The role of cytokines and hematopoietic growth factors in the autocrine/paracrine regulation of inducible hematopoiesis

 To elucidate the role of hematopoietic growth factors (HGFs) and other cytokines in the autocrine or paracrine regulation of inducible hematopoiesis we studied cytokine gene expression in the bone marrow (BM) of patients after myelosuppressive treatment. Furthermore, we studied the cytokine gene expression profile in healthy individuals before and after bone marrow harvesting for the purpose of bone marrow transplantation. We speculated that the bone marrow harvesting procedure might induce changes in cytokine gene expression. No induction of G-CSF, GM-CSF, IL-1α, IL-3, IL-5, IL-8, IL-9, and IL-12 was observed in the BM of patients following intensive chemotherapy. Also, no up-regulation of expression of M-CSF, IL-1β, IL-4, IL-6, TNF-α, TGF-β, IGF-1, EDF, and EPA gene was found, illustrating that the investigated cytokines probably are not relevant in the presumed autocrine/paracrine regulation of the recovery of hematopoiesis following depletion of hematopoietic progenitor cells (HPCs). Concomitantly, elevated G-CSF plasma levels were found in these patients, suggesting that G-CSF has an endocrine regulatory role in inducible hematopoiesis. Induction of GM-CSF and IL-8, but not of G-CSF or IL-3 gene expression and up-regulation of IL-1β and IL-6 gene following BM harvesting was observed. This induction of GM-CSF and IL-8 may be attributed to tissue damage rather than to HPC depletion. Received: 18 June 1997/Accepted: 20 June 1997     

In vitro stimulatory effect of substance P on hematopoiesis

The neuropeptide Substance P (SP) is widely distributed in the peripheral nervous system. Its biologic effects have been extensively studied in the immune system. However, even though the bone marrow (BM) is innervated with SP-immunoreactive fibers and some of its cells not only express SP receptors (T and B cells, endothelial cells, and macrophages) but also produce SP (macrophages, eosinophils, and endothelial cells), the effects of SP on hematopoiesis are scanty. Furthermore, SP induces the production of hematopoietic growth factors (HGFs) (interleukin-1 [IL-1], IL-6, and tumor necrosis factor alpha) from human monocytes. In this study, we have found a potent in vitro stimulatory effect of SP (10(-8) to 10(-12) mol/L) on hematopoiesis for both erythroid and granulocytic progenitors in short-term methyl- cellulose BM cultures. SP alone, in the absence of exogenous HGFs, is able to sustain hematopoiesis in vitro. This stimulatory effect of SP is: (1) mostly mediated by the adherent cells; (2) completely abrogated by two SP receptor (SP-R) antagonists; and (3) partially reduced by anti-IL-1, IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Furthermore, it appears that the stimulatory effect of SP may be mediated by IL-3 and GM-CSF because we have also found that SP induces the release of these two cytokines from BM mononuclear cells. Considering that the SP effect occurs at concentrations as low as 10(-11) mol/L, and via a specific receptor, it appears that SP may play a physiologic role in regulating hematopoiesis, at least partially through the adherent BM cells and the release of HGFs, and may place SP, a neuropeptide, in a new category of hematopoietic regulators.     

Hepatocyte growth factor plays roles in the induction and autocrine maintenance of bone marrow stromal cell IL-11, SDF-1 alpha, and stem cell factor

OBJECTIVE: Bone marrow (BM) stroma provides the microenvironment required for long-term hematopoiesis, and this is supported by direct interaction between stromal cells and hematopoietic cells, mediated by adhesion molecules, and through cytokine releases from the BM stroma. In a previous study, we demonstrated that hepatocyte growth factor (HGF) is one of the cytokines constitutively produced from BM stromal cells, promoting hematopoiesis mainly in an indirect way. We also showed that stromal cells themselves express HGF receptor c-MET. It was therefore postulated that HGF exerts its effect on hematopoiesis and maintenance of the hematopoietic microenvironment in a paracrine and autocrine manner. METHODS: The effect of HGF on stromal cells was analyzed by neutralizing intrinsic HGF. RESULTS: Addition of neutralizing anti-HGF antibody inhibited the ability of BM stromal cells to support colony formation from CD34(+) cells and reduced production of significant cytokines from stromal cells, interleukin-11 (IL-11), stromal cell-derived factor-1 alpha (SDF-1 alpha), and to a lesser extent, stem cell factor (SCF). Furthermore, this neutralizing antibody reduced proliferation of stromal cells and inhibited adhesion of stromal cells to collagen type IV and fibronectin. Inhibition of adhesion to fibronectin was mediated by inhibition of alpha(5)beta(1)-integrin. CONCLUSION: These findings indicate that HGF constitutively produced from BM stromal cells is an autocrine regulator, which is able to maintain the hematopoietic microenvironment through stimulating proliferation and adhesion to the extracellular matrix and promoting hematopoiesis through inducing constitutive production of IL-11, SDF-1 alpha, and SCF by stromal cells themselves.     

Extramedullary hematopoietic growth factor and cytokine gene expression indicate endocrine regulation of hematopoiesis in patients with acute appendicitis

 To analyze the role of hematopoietic growth factors (HGFs) and other cytokines in the regulation of hematopoiesis in vivo, we investigated HGFs and cytokine gene expression in appendices obtained from patients who underwent surgery for suspected appendicitis. Concomitantly, HGF gene expression was studied in bone marrow (BM) biopsy specimens and plasma HGF levels were measured. G-CSF gene expression was detected in inflamed but not in normal appendices. With one exception, GM-CSF was detectable in all appendices whether inflamed or not, whereas IL-3, except for one case, was not expressed in appendices. None of the investigated HGFs appeared to be expressed in BM biopsy specimens concurrently obtained with the appendices. Plasma G-CSF levels were significantly elevated in patients with appendicitis compared with patients without inflamed appendices. Circulating levels of GM-CSF and IL-3 were not increased. Significant up-regulation of IL-8 and IL-6 gene expression was observed in response to inflammation, in contrast to IL-1α and IL-1β expression, which appeared not to be influenced by the inflammatory state. These data indicate that G-CSF, and not GM-CSF or IL-3, is essential for the regulation of inducible granulopoiesis in acute inflammatory conditions, and that G-CSF acts in an endocrine fashion. Received: 1 September 1997 / Accepted: 19 September 1997     

Enrichment of murine bone marrow natural suppressor activity in the fraction of hematopoietic progenitors with interleukin 3 receptor-associated antigen.

Natural suppressor (NS) activity has been identified in several sites of active hematopoiesis. In this study we characterized NS activity in murine bone marrow (BM) using monoclonal antibodies (mAbs) to interleukin 3 (IL-3) receptor-associated antigen (IL-3RAA) and various cytokines that exert a strong influence on hematopoiesis or lymphocyte interaction. NS activity of BM cells of relatively low density was enhanced by IL-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF). When the BM cells were separated into IL-3RAA+ cells and IL-3RAA- cells, the IL-3RAA+ cells demonstrated potent NS activity, whereas IL-3RAA- cells had either no or weak NS activity. The IL-3RAA+ cells showed non-T- and non-B-cell phenotype and had high affinity to wheat germ agglutinin (WGA), a marker for hematopoietic progenitors. In assays for hematopoietic activity, it appeared that the early differentiating progenitors (day 8 spleen colony-forming units [CFU-S], granulocyte-macrophage colony-forming units [CFU-GM]) were enriched in the IL-3RAA+ cell population, whereas more immature multipotent progenitors (day 12 CFU-S, granulocyte erythrocyte macrophage megakaryocyte colony-forming units [CFU-GEMM]) were contained in the IL-3RAA- cell population. Both suppressor cells and IL-3RAA+ cells spontaneously developed from the IL-3RAA- cell population. These findings suggest that NS cells in murine BM are early hematopoietic progenitors and are probably committed to the myeloid lineage. Hybridoma cells established between the IL-3RAA+ cells and BW5147 cells produced suppressor factor(s). This finding suggests that the NS cells produce soluble mediator(s) that may be responsible for their suppressive action.     

T lymphocytes downregulate granulocyte-macrophage colony-stimulating factor secretion from stimulated monocytes by increasing the secretion of monocyte-derived interleukin-10

In previous studies we characterized the cytokine regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) secretion by endothelial cells and monocytes and found differences in secretion pattern within and between these cell systems. In this study, the regulatory effect of T lymphocytes on CSF secretion was examined. T lymphocytes had no effect on CSF secretion by endothelial cells. In contrast, the addition of T lymphocytes significantly and dose dependently downregulated GM-CSF, but not G-CSF, secretion by monocytes. In one of our previous studies it was shown that interleukin-4 (IL-4) and interleukin-10 (IL-10) were the most potent inhibitory cytokines of CSF secretion by monocytes. Both these cytokines are produced by T lymphocytes. However, the downregulating effect on monocyte GM-CSF secretion was not due to increased secretion of T-lymphocyte-derived IL-4 or IL-10. Instead, the presence of T lymphocytes increased the secretion of monocyte-derived IL-10. It was shown earlier than IL-10 regulates CSF secretion by monocytes in an autocrine manner. Our data indicate that T lymphocytes might interfere with this autocrine regulation and thereby influence monocyte function in immune response and cell proliferation.     

Interleukin-3 in vivo: kinetic of response of target cells

Human recombinant interleukin-3 (IL-3; Sandoz AG, Basel, Switzerland) was administered for 7 days to patients with neoplastic disease and normal hematopoiesis. The purpose of the study was to assess IL-3 toxicity, to identify target cells, to define their kinetics of response at different dose levels, and to determine if IL-3 in vivo increased the sensitivity of bone marrow (BM) progenitors to the action of other hematopoietic growth factors. A total of 21 patients entered the study; the dosage ranged from 0.25 to 10 micrograms/kg/d. The effect on peripheral blood cells during treatment showed no significant changes in the number of platelets, erythrocytes, neutrophils, or lymphocytes (and their subsets). A mild monocytosis and basophilia occurred. Eosinopenia, present in the first hours of treatment, was followed by a dose-and time-dependent eosinophilia. IL-3 treatment affected BM cell proliferation by increasing the percentage of BM progenitors engaged in the S-phase of the cell cycle. The effect was dose dependent, with the various progenitors showing different degrees of sensitivity. The most sensitive progenitors were the megakaryocyte progenitors (colony-forming unit-megakaryocyte), then the erythroid progenitors (burst-forming unit-erythroid), and finally the granulo- monocyte progenitors (colony-forming unit-granulocyte-macrophage) whose proliferative activity was stimulated at the higher doses of IL-3. Only a slight increase in the proliferative activity of myeloblasts, promyelocytes, and myelocytes was observed, whereas the activity of erythroblasts was unchanged. The priming effect was such that BM progenitors, purified from patients treated with IL-3, produced more colonies in vitro in the presence of granulocyte colony-stimulating factor (G-CSF; granulocyte colonies), IL-5 (eosinophil colonies), and granulocyte-macrophage CSF (GM-CSF; predominantly eosinophil colonies). These data indicate that even in vivo IL-3 acts essentially as a primer for the action of other cytokines. Therefore, optimum stimulus of myelopoiesis will require either endogenous or exogenous late-acting cytokines such as G-CSF, erythropoietin, GM-CSF, and IL-6 for achieving fully mature cells in peripheral blood. If exogenous cytokines are used with IL-3, it is likely that G-CSF will yield more neutrophils, whereas GM-CSF may enhance eosinophils, monocytes, and neutrophils. Attention to the clinical relevance of each cell type will be necessary and should determine the selection of the combination of cytokines.     

Hematopoietic growth factors upregulate the p65 type II interleukin-1 receptor on bone marrow progenitor cells in vitro.

Having previously shown that interleukin-1 (IL-1) induces the expression of IL-1 receptors (IL-1Rs) on bone marrow (BM) cells in vivo through an indirect mechanism, we studied whether hematopoietic growth factors (HGFs) could induce the expression of IL-1R on BM cells in vitro. In vitro treatment of light-density murine BM (LDBM) cells with either IL-3, IL-6, granulocyte--colony-stimulating factor (CSF), or granulocyte-macrophage--CSF caused a 5- to 10-fold upregulation of IL-1R expression, whereas IL-1, IL-5, IL-7, and macrophage-CSF had no effect. Scatchard analysis showed one class of IL-1Rs on LDBM cells with an average of 66 +/- 20 sites per cells. After 24 hours of treatment with IL-3, the number of IL-1Rs increased to 413 +/- 125, without effecting the affinity. This effect required protein synthesis, but was independent of cell division. Purified lineage-negative progenitor cells (Lin-) did not express detectable levels of IL-1R, but 24 hours of treatment with IL-3, GM-CSF, and G-CSF stimulated IL-1--specific binding. Autoradiographic analysis of Lin- cells showed that IL-1R induction by IL-3 occurs on undifferentiated blast cells. Affinity labeling of Lin- cells treated with HGFs showed an increase in a 65-Kd IL-1 binding protein that did not bind or compete with an anti-type I IL-1R antibody, suggesting that these cells expressed type II IL-1R. These data suggest that IL-1 stimulation of myelopoiesis occurs by a mechanism involving IL-1R upregulation on hematopoietic progenitor cells by HGFs.     

In vivo interleukin-1 (IL-1) administration indirectly promotes type II IL-1 receptor expression on hematopoietic bone marrow cells: novel mechanism for the hematopoietic effects of IL-1.

Interleukin-1 (IL-1) has profound stimulatory effects on hematopoiesis but the mechanism(s) of action remain unknown. The direct action of IL-1 on hematopoietic progenitor cells requires the presence of a specific IL-1 receptor (IL-1R). In this report, we tested the effect of in vivo IL-1 treatment on the expression of IL-1R on bone marrow (BM) cells. Injection of mice with IL-1 results in a marked upregulation of IL-1R on light-density BM cells as on a subpopulation enriched for myeloid precursors. Pretreatment of mice with anti-type I IL-1R antibody (35F5), which has been shown to prevent the radioprotective effect of IL-1, also blocked IL-1-induced IL-1R expression on BM cells. This antibody did not directly bind and block IL-1 binding to the type II IL-1R expressed on hematopoietic cells, suggesting that IL-1R upregulation by IL-1 is indirect. It is therefore possible that IL-1 acts on type I IL-1R-expressing accessory cells such as stromal cells or T cells to induce production of hematopoietic growth factors (HGFs). In support of this, granulocyte colony-stimulating factor administration can induce the increase of IL-1R on BM cells. Thus, the increased expression of IL-1R on hematopoietic BM cells by IL-1 is indirect, probably mediated in part through endogenous HGF production. These results also suggest that the restorative hematopoietic effect of IL-1 occurs through both indirect and direct mechanisms.     

Induction of colony-stimulating factor receptor expression on hematopoietic progenitor cells: proposed mechanism for growth factor synergism.

In many cells systems, the cellular interaction between two or more humoral factors leads to a synergistic response in terms of cellular growth and function. In particular, the growth and differentiation of hematopoietic progenitor cells involves numerous synergistic interactions between colony-stimulating factors (CSFs) that individually stimulate hematopoiesis (granulocyte-CSF, granulocyte-macrophage-CSF, and interleukin-3 [IL-3]), as well as between these factors and other cytokines that individually have no proliferative effect on progenitor cell growth (IL-1 and IL-6). The present study investigated whether hematopoietic growth factor (HGF) synergy could be mediated by upregulation of CSF receptors. Synergistic effects on bone marrow (BM) progenitor cell colony formation, regardless of the combination of factors used, were consistently preceded by increased CSF receptor expression on highly enriched BM progenitor cells, but not on unfractionated BM cells. Induction of CSF receptors preceded detectable differentiation and did not require cell division because nocodazole, an inhibitor of mitosis, blocked CSF-mediated cell proliferation, but not receptor upregulation. Furthermore, combinations of cytokines that did not synergize also failed to affect the level of CSF receptors on BM progenitors. These results have led us to propose a model for HGF synergy whereby one mechanism of action the investigated synergistic cytokines might be the ability to induce increased expression of CSF receptors.     

Regulation of cytokine expression by interferon-alpha in human bone marrow stromal cells: inhibition of hematopoietic growth factors and induction of interleukin-1 receptor antagonist

We investigated the effects of interferon-alpha (IFN-alpha) on the expression of cytokines by human bone marrow stromal cells. Production of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-CSF (G-CSF), and interleukin-1 beta (IL-1 beta) in stromal cell layers was induced by incubation with IL-1 alpha, tumor necrosis factor (TNF), or lipopolysaccharide (LPS). Addition of IFN-alpha to such stimulated cultures resulted in a strong downregulation of mRNA expression of GM-CSF and IL-1 beta. Similarly, the protein levels of GM- CSF and IL-1 beta were significantly reduced by IFN-alpha, whereas G- CSF production was only moderately inhibited. In contrast, IFN-alpha markedly stimulated the production of IL-1 receptor antagonist (IL-1RA) by stromal cells. The inhibition of cytokine expression resulted in a reduced hematopoietic activity of stromal cells, indicated by a reduced proliferation of the factor dependent cell line MO7e on IFN-alpha- treated stromal cells. In the presence of cycloheximide (CHX), IFN- alpha failed to inhibit IL-1 mRNA expression, whereas the regulation of GM-CSF and IL-1RA by IFN-alpha was not affected. Our results indicate that the myelosuppressive effects of IFN-alpha, as observed in therapeutic applications or associated with viral infections, are, in part, indirectly mediated by inhibition of the paracrine production of hematopoietic growth factors.     

Interleukin-2-activated natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo.

Purified natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID) to ascertain their effect on hematopoiesis. When activated and propagated with recombinant human interleukin-2 (rhIL-2) in vitro, SCID spleen cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. When added with syngeneic bone marrow cells (BMC) in soft agar, the activated NK cells could support hematopoietic growth in vitro without the addition of exogenous hematopoietic growth factors. However, when syngeneic BMC were added along with cytokines to produce optimal growth conditions, the addition of NK cells was then inhibitory for hematopoietic colony formation. Antibodies to interferon-gamma (IFN-gamma) partially reversed the inhibitory effects. Supernatants from the NK-cell cultures could also exert these effects on hematopoiesis, although to a lesser extent. Analysis of the NK cell RNA demonstrated that activated NK cells express genes for hematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL-1 beta. The NK cells were also found to express IFN-gamma, transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNA. Analysis of the NK-cell supernatants using factor-dependent myeloid progenitor cell lines showed that the NK cells were producing G-CSF and growth-promoting activity that could not be attributed to IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, G-CSF, macrophage CSF (M-CSF), or stem cell factor. The transfer of activated NK cells with BMC into lethally irradiated syngeneic mice resulted in greater BMC engraftment in the recipients. Thus, these results using a pure population of activated NK cells indicate that when activated, these cells can produce a variety of growth factors for hematopoiesis and exert significant hematopoietic growth-promoting effects in vivo.     

Primitive interleukin 3 null hematopoietic cells transduced with BCR-ABL show accelerated loss after culture of factor-independence in vitro and leukemogenic activity in vivo

Primitive chronic myeloid leukemia cells display a unique autocrine interleukin 3 (IL-3)/granulocyte-colony-stimluating factor (G-CSF) mechanism that may explain their abnormal proliferation and differentiation control. Here we show that BCR-ABL transduction of primitive Sca-1(+) lin(-) mouse bone marrow (BM) cells causes immediate activation of IL-3, G-CSF, and granulocyte macrophage-colony-stimulating factor (GM-CSF) expression in these cells. Their autocrine IL-3-mediated growth dependence is thus demonstrable only in clonal cultures where paracrine effects are reduced. Interestingly, upon continued culture, these cells produce large populations of rapidly proliferating mast cells in which only the IL-3 autocrine mechanism is consistently maintained, together with evidence of hyperphosphorylation of p210(BCR-ABL) and STAT5 and retention of a multilineage but attenuated in vivo leukemogenic potential characterized by a prolonged latency. BCR-ABL transduction of IL-3(-/-) Sca-1(+) lin(-) BM cells initially activates GM-CSF and G-CSF production, factor independence, and the ability to generate phenotypically indistinguishable populations of mast cells. However, maintenance of factor independence, and p210(BCR-ABL) and STAT 5 activation beyond 4 to 6 weeks, requires rescue with an IL-3 transgene. The cultured BCR-ABL-transduced IL-3(-/-) cells also lack leukemogenic activity in vivo. These findings provide new evidence that IL-3 production is a rapid, sustained, and biologically relevant consequence of BCR-ABL expression in primitive hematopoietic cells with multilineage leukemogenic activity.     

Interleukin-6 reduces the optimal growth in vitro of leukemic blast progenitors from acute myeloblastic leukemia patients.

The combined effects of five cytokines; recombinant human (rHu) granulocyte colony-stimulating factor (G-CSF), rHu granulocyte-macrophage colony-stimulating factor (GM-CSF), rHu interleukin-1 beta (IL-1 beta), rHu interleukin-3 (IL-3), and rHu interleukin-6 (IL-6) on blast colony formation in methylcellulose by leukemic blast progenitors from 10 patients with acute myeloblastic leukemia (AML) were studied. Combination of G-CSF, GM-CSF, IL-1 beta, and IL-3 stimulated maximum blast colony formation in 9 patients. Further addition of IL-6 reduced the combined effect of the four cytokines on blast colony formation. IL-6 regulates the proliferation of leukemic blast progenitors and may play an important role in the regulation of hematopoiesis.     

Autologous stem cell transplantation: release of early and late acting growth factors relates with hematopoietic ablation and recovery

We have monitored the serum concentrations of hematopoietic growth factors (HGFs; ie, stem cell factor [SCF], leukemia inhibitory factor [LIF], interleukin-3 [IL-3], IL-6, IL-8, and granulocyte colony- stimulating factor [G-CSF]) in 15 lymphoma/leukemia and 6 ovarian cancer patients undergoing autologous bone marrow (BM) or peripheral blood (PB) stem cell transplantation (SCT). Thus, the analysis was performed during and after high-dose chemotherapy (from day -6 to day - 1), at the time of SCT (day 0), and thereafter (through day +17). Despite the heterogeneity of these patients and their conditioning regimens, a consistent kinetic pattern was observed for all analyzed cytokines. Particularly, (1) SCF serum concentration did not significantly fluctuate. (2) High levels of LIF (approximately 250 to 450 pg/mL) before chemotherapy rapidly declined to markedly lower concentrations (approximately 10 ng/mL) starting from day -1 through day +17; (3) conversely, IL-3 level was low before treatment, sharply increased during chemotherapy, and rapidly returned to base-line level after SCT. Hypothetically, the sharp LIF decrease and IL-3 increase during chemotherapy may underlie the induction of stem cell cycling and differentiation caused by hematopoietic ablation. Furthermore, (4) IL-6 concentration was low before and immediately after chemotherapy, but increased starting from day +5, peaked at day +6 through 9 and then declined to baseline level from day +10 onward; (5) a strictly similar pattern was consistently observed for both G-CSF and IL-8 levels, in agreement with our previous studies. It is relevant that peak IL-6, G- CSF, and IL-8 concentrations were directly correlated to peak neutrophil numbers in the recovery phase, thus suggesting an important role for these cytokines in granulocyte rescue; in line with this interpretation, hematologic patients undergoing PBSCT (10 of 15) exhibited higher peaks of IL-6, G-CSF, and IL-8 and a more pronounced increase of neutrophil/platelet number than did hematologic cases undergoing BMSCT (5 of 15). Altogether, these studies indicate a coordinate pattern of cytokine release during hematopoietic ablation/recovery after chemotherapy and autologous SCT, the fluctuations of LIF and IL-3 levels during chemotherapy are seemingly related to stem cell recruitment, whereas the post-SCT increase of IL- 6, G-CSF, and IL-8 may underlie the neutrophil recovery.     

Severe congenital neutropenia: abnormal growth and differentiation of myeloid progenitors to granulocyte colony-stimulating factor (G-CSF) but normal response to G-CSF plus stem cell factor

Several mechanisms have been proposed to explain the pathogenesis of severe congenital neutropenia (SCN); however, the mechanism(s) still remains unknown. In particular, clinical observations suggest that abnormal responsiveness of myeloid progenitors to hematopoietic growth factors (HGFs) is a possible mechanism. Therefore, to better define the status of hematopoietic progenitors in the bone marrow (BM) of patients with SCN, the responsiveness of myeloid progenitors to HGFs from two SCN patients was compared with the responsiveness of progenitors from healthy individuals. BM cells (BMCs) from the first SCN patient required higher (10- to 100-fold) concentrations of granulocyte colony- stimulating factor (G-CSF) to achieve maximal and half-maximal colony growth in vitro compared with BMCs from controls. In contrast, the dose- response of interleukin-3 (IL-3) and granulocyte-macrophage-CSF (GM- CSF) in colony formation was normal. Interestingly, IL-3, GM-CSF, and G- CSF at optimal doses showed reduced ability to induce neutrophil differentiation of BMCs from a SCN patient compared with BMCs from controls. Despite an abnormal responsiveness of mature myeloid progenitors to G-CSF in this SCN patient, myeloid progenitors responsive to the combination of stem cell factor (SCF) and G-CSF showed normal dose-response. In contrast to G-CSF alone, the combination of G-CSF and SCF induced the formation of neutrophils almost to the same extent compared with cultures of normal BMCs. Furthermore, also on BM progenitor cells obtained from the second patient with SCN, SCF highly synergized with G-CSF to promote neutrophil progenitor cell growth and differentiation in vitro. Thus, these results indicate that one mechanism of the pathogenesis in SCN patients is reduced responsiveness of neutrophil progenitor cells to G- CSF and that SCF can enhance the responsiveness of these cells to G-CSF.     

Characterization of endogenous cytokine concentrations after high-dose chemotherapy with autologous bone marrow support

Endogenous cytokines are thought to mediate numerous biologic processes and may account for some adverse effects experienced following the administration of recombinant proteins. This study describes the pattern of endogenous cytokine exposure following high-dose chemotherapy. Blood concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), macrophage colony-stimulating factor (M-CSF), and erythropoietin (EPO) were measured by enzyme-linked immunosorbent assay (ELISA) methods in 68 patients receiving the same ablative chemotherapy regimen (cyclophosphamide, cisplatin, carmustine). Patients were grouped according to cellular support (autologous bone marrow [BM] CSF-primed peripheral blood progenitor cells [PBPCs]) and prescribed growth factor (recombinant human granulocyte or granulocyte-macrophage colony-stimulating factor [rHuG- CSF or rHuGM-CSF]). Leukocyte reconstitution was most accelerated in the groups treated with PBPCs and rHuG-CSF. IL-6, M-CSF, and TNF-alpha concentrations were higher in the groups treated with rHuGM-CSF and without PBPCs. Maximal endogenous cytokine concentrations occurred approximately 12 days after BM reinfusion. High concentrations of EPO occurred in patients experiencing significant hypotension despite routine transfusions for hematocrit < 42%. High M-CSF and IL-6 levels were associated with increased platelet transfusion requirements. Concentrations of all four cytokines were significantly higher in patients experiencing renal or hepatic toxicity, with elevations occurring in a predictable sequence and M-CSF elevations occurring first. This report shows that endogenous cytokine concentrations may be influenced by either cellular or CSF support and are associated with differences in platelet reconstitution and organ toxicity.     

Human cytomegalovirus increases constitutive production of interleukin- 6 and leukemia inhibitory factor by bone marrow stromal cells

Human cytomegalovirus (CMV) infection is often associated with myelosuppression and acute inflammatory reaction in immunocompromised patients. We have previously documented that CMV exposure of bone marrow (BM) stromal cells reduces the capacity of these cells to support hematopoiesis because of a decreased production of colony- stimulating factors. This study examines the potential role of CMV on constitutive and lipopolysaccharide (LPS)-stimulated production of cytokines involved in inflammatory reaction, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) by BM stromal cells. The release of IL- 6 was already detectable 2 hours post CMV-infection (2.5-fold increase in production) and the cumulative production of IL-6 after 5 days of infection was 23 +/- 1.2 ng/mL (ninefold increase in production). CMV was also able to induce a time-dependent production of LIF that was maximal 8 hours after CMV infection (2.5-fold increase in production). Concomitantly, there was no detectable release of granulocyte colony- stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) by CMV-infected stromal cells. The similar IL-6 and LIF production in the presence of polymyxin B ruled out the possibility that this increase could be caused by contamination of the viral stock by endotoxin. In addition, ultraviolet-inactivated virus behaved similarly to live virus and caused the release of IL-6 and LIF. However, heat-inactivated CMV was unable to induce IL-6 and LIF secretion by BM stromal cells. The production of IL-6 and LIF was also evaluated after stimulation by LPS. After 5 days of CMV exposure, the LPS-stimulated production of IL-6 and LIF was significantly lower than uninfected controls. This LPS-induced release of cytokine production was found to be dependent of viral replication. The experiments have shown that CMV is a potent inducer of IL-6 and LIF with differential effect on constitutive and LPS- stimulated cytokine production by stromal cells; we suggest that CMV induction of IL-6 and LIF during the first hours of infection could play a role in CMV-induced inflammatory reaction. Moreover, our results show that human CMV can disturb the balanced cytokine network involved in the regulation of hematopoiesis.