disease (GVHD) was investigated in B6AF1 mice that were injected with parental A/J lymphocytes. Splenocytes from GVH mice exhibited an increased capacity to produce interleukin (IL)-1, IL-6, and TNF-a when stimulated in culture with lipopolysaccharide (LPS). This enhanced capacity was diminished following in vivo treatment with immunosuppressive drugs. Concanavalin A-stimulated GVH spleen cells produced significantly lower levels of IL-2 but higher levels of interferon-gamma (IFN-gamma) than did syngeneic spleen cells. Immunosuppressive therapy in vivo increased the capacity of GVH spleen cells to produce IL-2. However, immunosuppressants differed in their effects on IFN-gamma production. Sch 24937 (6-bromo-5-chloro-2-[1-(methylsulfonyl)acetyl] 3-(2-pyridyl)indole) enhanced or had no effect while cyclosporin A consistently decreased the capacity of splenocytes to produce this lymphokine. These results indicate that the capacity of GVH splenocytes for cytokine production can be differentially affected by the actions of some pharmacological agents. The data also indicate that there may be differential regulation of the production of IL-2 and IFN-gamma by the Th1 subset in the GVH spleen.     

IL-28 elicits antitumor responses against murine fibrosarcoma

IL-28 is a recently described antiviral cytokine. In this study, we investigated the biological effects of IL-28 on tumor growth to evaluate its antitumor activity. IL-28 or retroviral transduction of the IL-28 gene into MCA205 cells did not affect in vitro growth, whereas in vivo growth of MCA205IL-28 was markedly suppressed along with survival advantages when compared with that of controls. When the metastatic ability of IL-28-secreting MCA205 cells was compared with that of controls, the expression of IL-28 resulted in a potent inhibition of metastases formation in the lungs. IL-28-mediated suppression of tumor growth was mostly abolished in irradiated mice, indicating that irradiation-sensitive cells, presumably immune cells, are primarily involved in the IL-28-induced suppression of tumor growth. In vivo cell depletion experiments displayed that polymorphonuclear neutrophils, NK cells, and CD8 T cells, but not CD4 T cells, play an equal role in the IL-28-mediated inhibition of in vivo tumor growth. Consistent with these findings, inoculation of MCA205IL-28 into mice evoked enhanced IFN-gamma production and cytotoxic T cell activity in spleen cells. Antitumor action of IL-28 is partially dependent on IFN-gamma and is independent of IL-12, IL-17, and IL-23. IL-28 increased the total number of splenic NK cells in SCID mice and enhanced IL-12-induced IFN-gamma production in vivo and expanded spleen cells in C57BL/6 mice. Moreover, IL-12 augmented IL-28-mediated antitumor activity in the presence or absence of IFN-gamma. These findings indicate that IL-28 has bioactivities that induce innate and adaptive immune responses against tumors.     

Regulation of mucosal B cell immunoglobulin secretion by intestinal epithelial cell-derived cytokines

Intestinal epithelial cells (IEC) secrete a variety of cytokines and, because of their close proximity to B cells in the lamina propria, may affect local antibody production via these cytokines. However, studies have not yet addressed which and to what extent these IEC-derived cytokines may affect B cell antibody production. In this study, rat mesenteric lymph node B cells were cultured with culture supernatants from the rat IEC-6 intestinal epithelial cell line to determine their effect on immunoglobulin (Ig) secretion. Unstimulated IEC-6 cells were found to secrete sufficient levels of IL-6 to enhance IgA, IgG and IgM secretion by unstimulated B cells. However, culture of lipopolysaccharide (LPS)-stimulated B cells with the unstimulated IEC-6 supernatant resulted in an enhancement of IgA secretion while IgM secretion was significantly suppressed. Depletion of the IEC-6 supernatant using cytokine specific antibodies revealed that both interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta) were responsible for the enhanced IgA secretion while TGF-beta suppressed IgM secretion. More importantly, culture supernatants from LPS stimulated IEC-6 cells contained enhanced levels of IL-6 which enhanced both IgG and IgA production and partially overcame the suppressive effect of TGF-beta on IgM secretion. These results suggest that intestinal epithelial cells may secrete IL-6 and TGF-beta to regulate local B cell antibody secretion and their effect may be highly dependent upon the activation state of the epithelial cells.     

Superantigen-induced apoptotic death of tumor cells is mediated by cytotoxic lymphocytes, cytokines, and nitric oxide.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is a potent inducer of CTL activity and cytokine production in vivo. Protein A (PA) of Staphylococcal aureus has been found to have diverse biological response modifying properties and to possess antitumor, antitoxic and antiparasitic effects. In this study we examined the anti-tumor effect of these two superantigens used separately as well as in combination in mice carrying the Ehrlich ascites tumor. With combined treatment, DNA cell cycle analysis of tumor cells showed a significant (P < 0.05) percentage of tumor cell death. Levels of the soluble mediators TNF-alpha, IFN-gamma and IL-1 as well as NO were elevated. Additionally, CD4(+) and CD8(+) specific T cells in spleen, thymus and PBMC in tumor carrying mice were increased (P < 0.01). Our data altogether suggests that enhanced tumor cell death is caused by the increased CTL activity, cytokine and nitric oxide levels, in response to the combined effect of SEA + PA.     

Activation of the production of the tumor-necrosis factor by the combined action of lipopolysaccharide and muramyl dipeptide in vitro and in vivo

The effect of bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP) and their combination on the production of tumour necrosis factor by spleen cells in vitro and on tumour regression in vivo has been studied. TNF activity was detected in spleen cell supernatants and serum of mice treated with drugs, using L929 cells as targets. The combination of LPS and MDP was more effective in TNF production than each of the drugs used alone in vitro and in vivo. The injection of LPS and MDP to A/Sn mice with subcutaneous nodes of sarcoma SA-I resulted in total tumour necrosis. The treatment of mice with these drugs in water solutions was more effective, however, more toxic than the administration of LPS-treated splenocytes in MDP solution.     

In vivo and in vitro activation of pulmonary macrophages by IFN-gamma for enhanced killing of Paracoccidioides brasiliensis or Blastomyces dermatitidis

The fungicidal capacity of murine pulmonary macrophages (PuM) activated in vitro with IFN or lymphokines or in vivo with IFN was studied. PuM treated overnight with IFN (1000 U/ml), Con A-stimulated spleen cell culture supernatants, or lymph node cells plus Con A significantly killed yeast cells of the Gar w isolate of Paracoccidioides brasiliensis 45.5 +/- 2.1%, 72.0 +/- 4.2%, and 51.5 +/- 0.7% respectively. Two other isolates of P. brasiliensis (Ru and LA) were also killed (45 and 34%) by PuM activated by lymph node cells plus Con A. Control PuM had lesser but significant capacity for killing of P. brasiliensis isolates, ranging from 15 to 22%. Killing of P. brasiliensis by PuM activated by Con A-stimulated spleen cell culture supernatants could not be significantly inhibited by superoxide dismutase, catalase, or azide. When mice were treated in vivo with 4 X 10(5) IFN U i.p. and PuM isolated 24 h later, the PuM had significantly enhanced ability to kill P. brasiliensis (47.0 +/- 6.3%) compared with PuM from control mice (25.0 +/- 4.2%). PuM thus activated also showed enhanced killing (43%) of a second isolate compared with control PuM (22%). PuM from IFN-treated mice were able to significantly kill Blastomyces dermatitidis (37.5 +/- 0.7%) compared with control PuM (4.5 +/- 6.3%). These results show that PuM can be activated in vitro and in vivo by IFN for enhanced fungicidal activity against two pulmonary fungal pathogens and suggests that immunologic production of IFN could be an important factor in host defenses against these diseases.     

Anti-CD40 antibody induces antitumor and antimetastatic effects: the role of NK cells

We assessed the effect of the stimulatory anti-CD40 Ab on NK cell activation in vivo and the therapeutic potential of activated NK cells in tumor-bearing mice. Single-dose i.p. injection of the anti-CD40 Ab resulted in production of IL-12 and IFN-gamma in vivo, followed by a dramatic increase in NK cell cytolytic activity in PBLs. NK cell activation by anti-CD40 Ab was also observed in CD40 ligand knockout mice. Because NK cells express CD40 ligand but not CD40, our results suggest that NK activation is mediated by increased cytokine production upon CD40 ligation of APCs. Treatment of tumor-bearing mice with anti-CD40 Ab resulted in substantial antitumor and antimetastatic effects in three tumor models. Depletion of NK cells with anti-asialo GM1 Ab reduced or abrogated the observed antitumor effects in all the tested models. These results indicate that a stimulatory CD40 Ab indirectly activates NK cells, which can produce significant antitumor and antimetastatic effects.     

IL-27 mediates complete regression of orthotopic primary and metastatic murine neuroblastoma tumors: role for CD8+ T cells

We have shown previously that IFN-gamma-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8(+) T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-gamma. In addition, marked enhancement of local IFN-gamma gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8(+), but not CD4(+) T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.     

Correlation of T-cell response and lymphokine profile with RESA peptides of Plasmodium falciparum containing a universal T-cell epitope and an immunopotentiator, polytuftsin.

Two RESA repeat sequences, (EENVEHDA)2 and (DDEHVEEPTVA)2, were chemically linked to a universal T-cell epitope, CS.T3 and polytuftsin, and a natural immunopotentiator, was physically mixed with these conjugates. The immunogens were studied for in vitro antigen-induced T-cell proliferation, and cytokine levels were measured in the culture supernatants. The RESA peptide(s)-CS.T3 conjugate containing polytuftsin showed the highest stimulation index (SI) as compared to the RESA peptide-CS.T3 conjugates or RESA peptides alone. Spleen cells from mice primed with either RESA peptide(s)-CS.T3 conjugate or RESA peptide-CS.T3 conjugate containing polytuftsin, when pulsed in vitro with the respective RESA peptide, showed a higher proliferation index as compared to spleen cells primed and pulsed in vitro with the respective RESA peptides. This observation has an important relevance during natural reinfection for boosting the immune response. The culture supernatants from the cells primed and pulsed in vitro with RESA peptide-CS.T3 conjugate and RESA peptide-CS.T3 conjugate containing polytuftsin showed higher IL-2 and IFN-gamma levels as compared to the RESA peptides alone. Very low IL-4 levels were detected with the above formulations. The cytokine profile is suggestive of a CD4+ TH1 type of immune response, which is ideal for the killing of intracellular pathogens like the malarial parasite.     

Demonstration of a macrophage migration enhancement factor in the sera of young rabbits

Alveolar macrophages (AM), harvested from the lungs of untreated normal young rabbits (New Zealand White) 14 days to 8 weeks of age, exhibited a state of migration stimulation compared to AM from normal adult rabbits (5 to 6 months of age). Migration of AM from normal adult rabbits (New Zealand White) was stimulated 2.0- to 2.5-fold when incubated with sera from 39- to 46-day-old rabbits compared with sera from normal adult rabbits. Furthermore, 4-day spleen cultures obtained from animals 28 to 59 days of age yielded supernatants that also stimulated the migration of adult AM. The spleen cell culture supernatants from 42- to 49-day-old animals had the greatest activity and stimulated the migration of adult AM 2.5- to 3.2-fold compared to the supernatants from adult normal rabbits. The peak production of migration enhancement factor (MEF) by splenic lymphoid cells coincided with the peak activities found in the sera. It was observed that nonadherent peanut agglutinable lymphoid cells produced MEF. When sera or culture supernatants containing MEF were mixed with MIF-containing adult sera or spleen cell culture supernatants, the respective activities were neutralized. The large migrations of normal neonatal AM were diminished by the addition of MIF-containing sera obtained from BCG-sensitized/challenged rabbits. In contrast, AM from BCG-sensitized rabbits, which exhibited a state of reduced migration, were enhanced by MEF-containing sera from untreated young rabbits. Three peaks of MEF activity were detected in Sephadex G-100 column fractionated sera from 42-day-old rabbits having MWs of approximately (Peak I) 80,000, (Peak II) 43,000, and (Peak III) 8000 to 18,000; most of the activity was found in peaks II and III. Two peaks of MEF activity were detected in Sephadex G-100 column-fractionated spleen cell culture supernatants from 42-day-old rabbits having MWs of approximately (Peak I) 35,000 to 43,000 and (Peak II) 10,000 to 14,000; most of the activity was in peak I which corresponds to peak II of the serum fractionation experiment. Collectively, these data indicate that MEF is a lymphokine that could be important in the modulation of cell-mediated immune effector responses.     

Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes. An essential role for keratinocyte-derived IL-10.

Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, we examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. IL-10 mRNA expression was enhanced in UV-irradiated keratinocytes. The secretion of IL-10 by the irradiated keratinocytes was determined by Western blot analysis. A band reactive with anti-IL-10 mAb was found in supernatants from the UV-irradiated but not the mock-irradiated cells. IL-10 biologic activity was determined by the ability of the supernatants from the UV-irradiated keratinocytes to suppress IFN-gamma production by Ag-activated Th 1 cell clones. Anti-IL-10 mAb neutralized the ability of supernatants from UV-irradiated keratinocytes to suppress the induction of delayed-type hypersensitivity in vivo. Furthermore, injecting UV-irradiated mice with antibodies against IL-10 partially inhibited in vivo immunosuppression. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by UV-irradiated keratinocytes plays an essential role in the induction of systemic immunosuppression after total-body UV exposure.     

Stimulated rat T cell-derived inhibitory factor for cellular DNA synthesis (STIF). II. Kinetics of the production and characterization of the producer

Kinetics of the production of a stimulated T cell-derived inhibitory factor for cellular DNA synthesis (STIF) (1) from Con A-stimulated SD rat spleen cells, and the cells involved in the production of the factor, were examined comparatively with those of interleukin 2 (IL 2) and interferon (IFN). STIF activity in the culture supernatants reached a plateau 24 hr after the culture, and the plateau level was maintained during an additional culture for 72 hr. Characterization of the cells involved in STIF production by means of negative selection of unfractionated or nylon-fractionated spleen cells with anti-rat T cell serum or monoclonal antibodies plus complement revealed that the cells are nylon-nonadherent T cells bearing a suppressor T cell marker. The nylon-nonadherent T cell population did not require additional macrophages for STIF production. In vivo pretreatment of rats with cyclophosphamide (25 to 100 mg/kg) reduced or abolished the production of STIF from the Con A-stimulated spleen cells of the rats. The STIF production from Con A-stimulated spleen cells was inhibited by the addition of indomethacin at 10 ng/ml, indicating a regulatory role of prostaglandin(s) in STIF production. The above characteristics distinguish a T cell subset for STIF production from those for IL 2 and IFN production.     

Increased levels of apoptosis of leukocyte subsets in cultured PBMCs compared to whole blood as shown by Annexin V binding: relevance to cytokine production

Most of the investigatory studies of cytokine production by cells have been performed on purified cells or cell lines by measuring the secreted cytokine levels in the bulk culture supernatant. However, results of cytokine production from isolated peripheral blood mononuclear cells (PBMCs) cultivated in synthetic media, have been reported to be inaccurate and of low reproducibility. Isolation procedures have been shown to be toxic to certain cells. We hypothesised that purified cell culture techniques may result in increased levels of apoptosis of cells compared with whole blood culture techniques. To compare the effects on cell viability between PBMCs and whole blood techniques, an Annexin V binding assay was utilised. The effect of different cell concentration and serum/plasma concentrations on apoptosis levels in the various leukocyte subsets in PBMC and whole blood cultures following stimulation was investigated. There were significantly increased levels of apoptosis of cells in PBMC compared to whole culture at similar plasma concentrations, suggesting that cell viability was plasma concentration-dependent. There were significantly increased levels of apoptosis in PBMC cultures at the same cell concentration to whole blood techniques, suggesting that interaction between all cellular elements (as in whole blood techniques) is important in maintaining cell viability. These results suggest that whole blood culture techniques provide the best conditions for study of leukocyte cytokine production. If PBMC culture is performed, similar plasma and cell concentration to whole blood will best preserve cell viability.