Effects of IFN on human eosinophils in comparison with other cytokines. A novel class of eosinophil activators with delayed onset of action.

Extracellular killing is regarded as one of the main functions of eosinophils. Therefore, a cytotoxicity assay against antibody-coated Daudi-lymphoma cells was established to measure cytokine effects on peripheral blood eosinophils from healthy volunteers. Optimal time of exposure to cytokines and half optimal concentrations (EC50) were determined and the capability of various cytokines to enhance cytotoxicity of eosinophils was compared. Thus, after 24 h with cytokine, the highest activation of eosinophils was observed with recombinant human rhIFN-gamma (EC50 = 0.2 U/ml), followed by the known activators of eosinophils recombinant human granulocyte/macrophage CSF (rhGM-CSF), rhIL-3, and murine IL-5 (mIL-5). rhIFN-alpha and natural human IFN-beta (nhIFN-beta) enhanced cytotoxicity as well. On the other hand, in short term assays, eosinophils were not stimulated by IFN and the strongest stimulator was rhGM-CSF (EC50 = 0.2 U/ml), followed by rhIL-3, mIL-5, rhTNF, and rhIL-4. With rhTNF-alpha enhancement was more pronounced on freshly isolated eosinophils (EC50 = 0.6 U/ml) and declined with time. No significant stimulation was detected with rhG-CSF, rhIL-1 beta, rhIL-2, rhIL-6, and rhIL-8. On neutrophils, rhIL-8 enhanced cytotoxicity, but the stimulation was weak in relation to other neutrophil activators. Studies on the mechanism of cytotoxic activity revealed that cytotoxicity required opsonization of targets with specific antibody. FMF analysis was performed demonstrating that freshly isolated eosinophils express Fc-gamma RII (CD32), small amounts of Fc-gamma RIII (CD16), but not Fc-gamma RI (CD64). In experiments with blocking antibodies to Fc-gamma R cytotoxicity was restricted to Fc-gamma RII. Expression of Fc-gamma RII was not enhanced by rhGM-CSF, rhTNF-alpha, and mIL-5, but a significant increase in the number of positive cells was observed after incubation with rhIFN-gamma for 24 h (p less than 0.05). In addition, enhanced viability of eosinophils was observed when cultured in the presence of rhIFN-gamma, rhIFN-alpha, rhGM-CSF, and rhTNF-alpha, but not of rhG-CSF and rhIL-2. Thus, IFN appear to be another class of activators of eosinophils, characterized by their delayed type of action.     

Paradoxical synergistic effects of tumour necrosis factor and interleukin 1 in murine gut-derived sepsis with Pseudomonas aeruginosa.

The authors evaluated the synergistic effect of tumour necrosis factor (TNF) and interleukin 1 (IL-1) in gut-derived sepsis in mice. After colonization of Pseudomonas aeruginosa strain D4 in the gastrointestinal tract, cyclophosphamide was administered to induce bacterial translocation of the P. aeruginosa and thereby to cause gut-derived sepsis. In this model, treatment either with 8 microg/kg of recombinant human TNF-alpha (rhTNF-alpha) or 2 microg/kg of recombinant human interleukin 1alpha (rhIL-1alpha) solely did not affect the mortality, whereas combined administration of the same doses of rhTNF-alpha and rhIL-1alpha significantly increased the mortality rate in comparison with saline-treated mice. Bacterial counts in liver and blood were significantly higher in rhTNF-alpha and rhIL-1alpha treated mice than in saline-treated mice. Endogenous TNF-alpha and IL-1beta productions were stimulated after combined treatment with rhTNF-alpha and rhIL-1alpha. On the contrary to these adverse effects, combined treatment with 500 microg/kg of rhTNF-alpha and 50 microg/kg of rhIL-1alpha on the day before the administration of cyclophosphamide significantly reduced the mortality from septic infection. We conclude that TNF and IL-1 synergistically affect the mortality of mice after gut-derived sepsis due to P. aeruginosa in mice and the timing of treatment with these cytokines causes both extremes in their effects.     

Involvement of the liver, but not of IL-6, in IL-1-induced desensitization to the lethal effects of tumor necrosis factor.

C57BL/cnb mice were found to be protected against a lethal combination of recombinant murine (m) TNF and GalN by pretreatment with several cytokines. At certain doses, rmTNF and human (h) TNF protected completely. The clearest protection was induced by rIL-1: all four rIL-1 species (both m and h, as well as alpha and beta) protected when given 12 h before the challenge. LPS and rmIFN-gamma protected weakly, whereas rmIL-6 and rhIL-6 did not protect at all. Also adrenocorticotropic hormone, dexamethasone, or dexamethasone in combination with rhIL-6 could not protect. A single IL-1 injection also completely protected mice against a lethal dose of mTNF in the absence of GalN sensitization. The desensitization by IL-1 cannot be explained by a faster clearance of the challenge TNF. In addition, we demonstrate that the IL-1-induced desensitization was only observed when a functioning liver was present, that IL-1-pretreated animals did not show decreased numbers of hepatocyte TNF receptors, and that the amount of TNF-induced IL-6 was not reduced.     

重组白介素6-假单胞菌外毒素融合蛋白对正常BN大鼠骨髓粒系造血的作用

本文报道了重组白介素6-假单胞菌外毒素融合蛋白(IL-6-PE40)对正常BN大鼠骨髓粒系造血的体外效应。10ng/ml的IL-6-PE40对高表达IL-6受体的U266骨髓瘤细胞的蛋白质合成抑制率为50％,1000ng/ml则为100％。1～1000ng/mlIL-6-PE40对正常骨髓未纯化细胞的CFU-GM集落形成和DNA合成无明显抑制,但IL-6却具有一定的刺激效应。提示正常骨髓粒系祖细胞和骨髓细胞可能不表达IL-6受体,IL-6-PE40对粒系造血仍具有某些细胞毒作用,但被IL-6-PE40中的IL-6极大地削弱了。     

Early antibiotic administration but not antibody therapy directed against IL-6 improves survival in septic mice predicted to die on basis of high IL-6 levels

Elevated interleukin (IL)-6 levels correlate with increased mortality following sepsis. IL-6 levels >14,000 pg/ml drawn 6 h after cecal ligation and puncture (CLP) are associated with 100% mortality in ND4 mice, even if antibiotic therapy is initiated 12 h after septic insult. Our first aim was to see whether earlier institution of antibiotic therapy could improve overall survival in septic mice and rescue the subset of animals predicted to die on the basis of high IL-6 levels. Mice (n = 184) were subjected to CLP, had IL-6 levels drawn 6 h later, and then were randomized to receive imipenem, a broad spectrum antimicrobial agent, beginning 6 or 12 h postoperatively. Overall 1-wk survival improved from 25.5 to 35.9% with earlier administration of antibiotics (P < 0.05). In mice with IL-6 levels >14,000 pg/ml, 25% survived if imipenem was started at 6 h, whereas none survived if antibiotics were started later (P < 0.05). On the basis of these results, we examined whether targeted antibody therapy could improve survival in mice with elevated IL-6 levels. A different cohort of mice (n = 54) had blood drawn 6 h after CLP, and then they were randomized to receive either monoclonal anti-IL-6 IgG or irrelevant rat IgG. Anti-IL-6 antibody failed to improve either overall survival or outcome in mice with IL-6 levels >14,000 pg/ml. These results demonstrate that earlier systemic therapy can improve outcome in a subset of mice predicted to die in sepsis, but we are unable to demonstrate any benefit in similar animals using targeted therapy directed at IL-6.     

Pig MAP/ITIH4 and haptoglobin are interleukin-6-dependent acute-phase plasma proteins in porcine primary cultured hepatocytes.

The acute-phase expression of pig MAP (major acute-phase protein)/ITIH4 (inter-alpha-trypsin inhibitor heavy chain 4) and haptoglobin were analysed in primary cultures of isolated pig hepatocytes in response to recombinant human (rh) cytokines: tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), as well as to bacterial lipopolysaccharide (LPS). Analysis of pig MAP/ITIH4 and haptoglobin mRNAs was carried out by RT-PCR amplification. Secreted proteins from the cytokine-treated hepatocytes were quantified by immunochemical techniques. Time-course and dose-response experiments show that pig MAP/ITIH4 and haptoglobin belong to the type II acute-phase proteins, as they are specifically induced by rhIL-6 and not by rhTNF-alpha or rhIL-1. Stimulation of cultured pig hepatocytes with rhIL-6 for 48 h at doses of 1000 U.mL-1 showed a fourfold to fivefold increase in pig MAP/ITIH4 concentration in the medium, while the concentration of haptoglobin only increased twofold. A similar increase in the concentration of pig MAP/ITIH4 was also observed in media of LPS-treated hepatocytes with the simultaneous generation of IL-6 by the Kupffer cells present in the cultures. Albumin secretion decreased after stimulation with doses of 100 or 1000 U.mL-1 rhTNF-alpha, rhIL-1 or rhIL-6. Therefore, it can be concluded that pig MAP/ITIH4 behaves as a major acute-phase protein produced by porcine hepatocytes under the effect of inflammatory cytokines.     

IL-1 stimulates IL-6 production in endothelial cells

Leukocytes and vascular cells interact closely in inflammation and immunity and lymphokines are important mediators of this interaction. The present study was designed to define the possible role of IL-6 as a communication signal between vascular and immunocompetent cells. IL-6 was measured as hybridoma growth factor (HGF) on the 7TD1 cell line in the supernatants of human endothelial cells (HEC). HEC released appreciable levels of HGF activity in the absence of deliberate stimulation. In vitro exposure to recombinant IL-1 beta markedly increased (usually 10 to 15-fold) HGF production by HEC. Optimal stimulation was observed with 0.1 to 50 U/ml for 4 to 20 h of incubation. Human and murine rIL-1 alpha stimulated HGF production in HEC. Anti-IL-6 antibodies inhibited the HGF activity of the HEC supernatants, thus confirming, together with the cytokine specificity of the assay, the nature of HEC-produced cytokine. IL-1-treated HEC expressed high levels of IL-6 mRNA as detected by Northern blot analysis. Inasmuch as IL-1 elicits a complex series of changes in HEC, it was important to assess whether IL-6, produced after exposure to IL-1, modified HEC function. Natural or rIL-6 did not affect the functional status of HEC as assessed by proliferative capacity, production of procoagulant activity and prostacyclin, ability to induce adhesion of polymorphonuclear leukocytes. The capacity to produce IL-6 may represent an important mechanism by which endothelial cells participate in inflammatory and immune reactions.     

Effect of recombinant human interleukin-6 on nitrite production of mouse myeloid leukemia cells

The effect of recombinant human interleukin-6 (rhIL-6) on induction of nitrite (NO(-2)) production was investigated in a mouse myeloid leukemia cell line (M1) and a subclone (Mm1). NO(-2) was induced by rhIL-6 (greater than 50 U/ml) in these cell lines. Pretreatment with rhIL-6 (100 U/ml) for 1 day synergistically enhanced the production of NO(-2) in Mm1 by LPS. Furthermore, pretreatment with IL-6 (100 U/ml) shortened the lag time of induction. These results indicate that IL-6 is involved in the regulation of the NO(-2) production in macrophage-like cells.     

Sequential appearance of IL-1 and IL-6 activities in rat carrageenin-induced pleurisy.

IL-1 and IL-6 activities were measured in the pleural exudate of rats during carrageenin-induced pleurisy to examine the relationship of the local production of cytokines to the inflammatory reaction. Time courses of appearance of the cytokines and inflammatory parameters in the exudate were compared. IL-1 activity and exudate volume started to increase at 1 h after the carrageenin injection, and then slightly later IL-6 activity and leukocyte number began to increase. IL-1 showed peak activity of approximately 700 U at 3 h and IL-6, of 6000 U at 5 h in the exudate, whereas exudate volume and number of polymorphonuclear leukocytes continued to increase thereafter. Furthermore, IL-6 level in the plasma of the carrageenin-injected rats showed a peak at 4 h (30 U/ml), and when rhIL-1 alpha (100,000 U) was intrapleurally injected, the more rapid increase in plasma IL-6 level was demonstrated at 1 h (30 U/ml). This latter rise was neutralized with simultaneous injection of anti-rhIL-1 alpha antibody. These facts indicate the possibility that IL-1 produced in the exudate or injected could rapidly propagate a signal to induce IL-6 production in the circulation. It took several hours to transmit an inflammatory signal that stimulated the liver to synthesize the acute-phase protein, T-kininogen. The time lag from the peak induction of IL-1 to the T-kininogen-increase in the pleurisy corresponded well to the interval for T-kininogen-increase by exogenous rhIL-1 alpha injection. These results strongly suggest that the initial inflammatory stimulus induces sequentially IL-1, IL-6, and T-kininogen production in this carrageenin inflammation.     

IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release

IL-6 enhances the differentiation of pluripotent hematopoietic stem cells but predominantly affects the differentiation of hematopoietic cells in the granulocyte-macrophage lineage. We have previously shown that multinucleated cells (MNC) with many features of the osteoclast phenotype form in long term human marrow cultures. Addition of rhIL-6 (10 to 100 pg/ml) to these cultures significantly increased MNC formation, with greater than 80% of the MNC expressing an Ag that cross-reacts with the mAb 23c6. This antibody preferentially binds to osteoclasts. rhIL-6 did not enhance MNC formation in marrow cultures treated with 1,25 dihydroxyvitamin D3, a potent stimulator of MNC formation, but significantly increased the percentage of MNC that cross-reacted with the 23c6 mAb. Addition of antihuman IL-1 to cultures treated with rhIL-6 totally inhibited the increase in MNC formation stimulated by rhIL-6. In contrast, anti-IL-1 did not affect MNC formation stimulated by 1,25 dihydroxyvitamin D3. Further, conditioned media from marrow cultures exposed to rhIL-6 contained elevated levels of IL-1 beta (500 pg/ml compared to 23 pg/ml in control cultures 15 h after IL-6 addition). These results suggest that the capacity of rhIL-6 to stimulate formation of MNC which cross-react with 23c6 is mediated by induction of release of IL-1 beta.     

IL-6 enhances plasma IL-1ra,IL-10, and cortisol in humans

The purpose of the present study was to test the hypothesis that a transient increase in plasma IL-6 induces an anti-inflammatory environment in humans. Therefore, young healthy volunteers received a low dose of recombinant human (rh)IL-6 or saline for 3 h. Plasma IL-6 levels during rhIL-6 infusion were approximately 140 pg/ml, corresponding to the levels obtained during strenuous exercise. The infusion of rhIL-6 did not induce enhanced levels of the proinflammatory cytokine TNF-alpha but enhanced the plasma levels of the two anti-inflammatory cytokines IL-1 receptor agonist (IL-1ra) and IL-10 compared with saline infusion. In addition, C-reactive protein increased 3 h post-rhIL-6 infusion and was further elevated 16 h later compared with saline infusion. rhIL-6 induced increased levels of plasma cortisol and, consequently, an increase in circulating neutrophils and a decrease in the lymphocyte number without effects on plasma epinephrine, body temperature, mean arterial pressure, or heart rate. In conclusion, this study demonstrates that physiological concentrations of IL-6 induce an anti-inflammatory rather than an inflammatory response in humans and that IL-6, independently of TNF-alpha, enhances the levels not only of IL-1ra but also of IL-10. Furthermore, IL-6 induces an increase in cortisol and, consequently, in neutrocytosis and late lymphopenia to the same magnitude and with the same kinetics as during exercise, suggesting that muscle-derived IL-6 has a central role in exercise-induced leukocyte trafficking.     

Effects of IL-1 on hematopoietic progenitors after myelosuppressive chemoradiotherapy

Recently it has been recognized that IL-1 plays an important role in hematopoietic regulation. Administration of 5-fluorouracil (5-FU) to mice causes prolonged neutropenia. rHIL-1 injected to mice after 5-FU, accelerated the recovery of hematopoietic progenitors and blood neutrophils. The combination of rhIL-1 and rhG-CSF reduced the neutropenic period significantly. Sublethal irradiation of mice induced profound neutropenia for 3 weeks which was associated with 80% mortality. Administration of rhIL-1 20 hours prior to or 2 hours post irradiation resulted in a significantly improved survival and rapid recovery of the neutrophil count. IL-1 administered alone or in combination with other colony stimulating factors to spontaneous breast tumor bearing mice following 5-FU therapy resulted in a rapid recovery of neutrophils, improved survival, and markedly reduced the tumor mass. Experiments in primates demonstrated that rhIL-1 administered to 5-FU treated animals shortened the neutropenic period from 30 to 17 days and increased the number of marrow progenitors responsive to other CSFs. Prolonged administration of IL-1 (14 days) to these animals resulted in a delayed neutrophil recovery as compared to animals receiving short courses of IL-1. rhIL-1 administered to primates receiving marrow grafts after lethal irradiation, did not result in rapid hematopoietic recovery. In humans, studies with CD-34 positive marrow cells showed that IL-1 had a radioprotective effect on a committed and early marrow progenitors. These data show the therapeutic potential of IL-1 in the treatment of chemoradiotherapy induced myelosuppression.     

Effects of treatment with interleukin-1 receptor antagonist on endogenous interleukin-1 levels in normal and irradiated mice

The in vivo effects of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) administration on endogenous IL-1 levels in the circulation and conditioned media (CM) from different immunohematopoietic organ/tissues were studied in CBA mice under steady state and postirradiation conditions. In normal mice, constitutive IL-1 levels were demonstrated in the plasma, CM of peritoneal exudate cells and full-thickness skin explants with low or undetectable levels in CM of splenic and bone marrow cell suspensions. In irradiated mice (2 Gy, X rays) on day 3 post exposure a significant increase of IL-1 levels was seen in the circulation and CM of peritoneal exudate cells, with no significantly different levels in postirradiation bone marrow, spleen and skin. After rhIL-1Ra treatment of the animals (2 x 50 microg/mouse, i.p.), significantly elevated IL-1 levels were observed in the skin and CM of peritoneal exudate cells in normal mice, whereas slightly increased levels were detected in CM of splenic cells. The rhIL-1Ra administration in irradiated mice led to decreased IL-1 concentrations in the circulation, and CM of peritoneal exudate cells and skin. The results pointed out the importance of IL-1 secretion and receptor expression in the maintenance of homeostasis in steady state, as well as during recovery after irradiation. Modulatory effects of IL-1Ra on IL-1 production were dependent on basic endogenous IL-1 concentration.     

Modulation of IL-1 inflammatory and immunomodulatory properties by IL-6.

Among the major cytokines present in inflammatory lesions interleukin-1 (IL-1), tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) share many biological activities. Since IL-1 alpha, IL-1 beta and TNF alpha have been previously demonstrated to play an important role in connective tissue destruction by stimulating the production of prostaglandin E2 (PGE2) and collagenase, these functions were investigated in the presence or absence of natural human IL-6 (nhIL-6) or recombinant human IL-6 (rhIL-6). IL-6 was found 1 degree to stimulate immunoglobulin A production by the CESS B cell line up to 19 fold without being affected by the presence of IL-1 beta and 2 degrees to stimulate murine thymocytes proliferation up to 2-4 fold, with an increase up to 60-fold in costimulation with either IL-1 alpha or beta. IL-6 alone, even at very high concentrations (up to 200 U/ml and 50 ng/ml), did not induce PGE2 production by fibroblasts and synovial cells. However, IL-1 alpha or beta induced PGE2 production by human dermal fibroblasts and by human synovial cells was inhibited (in 5/8 experiments) up to 62% by addition of IL-6. On the contrary in 2/4 experiments TNF alpha-induced PGE2 production was increased (approximately 2 fold) by the addition of IL-6. IL-1 and TNF alpha-induced collagenase production in synovial cells remained unchanged in the presence of IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytokines in normal and abnormal parturition: elevated amniotic fluid interleukin-6 levels in women with premature rupture of membranes associated with intrauterine infection

The participation of interleukin-6 (IL-6) in the pathophysiology of normal and abnormal human parturition was evaluated by determining IL-6 concentrations in amniotic fluid (AF). Biologically active IL-6 was determined (in U/ml) using the B9 hybridoma growth factor assay, while the concentrations of immunoreactive IL-6 species (in pg/ml) were assessed using a monoclonal antibody (moAb)-based ELISA. Two hundred and twenty-seven AF samples from women in normal labor and from those presenting with a clinical diagnosis of premature rupture of membranes (PROM) were assayed. In selected instances, IL-6 levels were evaluated simultaneously in AF and in maternal and fetal plasma. Women with a normal pregnancy had low titers of biologically active IL-6 in AF both at midtrimester (group 1, n = 27; median IL-6 concentration = 16 U/ml) and at term (group 2, n = 33; median = 15 U/ml). There was an increase in the IL-6 bioactivity in AF from women in normal labor at term (group 3, n = 40; median = 74 U/ml; p less than 0.001). In order to distinguish between the relative contributions of parturition per se and of intrauterine infection to the elevation of biologically active IL-6 levels in AF, IL-6 titers were compared in four different groups of women with PROM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced oral tolerance in transgenic mice with hepatocyte secretion of IL-10

Several cytokines derived from Th3 and Tr1 cells, including IL-10, are believed to regulate oral tolerance, but direct evidence is lacking. We have explored the potential role of IL-10 by generating transgenic (TG) mice with sustained hepatocyte-specific expression of rat IL-10. TG mice expressed rat IL-10 downstream of a transthyretin promoter, which led to serum levels that were increased 10- to 100-fold compared with normal animals. Animals were orally administered 1 mg of whole OVA for 5 consecutive days, with control animals receiving PBS. There were six animal groups: Either OVA or PBS were fed orally to rat IL-10 TG mice, non-TG wild-type mice without IL-10 administration, and non-TG wild-type mice administered rat IL-10 systemically. On day 8, all mice were immunized with two injections of OVA, and then analyzed on day 18. T cell proliferation responses were reduced by 65.8 +/- 14.3% after feeding of OVA in rIL-10 TG animals, compared with 39.4 +/- 15.6% in the non-TG mice (p = 0.02). Anti-OVA titers were expressed as fold increase over naive non-TG mice. After feeding, titers decreased by approximately 33% (from 3- to 2-fold) in TG animals and, to a lesser extent, in non-TG animals. IFN-gamma secretion by cultured popliteal lymphocytes decreased in TG animals by 83% after feeding and by 69% in non-TG animals. IL-4 secretion increased 4-fold in TG-fed mice, but did not significantly change in non-TG OVA-fed animals. In contrast to hepatic TG expression of rIL-10, systemic administration of rIL-10 had only a modest effect on tolerance. IL-10, when transgenically expressed in the liver enhances mucosal tolerance to an oral Ag.     

Chemoprotective effects of recombinant human IL-1 alpha in cyclophosphamide-treated normal and tumor-bearing mice. Protection from acute toxicity, hematologic effects, development of late mortality, and enhanced therapeutic efficacy

In this study, recombinant human IL-1 alpha (rhIL-1 alpha) was used to protect normal and tumor-bearing BALB/c mice from the acute toxicity caused by lethal doses of cyclophosphamide (Cy) and 5-fluorouracil. Pretreatment of mice for 7 days with 10,000 U/day of rhIL-1 alpha protected 70 to 100% of mice from the acute death induced by lethal doses of both Cy (380 mg/kg) and 5-fluorouracil (250 mg/kg). In contrast, post-treatment of mice with single or multiple doses of rhIL-1 alpha was not chemoprotective. Pretreatment of mice with rhIL-1 alpha increased the acute LD90 of Cy from 380 mg/kg to greater than 500 mg/kg in normal mice, an LD90 dose-modifying effect of at least 1.25, was accompanied by a more rapid recovery from neutropenia and a less severe reduction in the number of bone marrow single lineage monocyte, myeloid, or myelomonocytic colonies. Some of the mice (10 to 50%) that were successfully protected by pretreatment with rhIL-1 alpha died after day 50. These mice consistently presented with extensive pulmonary inflammation and fibrosis at death. Mice bearing murine renal cancer (Renca) were also protected from the acute toxic effects of Cy (450 mg/kg) by pretreatment with rhIL-1 alpha. Renca-bearing mice pretreated with rhIL-1 alpha and either sublethal (300 mg/kg) or lethal (450 mg/kg) doses of Cy exhibited enhanced survival times over those of untreated Renca-bearing mice. Interestingly, the cause of death in Renca-bearing mice that ultimately failed treatment with rhIL-1 alpha plus 300 mg/kg Cy was recurrent tumor, whereas most mice treated with rhIL-1 alpha plus 450 mg/kg Cy had no detectable tumor, although several died from late pulmonary inflammation and fibrosis. Thus, the dose escalation of Cy in rhIL-1 alpha-pretreated mice results in greater antitumor effects of Cy. However, the dose escalation of some cytotoxic agents allowed by the use of myelostimulatory agents can result in late fatal complications not detected in acute toxicity testing.     

Relationship between circulating interleukin-10 (IL-10) with interleukin-6 (IL-6) type cytokines (IL-6, interleukin-11 (IL-11), oncostatin M (OSM)) and soluble interleukin-6 (IL-6) receptor (sIL-6R) in patients with multiple myeloma

We investigated the serum concentration of the interleukin-10 (IL-10), along with cytokines of interleukin-6 (IL-6) family (IL-6, IL-11 and oncostatin M - OSM), as well as soluble receptor for IL-6 (sIL-6R), in 121 patients with multiple myeloma (MM) and 28 healthy subjects. We studied the interactions between IL-10 and other cytokines, and the receptor. The correlation between IL-10 and some clinical and laboratory parameters associated with the disease activity were also analysed. The IL-10 was detectable in all patients with multiple myeloma and in all controls. The IL-10 concentration was significantly increased in myeloma patients compared with healthy persons (mean - 7.09 and 2.1 pg/ml, respectively) (p = 0.008). The level of IL-10 correlated positively with the advanced stage of disease estimated according to the Salmon and Durie classification (I versus III stage - p = 0.03). Higher values of IL-10 were found in patients with the light chain disease, hypercalcaemia, and correlated with the elevated concentrations of C-reactive protein (CRP). IL-6 was detected in 117 of the 121 patients and in all controls. The concentration of IL-6 was statistically increased in MM patients compared with control group (mean - 16.06 and 4.49 pg/ml, respectively) (p = 0.01). We found a positive correlation between IL-10 and IL-6 serum levels in MM patients. The relationship, expressed as Spearman's rank sum coefficient (rho = 0.249, p = 0.006) was significant. IL-11 was detected in 26 of the 121 MM patients and in 3 of the 28 healthy subjects at the mean concentration of 1.2 and 0.6 pg/ml respectively (p > 0.05). OSM was at detectable levels in 51 of the 121 patients and in only 4 of the 28 controls (mean - 3.84 and 0.1 pg/ml, p = 0. 002). The correlation between IL-10 and IL-11 levels in MM patients was not significant, but there was a strong statistical correlation between IL-10 and OSM concentrations (rho= 0.327, p = 0.0002). The serum concentration of sIL-6R was measurable in all patients and all controls (mean - 66.00 and 39.57 ng/ml respectively), but the difference between these groups was not significant. We found significant, positive correlation between the levels of IL-10 and sIL-6R (rho= 0.233, p = 0.01). In conclusion, we state that the serum concentrations of IL-10, IL-6, OSM and sIL-6R in MM patients may be a useful markers for the evaluation of the disease activity.     

Adoptive immunotherapy of microscopic and advanced visceral metastases with in vitro sensitized lymphoid cells from mice bearing progressive tumors

Lymphocytes from mice bearing the weakly immunogenic MCA 105 sarcoma contained pre-effector cells that could be sensitized and expanded in vitro to acquire anti-tumor reactivity. The in vitro sensitization (IVS) required antigenic stimulation by tumor cells and the presence of IL-2 for cellular proliferation. Recent work has also demonstrated that IVS cells could be generated in an IL-2 concentration as low as 2 U/ml. In the present study, we have evaluated therapeutic efficacy of IVS cells generated in different concentrations of IL-2 against advanced metastases established in the lung as well as in the liver. Treatment of microscopic or grossly visible pulmonary metastases established for 3 or 10 days by systemic transfer of IVS cells resulted in significant reductions of the numbers of metastases. On a per cell basis, IVS cells generated in 2 U/ml of IL-2 exhibited at least twofold greater reactivity than cells generated in 1000 U/ml of IL-2. In survival experiments, treatment of established microscopic (day 3) and visible (day 10) pulmonary metastases with 1.5 x 10(7) and 3 x 10(7) IVS cells generated in 2 U/ml of IL-2 resulted in prolongation of survival and cure of the disease in 60 and 30% of animals, respectively. The systemic anti-tumor effect of IVS cells was further investigated in mice with hepatic metastases. Treatment of day 3 microscopic hepatic metastases revealed that as little as 1.2 X 10(7) IVS cells generated in 2 U/ml of IL-2 reduced the mean number of metastases from more than 250 in various control groups to 32. Evaluation by survival demonstrated that transfer of 1.7 x 10(7) and 3.8 x 10(7) IVS cells was capable of prolonging the survival and curing 40 and 30% of mice bearing day 3 and day 9 hepatic metastases, respectively. Again, IVS cells generated in 2 U/ml of IL-2 were more effective therapeutically than cells generated in 1000 U/ml of IL-2. In all experiments, mice were also given IL-2 to enhance the in vivo reactivity of IVS cells. However, the doses of IL-2 alone had no therapeutic benefit. Taken together, our results show that adoptive immunotherapy with IVS cells generated from tumor-bearing mice was an effective means of eliminating advanced metastases in various visceral organs.