Susceptibility of monocytes to lymphokine-activated killer cell lysis: effect of granulocyte-macrophage colony-stimulating factor and interleukin-3

Cultured human monocytes have been shown to be susceptible to lysis by autologous lymphokine-activated killer (LAK) cells. To determine factors that might modulate the sensitivity of monocytes to lysis, we cultured adherent peripheral blood leukocytes (PBL) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) since these cytokines have been reported to affect both functional and physical characteristics of monocytes. Both recombinant human GM-CSF and IL-3 were found to significantly enhance the susceptibility of monocytes to lysis by LAK cells in a dose- dependent manner, with GM-CSF being slightly more effective. In a kinetics study, the lysability of monocytes increased after two days of incubation with either cytokine, with maximal susceptibility occurring after four to six days of culture. The effects of GM-CSF and IL-3 appeared to be specific for monocytes since culture of either nonadherent cells or granulocytes, which are normally resistant to LAK- mediated lysis, did not induce sensitivity. While the effects of GM-CSF and IL-3 have been shown to be synergistic in some cases, they did not act synergistically to induce monocyte susceptibility to LAK lysis. In cold target experiments cytokine-treated monocytes reciprocally blocked lysis, suggesting that similar target structures were modulated with either factor. FACS analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated comparable modulation of surface antigens with either GM-CSF or IL-3. Thus, these cytokines can serve to augment susceptibility of monocytes to LAK cells, emphasizing the complex interactions that occur in the immune system.     

Lysis of human malignant mesothelioma cells by natural killer (NK) and lymphokine-activated killer (LAK) cells

Malignant mesothelioma is an aggressive tumor of the pleura for which, at present, there is no effective therapy. As interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells lyse many solid tissue malignancies that are unresponsive to conventional forms of therapy, the aim of this study was to evaluate the susceptibility of human malignant mesothelioma cells to lysis by natural killer (NK) and LAK cells. Using a 4-h 51Cr release assay, malignant mesothelioma cell lines grown from six different patients were found to be resistant to NK cell lysis (less than 10% lysis as compared to 50 +/- 3% lysis of the standard NK-sensitive target, K562, p less than 0.001). These malignant mesothelioma cells were, however, susceptible to lysis by LAK cells (58 +/- 4% lysis, p less than 0.001 compared to NK lysis). Similar results were seen using fresh mesothelioma cell targets (4 +/- 2% and 34 +/- 12% lysis for NK and LAK cells, respectively). Optimal LAK cell activation against these targets was achieved by incubating peripheral blood mononuclear cells (2 to 4 x 10(6)/ml) in culture medium containing 1,000 units/ml IL-2 for 3 to 14 days. The degree of LAK cell activation was dependent on the serum source used in culture, with autologous serum being more effective than pooled human AB serum or fetal calf serum at generating LAK cell activity in vitro (p less than 0.05). The results of this study demonstrate that although human malignant mesothelioma cells are resistant to NK cell lysis, IL-2-activated LAK cells effectively kill these targets.(ABSTRACT TRUNCATED AT 250 WORDS)     

The gamma subunit of the interleukin-2 receptor is expressed in human monocytes and modulated by interleukin-2, interferon gamma, and transforming growth factor beta 1

The interleukin-2 receptor gamma (IL-2R gamma) chain is a newly recognized component of the IL-2R of lymphoid cells that is required for their response to IL-2. We investigated the expression of IL-2R gamma protein in human monocytes by Western blot analysis using an antiserum specific for IL-2R gamma. We found that IL-2R gamma subunit is constitutively expressed in human monocytes and upregulated by the monocyte-activating factors IL-2 and interferon gamma (IFN gamma). Furthermore, we show that transforming growth factor beta 1 (TGF beta 1) downmodulates, in a dose-dependent manner, basal and IL-2-induced, but not IFN gamma-induced, IL-2R gamma chain expression, and this effect may be responsible for TGF beta 1 suppressive activity on IL-2- activated monocytes. Overall, these results show that the expression of the IL-2R gamma subunit in human monocytes is tightly regulated by the cytokine network, suggesting a critical role played by this protein on monocyte activation.     

Inhibitory cytokine circuits involving transforming growth factor-beta, interferon-gamma, and interleukin-2 in human monocyte activation

We have previously reported that transforming growth factor-beta 1 (TGF- beta 1) inhibits interleukin-6 (IL-6) induction by IL-2 and IL-1 in fresh human monocytes. We investigated the effects of TGF-beta 1 on the expression of tumoricidal activity induced by IL-2 or interferon-gamma (IFN-gamma) in human monocytes. We showed that TGF-beta 1 specifically inhibited, in a dose-dependent manner, IL-2-induced but not IFN-gamma- induced monocyte tumoricidal activity. The inhibitory effects of TGF- beta 1 on IL-2-activated monocytes were not caused by down-modulation of the IL-2 receptor beta (IL-2R beta) because the treatment of monocytes with IL-2 and TGF-beta 1 increased IL-2R beta mRNA expression. However, we found that TGF-beta 1 down-modulated IL-2- induced IL-2R gamma mRNA, which may be responsible for the TGF-beta 1 inhibition of monocyte activation by IL-2. The resistance of the IFN- gamma-induced activation to the inhibitory effects of TGF-beta 1 could be caused by the ability of IFN-gamma to decrease TGF-beta 1 receptor expression, as shown by cross-linking experiments. Overall, these results showed that TGF-beta 1 is a powerful inhibitor of IL-2- but not of IFN-gamma-induced activation of monocytes to a cytotoxic stage. This differential effect may be attributed to modulation of cytokine receptor expression.     

Cyclooxygenase-2 regulates granulocyte-macrophage colony-stimulating factor, but not interleukin-8, production by human vascular cells: role of cAMP

Vascular smooth muscle is now recognized as an important site of mediator generation under inflammatory conditions. Indeed, the release of leukocyte activators, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-8, by human arterial smooth muscle cells has recently been demonstrated. However, the potential for venous cells to release GM-CSF has not been addressed. We have shown that human vascular smooth muscle cells express the "inflammatory" form of cyclooxygenase (COX), cyclooxygenase-2 (COX-2), when stimulated with cytokines. In some nonvascular cell types, the COX activity has been shown to regulate the release of GM-CSF and IL-8, although the nature of the isoform responsible was not addressed. We show that human venous smooth muscle cells, like their arterial counterparts, release GM-CSF after stimulation with IL-1beta. Similarly, both cell types released IL-8. Under the same conditions, we found that COX-2 activity suppressed GM-CSF, but not IL-8, release by both types of human vascular cells. Moreover, the prostacyclin mimetic, cicaprost, and the cAMP analogue, dibutyryl cAMP, inhibited GM-CSF release from these cells. These observations suggest that COX-2 activity suppresses GM-CSF release via a cAMP-dependent pathway in human vascular cells and illustrates a novel mechanism by which this enzyme can modulate immune and inflammatory events.     

Effects of interleukin-2 on gene expression in human neutrophils

Recently, the interleukin-2 receptor (IL-2R) was shown to be present on human neutrophils, and IL-2-neutrophil interactions are believed to be important in both tumor rejection and increased susceptibility to bacterial infections. Furthermore, neutrophils have been shown to synthesize host defense proteins, such as cytokines. In this study, we analyzed the effects of IL-2 on the induction of de novo RNA and protein synthesis in this cell type. When cells were stimulated with IL- 2 alone, the level of incorporation of either [5-3H]-uridine or [35S]- methionine and [35S]-cysteine was similar to unstimulated cells. However, when cells were stimulated with the combination of a fixed concentration of granulocyte-macrophage colony-stimulating factor (GM- CSF), a dose-dependent effect of IL-2 was observed on the induction of both RNA and protein synthesis. In the presence of tumor necrosis factor-alpha or formyl-methionyl-leucyl-phenylalanine, however, IL-2 exerted no similar effect. Furthermore, the study of a large number of normal subjects (n = 55) showed reproducible categories of responders (low, intermediate, and high). The binding of IL-2 to the IL-2R complex on human neutrophils increased on GM-CSF-stimulated neutrophils compared with unstimulated cells. However, no increase in the level of expression of either the alpha or beta chains of this receptor complex was observed. This finding suggests that GM-CSF functionally activates the IL-2R, but does not regulate its level of expression. Finally, we found that human neutrophils constitutively express IL-2R gamma chain mRNA and thus have the potential to express the functional IL-2R complex. Our findings on IL-2-neutrophil interactions should lead to new avenues of research in understanding the responses of patients undergoing GM-CSF or IL-2 therapy.     

Lysis of neuroblastoma cell lines by human natural killer cells activated by interleukin-2 and interleukin-12

Neuroblastoma is the most common extracranial, solid tumor in children. Despite intensive chemotherapy and bone marrow transplantation, the 5- year projected survival rate is 20% to 25%. In vitro studies have shown enhanced natural killer cell (NK) lysis of tumor cells after exposure of NK cells to interleukin-2 (IL-2). In vivo studies have demonstrated similar immunologic effects but have also revealed severe toxicities associated with the use of IL-2. IL-12 is a newly described cytokine that has several properties, including the ability to act synergistically with IL-2 in generating lymphokine-activated killer cells (LAK) against known tumor targets. We investigated the role of IL- 12 in the generation of peripheral blood mononuclear cell (PBMC) lysis of neuroblastoma cell lines. PBMC were activated with IL-12 alone and in combination with IL-2. Whereas IL-12 alone produced only modest enhancement of NK cell cytotoxicity, the combination of IL-2 and IL-12 was most effective in activating NK cell lysis of neuroblastoma cell lines. Further, we showed that large granular lymphocytes were the effector cells involved in target cell lysis. Finally, the CD18 molecule was shown to be critical in the lysis of neuroblastoma cells by activated PBMC.     

Co-culture of synovial fibroblasts and differentiated U937 cells is sufficient for high interleukin-6 but not interleukin-1beta or tumour necrosis factor-alpha release

Inflamed synovium is characterized by high concentrations of cytokines [interleukin (IL)-6, IL-1beta and tumour necrosis factor (TNF)-alpha] and the abundant presence of infiltrated monocytes, many of which are found adjacent to the resident fibroblast-like synoviocytes. We have used a co-culture of fibroblast-like synoviocytes and differentiated U937 cells to study IL-6, IL-1beta and TNF-alpha release. After a 3 day co-culture, 35% of the U937 cells had adhered and were fully differentiated towards monocytes, as determined by expression of p47phox, CD14, MSE-1, Mac-1, collagenase and NADPH oxidase activity. IL- 6 release from fibroblast-like synoviocytes was induced 4-fold by co- culture with differentiated U937 cells. However, co-culture of differentiated U937 cells with fibroblast-like synoviocytes failed to release detectable levels of IL-1beta and TNF-alpha from the U937 cells. Addition of synovial fluid further increased IL-6 release, but again had no effect on IL-1beta or TNF-alpha, although U937 cells differentiated by phorbol ester were able to release these two cytokines and, in the case of the co-culture, mRNAs for both cytokines were highly expressed in the U937 cells. We postulate that the influx of monocytes into the synovium is instrumental in the elevation of IL-6 levels, but this is not sufficient to explain high levels of IL-1beta or TNF-alpha.      

Nonspecific cytotoxicity of recombinant interleukin-2 activated lymphocytes

The administration of interleukin-2 (IL-2) and lymphokine activated killer (LAK) cells to patients with advanced metastatic cancer has yielded encouraging results. The purported ability of LAK cells to be discriminatively tumoricidal, thus sparing normal host tissue, represents a major advance over conventional chemotherapy. However, IL-2 adoptive immunotherapy results in dose-limiting toxicity characterized by weight gain, dyspnea, ascites, and peripheral-pulmonary edema suggestive of a vascular leak syndrome. It is unclear whether the observed toxicity is directly related to IL-2 and/or LAK cells. The authors examined the cytolytic nature of human LAK cells against human endothelial, epithelial, and fibroblast cell lines. Bovine endothelial cells also were studied. Using a 51Cr release assay, the cytolytic potential, time course, and effect of reactive oxygen intermediate inhibitors were studied. LAK cells were uniformly toxic against all cell lines, in contrast to high dose rIL-2 and excipient. Significant cytolysis was observed within 30 minutes and increased over the first 2 hours of LAK cells coming in contact with target cells. Reactive oxygen intermediate inhibitors did not reduce cytolytic activity. The authors thus found human LAK cells to be rapidly cytolytic against a variety of human and bovine cell lines. This cytolysis was independent of reactive oxygen intermediates.     

HIV infection is associated with the spontaneous production of interleukin-1 (IL-1) in vivo and with an abnormal release of IL-1 alpha in vitro

Interleukin-1 (IL-1) is not constitutively produced by normal human monocytes. We have investigated the production of cell-associated IL-1 in uncultured unstimulated adherent monocytes from HIV-infected patients, which reflects ongoing generation of IL-1 by the cells in vivo. High levels of cell-associated IL-1 activity and of cell-associated IL-1 alpha and IL-1 beta antigens were found in monocytes from HIV-infected patients as compared with those found in monocytes from normal individuals. Amounts of cell-associated IL-1 were high in patients with AIDS and in patients from Centers for Disease Control groups II and III. Serum-free culture for 24 h of monocytes from HIV-infected individuals in the absence of lipopolysaccharides (LPS) resulted in spontaneous release of IL-1 activity from the cells whereas no release occurred upon culture of normal cells. Stimulation of monocytes with LPS induced the release of IL-1 alpha and IL-1 beta from cells of infected patients. Only IL-1 beta was released from cells of normal individuals. Thus, circulating monocytes from HIV-infected patients are triggered to produce IL-1 in vivo. The present study also indicates that HIV infection is associated with an acquired defect in the intracellular processes regulating IL-1 secretion.     

The neuroendocrine effects of interleukin-2 treatment

Observations of neuropsychiatric changes in patients receiving interleukin-2 (IL-2) led us to examine the effects of IL-2 administration on the stress-related hormones, beta-endorphin, ACTH, cortisol, and CRH. We evaluated 30 cancer patients who received immunotherapy with IL-2 or IL-2 plus lymphokine-activated killer (LAK) cells. Blood samples were taken immediately before and 4 and 8 h after infusion of IL-2 or IL-2 plus LAK cells. IL-2 stimulated increased hormone levels 4 h after infusion compared with those before therapy and with basal levels in normal volunteers at the following magnitudes: beta-endorphin, 10-fold; ACTH, 20-fold; and cortisol, 2-fold. The effect of IL-2 was not altered in patients also receiving LAK cells. An effect of treatment course was noted, with higher stimulated values seen 4 h after IL-2 in the second treatment course compared with those after the first course [change (delta) in beta-endorphin, 101 vs. 11 fmol/mL; delta ACTH, 138 vs. 8 pmol/L; delta cortisol, 414 vs. 218 nmol/L]. We conclude that IL-2 treatment induces the release of neuroendocrine hormones and that a significant increase in hormonal stimulation occurs upon reexposure to IL-2.     

Recombinant interleukin 3 induces interleukin 2 receptor expression on early myeloid cells in normal human bone marrow.

Human interleukin 3 (IL-3) is a multipotential cytokine that supports the growth of early hematopoietic progenitors and promotes their response to other, later-acting cytokines. We found that IL-3 was able to induce the expression of interleukin 2 (IL-2) receptor (IL-2R) (CD25) on a subset of early myeloid cells in normal human bone marrow that had been first depleted of mature hematopoietic cells and E-rosette-positive T cells by treatment with soybean lectin and sheep erythrocytes (SBA-E-BM). Immunofluorescence analysis revealed that the CD25+ cells were contained almost entirely within the lymphoblastoid gate of the IL-3-cultured marrow. CD25 was undetectable on freshly isolated marrow and less than 10% CD25+ cells could be detected following liquid culture at 37 degrees C in the presence of 10% human serum, 10% fetal calf serum, or under serum-free conditions. Addition of IL-3 (100 U/ml) significantly increased the expression of CD25 to 37%, 31%, and 24%, respectively. CD25 could also be induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), but no IL-2R was detectable following exposure to granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), interleukin 1 (IL-1), interleukin 4 (IL-4), or IL-2. Expression of CD25 was dependent on the dose of IL-3 or GM-CSF added and was maximal within 24 h of exposure. Two-color immunofluorescence analysis demonstrated that CD25 was not expressed by cells of lymphoid lineage or by mature monocytes, but rather was present on cells that coexpressed CD13, CD33, CD34, MY8, and HLA-DR, and that lacked CD14 or CD11b, thus placing the CD25+ cells at or near the myeloblast stage of differentiation. An identical phenotype was found for CD25+ cells induced by GM-CSF. Cycloheximide completely inhibited the IL-3-induced expression of CD25, indicating the necessity for protein synthesis, and although most of the CD25+ cells were in G0/G1 phase, 25% of the cells were in S or G2M phase, indicating that receptor expression was not cell-cycle dependent. The p75 chain of IL-2R was not detected on the CD25+ cells. IL-3 was also found to directly induce CD25 in greater than 46% of SBA-E-BM enriched for CD34+ cells by panning. Consistent with the expression of only p55 IL-2R, the functional activity of IL-2 on enriched CD34+ cells exposed to IL-3 could not be demonstrated in either granulocyte-macrophage colony-forming unit (CFU-GM) assays or proliferation assays.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adoptive immunotherapy with high-dose interleukin-2: kinetics of circulating progenitors correlate with interleukin-6, granulocyte colony-stimulating factor level.

Immunotherapy with interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells results in significant tumor regression in patients with advanced cancer. We have investigated the kinetics of circulating erythroid (BFU-E) and granulocytic-macrophage (CFU-GM) progenitors after IL-2 therapy in 11 cancer patients, mainly affected by metastatic melanoma and renal cell carcinoma. Administration of IL-2 from day 1 through day 5 constantly induced a dramatic decrease of the number of circulating BFU-E and CFU-GM, which then showed a striking rebound (up to values fourfold and sevenfold higher, respectively, than the pretherapy levels) on discontinuation of IL-2, ie, from day 5 through day 10. A similar kinetic pattern was observed during and after the second cycle of IL-2 administration. 3[H]-thymidine killing experiments showed that the cycling activity of the progenitors was virtually unmodified in the rebound phases. To explore the mechanism(s) underlying this kinetic pattern, we have analyzed the plasma concentration of several hematopoietic growth factors, including IL-1 beta, IL-3, IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and erythropoietin (Ep). No modifications in the levels of IL-3, GM-CSF, or IL-1 beta were observed, whereas a pronounced increase of IL-6 and G-CSF concentration was monitored, starting at day 3 and peaking at day 5 of treatment (a parallel, but modest, increase of Ep level was also observed). The elevation of IL-6 and G-CSF concentration is directly correlated with and may, at least in part, underlie the subsequent rebound of circulating hematopoietic progenitors. Furthermore, the increase in IL-4 level observed at day 10 of therapy may mediate the eosinophilia gradually starting at this stage of treatment.     

Activities of granulocyte-macrophage colony-stimulating factor and interleukin-3 on monocytes

We examined the actions of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) on human monocytes, using a serum-free culture system. GM-CSF and IL-3 did not promote the differentiation of monocytes into macrophages but rather into cells with a phenotype compatible with that of immature dendritic cells (DCs). The addition of fetal bovine serum to serum-free cultures with GM-CSF or IL-3 restored the differentiation of monocytes into macrophages. Cells generated with GM-CSF or IL-3 elicited phagocytic activity. Cells generated in the presence of GM-CSF or IL-3, followed by the addition of tumor necrosis factor-alpha, displayed a phenotype of mature DCs, and primed and stimulated immunogenic peptide-specific T lymphocytes. Surprisingly, GM-CSF and IL-3 inhibited macrophage colony-stimulating factor (M-CSF)-dependent differentiation of monocytes into macrophages and induced differentiation into immature DCs. We asked if the inhibition of M-CSF-dependent differentiation into macrophages by GM-CSF or IL-3 was associated with the expression of M-CSF receptors (M-CSFR). GM-CSF or IL-3 down-regulated the expression of M-CSFR. These data demonstrate that GM-CSF and IL-3 primarily support the differentiation of monocytes into DCs and inhibit M-CSF-dependent differentiation into macrophages by suppressing the expression of M-CSFR, thereby promoting differentiation into DCs.     

Functional reconstitution of the human interleukin-3 receptor.

The high-affinity receptors for human interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of alpha and beta subunits. The alpha subunits are primary ligand binding proteins specific for each ligand, whereas the three human receptors share a common beta subunit (beta c). In contrast to humans mice have two closely related genes, AIC2A and AIC2B, which are homologous to human beta c. The AIC2A gene encodes a low-affinity murine IL-3 binding protein, and the AIC2B protein is the beta subunit shared between murine GM-CSF receptors (mGMR) and IL-5 receptors (mIL-5R). To examine the function of these receptor components, we established various stable transfectants of murine IL-2-dependent CTLL-2 cells. CTLL-2 transfectants expressing both the alpha and beta subunits of the human IL-3 receptor (hIL-3R) proliferated in response to physiologic concentrations of hIL-3. Coexpression of hIL-3R alpha with AIC2B but not with AIC2A in CTLL-2 cells conferred a growth response to hIL-3. Although CTLL-2 transfectants expressing hIL-3R alpha alone did not proliferate in the presence of hIL-3, hIL-3-responsive sublines were repeatedly isolated. These sublines expressed endogenous AIC2B but not AIC2A. These results indicate that human beta c is essential for hIL-3 signaling and that AIC2B is a murine equivalent of human beta c. We also showed that hIL-3 and hGM-CSF induced tyrosine phosphorylation of several proteins in CTLL transfectants, similar to those observed in human factor-dependent TF-1 cells stimulated with hIL-3 and hGM-CSF.     

Effect of granulocyte-macrophage colony-stimulating factor on lymphokine-activated killer cell induction

The treatment of cancer with lymphokine-activated killer (LAK) cells in conjunction with high-dose interleukin-2 (IL-2) has been limited by the toxicity of IL-2 and the narrow range of tumors that respond to therapy. Cytokines that are capable of augmenting lower doses of IL-2 are, therefore, a major focus of research. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF) can augment low-dose IL-2 LAK induction from murine splenocytes. Anti-tumor necrosis factor alpha (anti-TNF alpha) or anti-interferon gamma (anti- IFN gamma) monoclonal antibodies did not inhibit (IL-2 + GM-CSF)- induced LAK generation, indicating that GM-CSF augmentation does not require TNF alpha or IFN gamma activity. Depletion of natural killer cells before culture did not inhibit low-dose IL-2-induced LAK generation or the ability of GM-CSF to augment LAK generation. In contrast, depletion of both CD4+ and CD8+ T cells before culture inhibited the generation of LAK activity. However, depletion of only CD4+ T cells, or only CD8+ T cells, did not inhibit the generation of IL-2 or (IL-2 + GM-CSF) LAK activity. These results suggest that LAK precursors are present in both the CD4+ and CD8+ T-cell populations and suggest that the addition of GM-CSF to low-dose IL-2 may result in the generation of T-derived LAK cells.     

Rapid priming of human monocytes by human hematopoietic growth factors: granulocyte-macrophage colony-stimulating factor (CSF), macrophage-CSF, and interleukin-3 selectively enhance superoxide release triggered by receptor-mediated agonists.

The effects of hematopoietic growth factors on human monocyte superoxide (O2-) release were investigated by using purified human monocytes in suspension. Among growth factors studied, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-CSF (M-CSF), and interleukin-3 (IL-3) primed human monocytes and enhanced O2- release stimulated by the receptor-mediated agonists, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A), but not by phorbol myristate acetate, which bypasses the receptors to stimulate the cells. The optimal priming was obtained by pretreatment of cells with 1 to 5 ng/mL (0.07 to 0.34 nmol/L) GM-CSF, 50 to 100 ng/mL (0.5 to 1.1 nmol/L) M-CSF, or 10 to 20 ng/mL (0.6 to 1.3 nmol/L) IL-3 for 10 minutes at 37 degrees C. Potency of the maximal priming effects on FMLP- or Con A-induced O2- release was GM-CSF greater than M-CSF = IL-3. The combination of the optimal concentrations of any two CSFs resulted in the effect of more potent priming agent alone. Enhancement of O2- release by GM-CSF was observed over the complete range of effective concentrations of FMLP (10(-8) to 10(-6) mol/L). The pretreatment of monocytes with granulocyte-CSF (50 ng/mL), interferon-gamma (1,000 U/mL), or IL-4 (20 ng/mL) for 10 minutes at 37 degrees C had no effect on O2- release stimulated by FMLP or Con A. These findings show that GM-CSF, M-CSF, and IL-3 selectively enhance O2- release in human monocytes triggered by receptor-mediated agonists after short-term preincubation.     

Toxicity and immunomodulatory effects of interleukin-2 after autologous bone marrow transplantation for hematologic malignancies

Autologous bone marrow transplantation (ABMT) for advanced hematologic malignancies is associated with high relapse rates. Interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells represent a potentially non-cross-resistant therapeutic modality that might prevent or delay relapses if used early after ABMT at a time when the tumor burden is minimal. However, high-dose chemoradiotherapy and ABMT might increase patients' susceptibility to IL-2 toxicity, and might interfere with immunologic responses to IL-2 in vivo. Therefore, to determine safety, tolerance, and immunomodulatory effects of IL-2 therapy early after ABMT, IL-2 was administered by continuous intravenous infusion to 16 patients 14 to 91 days (median, 33) after ABMT for acute leukemia, lymphoma, or multiple myeloma. Patients were sequentially assigned to escalating IL-2 "induction" doses (0.3 to 4.5 x 10(6) U/m2/d, days 1 to 5), and all patients received a nonescalating IL-2 "maintenance" dose (0.3 x 10(6) U/m2/d, days 12 to 21). Most patients exhibited mild to moderate fever, nausea, diarrhea, and/or skin rash with IL-2 infusions. The maximum tolerated "induction" dose was 3.0 x 10(6) U/m2/d; dose-limiting toxicities were hypotension and thrombocytopenia. All toxicities reversed on stopping the IL-2 infusions, and all patients completed "maintenance." Postinfusion lymphocytosis was exhibited by patients at all IL-2 dose levels. With the higher IL-2 doses, increased percentages of patients' PBMC expressed CD16 and CD56, with augmented lysis of K562 and Daudi, reflecting the induction of natural killer and circulating LAK effector activities. Increased LAK precursor activity was exhibited by patients at all IL-2 dose levels. Thus, the IL-2 therapy regimen was safely tolerated after ABMT, and pronounced immunomodulatory effects were observed with the higher IL-2 doses. These studies support the planned use of IL-2 and LAK cells after ABMT in an attempt to reduce relapses of advanced hematologic malignancies.