Chemokines and leukocyte activation in the fetal circulation during preeclampsia.

Preeclampsia is a potentially life-threatening disease for both mother and fetus. Endothelial dysfunction is pivotal in the pathogenesis of this disorder, possibly reflecting a state of persistent inflammation. In the present study, we examined whether signs of inflammation with production of chemokines and leukocyte activation were present in the fetal circulation during preeclampsia. Venous cord blood was sampled during cesarean sections from 36 neonates born after uncomplicated pregnancies and from 35 born after severe preeclamptic pregnancies with premature newborns. The expression of adhesion molecules on neutrophils and monocytes was analyzed by flow cytometry, and plasma levels of chemokines and soluble adhesion molecules were analyzed by enzyme immunoassay. Newborns of preeclamptic mothers had increased expression of CD15s (P=0.003), CD49d/CD29 (P=0.01/0.005), and CD31 (P=0.007) on neutrophils and CD15s (P<0.001), CD11c (P=0.009), and CD54 (P=0.001) on monocytes. This activation of neutrophils and monocytes was accompanied by raised plasma levels of the CXC chemokines interleukin-8 (P=0.007) and growth-related oncogene-alpha (P=0.01) and decreased plasma levels of soluble E-selectin (P=0.001) and L-selectin (P=0.002). Although raised levels of adhesion molecules on leukocytes or decreased levels of soluble adhesion molecules in plasma were not related to prematurity or the degree of preeclampsia, raised interleukin-8 levels were found only in neonates of preeclamptic mothers with the highest blood pressures. Our findings suggest the activation of neutrophils and monocytes in the fetus during preeclampsia involving enhanced chemokine activation, possibly contributing to the fetal morbidity of this disorder.

Human intestinal endothelium shows high susceptibility to cytomegalovirus and altered expression of adhesion molecules after infection.

Human cytomegalovirus (HCMV) causes gastro intestinal disease with ulcerations, apparently as a consequence of cytopathic damage to endothelial cells (EC) and subsequent microvascular obliteration. In this study we showed that cultured human intestinal microvascular endothelial cells (HIMEC) are much more susceptible to HCMV infection than human umbilical vein endothelial cells (HUVEC). When both cell types were challenged with a clinical isolate of HCMV (10 pfu per cell), 30% of HIMEC expressed HCMV immediate early proteins, but only 10% of HUVEC. Enhanced susceptibility was also reflected in the expression of early and late HCMV proteins. In addition, the interleukin-1beta (IL-1beta)-induced cellular expression of adhesion molecules differed between HIMEC and HUVEC after HCMV-infection. E-selectin was unaffected in HUVEC but increased in HIMEC, whereas vascular cell adhesion molecule (VCAM)-1 was increased in HUVEC but decreased in HIMEC. Furthermore, HCMV-infection enhanced the expression of intercellular adhesion molecule (ICAM)-1 in both cell types. In conclusion, the enhanced susceptibility to HCMV infection observed in HIMEC and the elevated expression of E-selectin and ICAM-1 observed in these cells may provide an indication to the liability of developing gastrointestinal HCMV disease and may have a possible relevance to the survival of intestinal transplants.

Human cytomegalovirus induced inhibition of hematopoietic cell line growth is initiated by events taking place before translation of viral gene products.

Bone marrow suppression with leukopenia is frequently observed during human cytomegalovirus (HCMV) infection, and in vitro the cell colony formation of bone marrow progenitors is directly inhibited by HCMV. To better understand the mechanisms of HCMV's ability to directly inhibit the cell colony formation of hematopoietic cells, we examined the effect of HCMV infection on four hematopoietic cell lines, ML-3, HL-60, KG-1, and U-937. Similarly to the observed effect on hematopoietic progenitors, HCMV significantly inhibited the cell colony formation of KG-1 and U-937 cells, 40% and 30% respectively. Following HCMV infection, uptake of HCMV pp65 was detected in all cell lines. In contrast, no immediate early protein production could be observed. When the cell line KG-1 was challenged with UV-inactivated HCMV or with HCMV dense bodies, containing pp65 and other matrix proteins, a 20% to 25% inhibition of cell colony formation was found. In addition, a dose-dependent inhibition of proliferation of the KG-1 cells challenged with intact or UV-inactivated HCMV, was observed. Transfection of this cell line with vectors containing genes for the HCMV matrix protein pp65, revealed no inhibitory effect. In contrast, the transfection with pp71 resulted in a 20% growth inhibition. These results indicate that HCMV can induce inhibition of cell colony formation of hematopoietic cells without transcription of HCMV regulatory proteins, and that at least one HCMV matrix protein may play an important role in this inhibitory effect.

Human cytomegalovirus induces apoptosis in the hematopoietic cell line MO7e.

Several studies have shown that human cytomegalovirus (HCMV) induces growth suppression of hematopoietic progenitors. In vitro studies have demonstrated that the HCMV-induced suppression is independent of viral protein production. Previous studies have indicated a link between HCMV infection and apoptosis in human cells. The purpose of our study was to investigate whether the observed inhibitory effect of HCMV on the human myeloid progenitors could be connected to the induction of apoptosis. The growth and cell death of the hematopoietic cell line MO7e was investigated following infection with HCMV virions and dense bodies. Both virions and dense bodies inhibited the growth of MO7e cells, and induced cell death measured by trypan blue staining. In addition, both HCMV virions and dense bodies caused an increased amount of apoptosis-characteristic DNA fragmentation in the MO7e cells compared to mock-treated cells. The HCMV virions were also able to induce an increased expression of phosphatidylserine on the cell surface, which is an early event in the initiation of apoptosis in most cell types. In conclusion, HCMV and HCMV dense bodies are able to induce apoptosis in the myeloid progenitor cell line MO7e.

CD47 signals T cell death.

Activation-induced death of T cells regulates immune responses and is considered to involve apoptosis induced by ligation of Fas and TNF receptors. The role of other receptors in signaling T cell death is less clear. In this study we demonstrate that activation of specific epitopes on the Ig variable domain of CD47 rapidly induces apoptosis of T cells. A new mAb, Ad22, to this site induces apoptosis of Jurkat cells and CD3epsilon-stimulated PBMC, as determined by morphological changes, phosphatidylserine exposure on the cell surface, uptake of propidium iodide, and true counts by flow cytometry. In contrast, apoptosis was not observed following culture with anti-CD47 mAbs 2D3 or B6H12 directed to a distant or closely adjacent region, respectively. CD47-mediated cell death was independent of CD3, CD4, CD45, or p56lck involvement as demonstrated by studies with variant Jurkat cell lines deficient in these signaling pathways. However, coligation of CD3epsilon and CD47 enhanced phosphatidylserine externalization on Jurkat cells with functional CD3. Furthermore, normal T cells required preactivation to respond with CD47-induced apoptosis. CD47-mediated cell death appeared to proceed independent of Fas or TNF receptor signaling and did not involve characteristic DNA fragmentation or requirement for IL-1beta-converting enzyme-like proteases or CPP32. Taken together, our data demonstrate that under appropriate conditions, CD47 activation results in very rapid T cell death, apparently mediated by a novel apoptotic pathway. Thus, CD47 may be critically involved in controlling the fate of activated T cells.

Microencapsulated octreotide pamoate in advanced gastrointestinal and pancreatic cancer: a phase I study.

Fourteen patients suffering from advanced colorectal (n = 7), pancreatic (n = 4) or gastric (n = 3) carcinomas received treatment with microencapsulated octreotide pamoate 90 mg i.m. every 4 weeks (n = 4), 160 mg i.m. every 4 weeks (n = 4) or 160 mg i.m. every 2 weeks (n = 6). Two patients had stable disease, one for 4 and one for 6 months. Plasma insulin-like growth factor (IGF)-I decreased by 49-53%, IGF-II by 27-37% and total IGF-binding protein (IGFBP)-3 by 16-19%, whereas IGFBP-1 increased by 35-55%. Insulin and C-peptide levels decreased by 29-38% and 41-46% respectively. A non-significant decrease in urinary GH secretion and an increase in the ratio of fragmented to intact IGFBP-3 as well as IGFBP-3 protease activity was seen. The increase in IGFBP-3 fragmentation correlated negatively with alterations in IGF-I and IGF-II (P < 0.05). We conclude that microencapsulated octreotide administered in doses up to 160 mg every 2 weeks is well tolerated and has pronounced effects on several components of the IGF system in plasma. In addition, changes in IGFBP-3 protease activity because of cancer may contribute to alterations in IGF-I and -II, indicating the importance of measuring this parameter in addition to IGFs and IGFBPs when evaluating alterations in IGF-I.

The TCR-binding region of the HLA class I alpha2 domain signals rapid Fas-independent cell death: a direct pathway for T cell-mediated killing of target cells?

TCR binding to an MHC class I/peptide complex is a central event in CTL-mediated elimination of target cells. In this study, we demonstrate that specific activation of the TCR-binding region of the HLA-A2 class I alpha2 domain induces apoptotic cell death. mAbs to this region rapidly induced apoptosis of HLA-A2-expressing Jurkat E11 cells, as determined by morphologic changes, phosphatidylserine exposure on the cell surface, and propidium iodide uptake. In contrast, apoptosis was not induced following culture with mAbs directed to other regions of the class I molecule. Death signaling by class I molecules is apparently dependent on coreceptor activation, as apoptosis is also signaled by HLA-A2 molecules, where the intracytoplasmic residues were deleted. HLA class I alpha2-mediated cell death appeared to proceed independent of the Fas pathway. Compared with apoptotic signaling by Fas ligation, HLA class I alpha2-mediated responses displayed a faster time course and could be observed within 30 min. Furthermore, class I alpha2-induced cell death did not involve observable DNA fragmentation. The apoptotic response was not affected significantly by peptide inhibitors of IL-1beta converting enzyme (ICE)-like proteases and CPP32. Taken together, activation of the TCR-binding domain of the class I alpha2 helix may result in apoptotic signaling apparently dependent on a novel death pathway. Thus, target HLA class I molecules may directly signal apoptotic cell death following proper ligation by the TCR.

Dysregulation of membrane-bound tumor necrosis factor-alpha and tumor necrosis factor receptors on mononuclear cells in human immunodeficiency virus type 1 infection: low percentage of p75-tumor necrosis factor receptor positive cells in patients with advanced disease and high viral load.

The correlation of persistent tumor necrosis factor-alpha (TNF-alpha) activation with disease progression in patients infected with human immunodeficiency virus type 1 (HIV-1), suggests a role for TNF-alpha in the pathogenesis of HIV-1 infection. In the present study, we examined by flow cytometry the expression of membrane-bound (m) components of the TNF system in 33 HIV-1-infected patients and 12 healthy controls. While peripheral blood mononuclear cells (PBMC) from asymptomatic and symptomatic non-acquired immune deficiency syndrome (AIDS) patients showed a significantly increased percentage of mTNF-alpha+ and mTNF receptor (TNFR)+ cells compared with controls, this was not found in the AIDS group. Compared with healthy controls, AIDS patients had a significantly decreased percentage of both monocytes and lymphocytes expressing p75-TNFR. PBMC from AIDS patients showed a higher p75-TNFR mRNA level and a higher spontaneous release of soluble p75-TNFR than healthy individuals, suggesting enhanced cell surface turnover of this TNFR. The low expression of TNFRs on both lymphocytes and monocytes in the AIDS group was associated with high numbers of HIV-1 RNA copies in plasma, low numbers of CD4+ lymphocytes, and high serum levels of soluble TNFRs. AIDS patients had a decreased percentage of CD8+ lymphocytes expressing TNFRs compared with healthy controls. In contrast, these patients, as well as symptomatic non-AIDS patients, had an increased percentage of TNF-alpha+ and TNFRs+ cells among remaining CD4+ lymphocytes. The pattern of abnormalities seen in AIDS patients suggests a role for persistent activation of the TNF system in the accelerated CD4+ lymphocyte destruction, the enhanced HIV-1 replication, and the markedly impaired antimicrobial defense in advanced HIV-1-related disease.

Effects of intravenous immunoglobulin in vivo on abnormally increased tumor necrosis factor-alpha activity in human immunodeficiency virus type 1 infection.

The effect of a single bolus injection (0.4 g/kg) of intravenous immunoglobulin (IVIG) on the tumor necrosis factor (TNF) system in human immunodeficiency virus type 1 (HIV-1)-infected patients was investigated. At 140 h after infusion, there was a significant decrease in levels of TNF-alpha and a significant increase in levels of soluble TNF receptors (sTNFR) in both plasma and lipopolysaccharide-stimulated peripheral blood mononuclear cells (PBMC). A rapid (within 1 h) decline in expression of membrane-bound TNF-alpha and p55-TNFR on PBMC persisted throughout the study. In contrast, there was an increased expression of membrane-bound p75-TNFR after 140 h. IVIG administration also resulted in significantly increased numbers of circulating CD4 lymphocytes, correlated with down-regulation of TNF-alpha activity in PBMC supernatants. Thus, down-regulation of the abnormally increased TNF-alpha activity may be achieved by IVIG administration. Studies evaluating the possible therapeutic role of long-term TNF-alpha suppression by IVIG may be warranted in HIV-1-infected patients.

Human cytomegalovirus suppression of and latency in early hematopoietic progenitor cells.

Bone marrow cells (BMC) are involved in the pathogenesis of human cytomegalovirus++ (HCMV) infections, and the hematopoietic cells are probable sites of HCMV latency in healthy donors. In vitro studies have indicated both a direct inhibitory effect of HCMV on proliferation and differentiation of myeloid bone marrow progenitors and an impairment of bone marrow stroma cell function by HCMV. The purpose of the present study was to establish whether the suppressing effect could be limited to subsets of immature CD34+ BMC and to investigate the role of immature cell populations as possible sites of HCMV latency. CD34+ cells from healthy HCMV-seropositive and -seronegative donors were sorted according to the expression of HLA-DR (CD34+ HLA-DR+ and CD34+ HLA-DR- cells). The progenitor growth of hematopoietic progenitor cells from seronegative donors was examined by colony and single-cell assays after in vitro infection with HCMV. To determine the susceptibility of the CD34+ cells to HCMV infection in vitro and in vivo, cells of both subsets from seronegative and seropositive donors were analyzed for the presence of HCMV DNA by polymerase chain reaction. HCMV infection in vitro inhibited the interleukin-1alpha (IL-1alpha)-, IL-3-, granulocyte colony-stimulating factor-, granulocyte-macrophage colony-stimulating factor-, and stem cell factor-induced proliferation in single-cell assays of CD34+ HLA-DR- cells by 34%. In contrast, the colony growth of the CD34+ HLA-DR+ subset was suppressed in cells from only 3 of the 8 donors. However, in vitro HCMV infection of the CD34+ HLA-DR+ progenitor cells inhibited the proliferation of all donors tested when hematopoietic growth factors were used individually to promote progenitor growth. In addition, the formation of burst-forming units-erythroid and colony-forming units-granulocyte, erythrocyte, monocyte, megakaryocyte was reduced 40% to 60% by HCMV in vitro. In contrast, the growth of high proliferative potential colony-forming cells was not inhibited after in vitro HCMV infection. Furthermore, HCMV DNA was detected in both CD34+ HLA-DR- and CD34+ HLA-DR+ progenitors from in vitro-infected HCMV-seronegative donors and cells from HCMV-seropositive donors. Taken together, the early progenitors defined as CD34+ HLA-DR- and CD34+ HLA-DR+ are directly suppressed in their proliferation by HCMV in vitro, and hematopoietic stem cells are also sites of HCMV latency in healthy HCMV-seropositive donors.

Direct growth suppression of myeloid bone marrow progenitor cells but not cord blood progenitors by human cytomegalovirus in vitro.

Recently, considerable interest has arisen as to use cord blood (CB) as a source of hematopoietic stem cells for allogenic transplantation when bone marrow (BM) from a familial HLA-matched donor is not available. Because human cytomegalovirus (HCMV) has been shown to inhibit the proliferation of BM progenitors in vitro, it was important to examine whether similar effect could be observed in HCMV-infected CB cells. Therefore, the effect of HCMV challenge on the proliferation of myeloid progenitors from BM and CB was compared using both mononuclear cells (MNC) and purified CD34+ cells. A clinical isolate of HCMV inhibited the colony formation of myeloid BM progenitors responsive to granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, macrophage-CSF, interleukin-3 (IL-3) and the combination of IL-3 and stem cell factor (SCF). In contrast, colony growth of CB progenitors was not affected. In addition, HCMV inhibited directly the growth of purified BM CD34+ cells responsive to IL-3 and SCF in single cell assay by 40%, wheras the growth of CD34+ progenitors obtained from CB was not suppressed. The HCMV lower matrix structural protein pp65 and HCMV DNA were detected in both CB and BM CD34+ cells after in vitro challenge. However, neither immediate early (IE)-mRNA nor IE proteins were observed in infected cells. Cell cyclus examination of BM and CB CD34+ cells revealed that 25.7% of BM progenitors were in S + G2/ M phase wheras only 10.7% of the CB progenitors. Thus, a clinical isolate of HCMV directly inhibited the proliferation of myeloid BM progenitors in vitro wheras CB progenitors were not affected. This difference in the susceptibility of CB and BM cells to HCMV may partly be caused by the slow cycling rate of naive CB progenitors compared to BM progenitors at the time of infection.

Role of the TCR binding region of the HLA class I alpha 2 domain in regulation of cell adhesion and proliferation.

In addition to Ag presentation for T cell surveillance, MHC molecules have been implicated in mediating regulatory signals. We have assessed biologic responses following engagement of the TCR accessible region of the HLA class I alpha 2 domain. mAbs directed to this domain specifically induced cell aggregation of normal hematopoietic and leukemic cells. The functional consequences were unique since other mAbs reactive with HLA class I residues outside the TCR binding domain did not induce cell aggregation. The adhesion response required ATP, mRNA, protein, and actin synthesis and did not depend on LFA-1/ICAM interactions. Cell aggregation was also induced when all but four of the intracytoplasmic residues of the class I molecule were deleted, indicating that transduction of signals leading to cell adhesion does not require this portion of the molecule. mAbs directed to HLA class I alpha 2 amino acid residues within the TCR binding domain were also able to inhibit proliferation of normal mitogen-stimulated T cells. Growth inhibition correlated with down-regulated expression of CD25, CD28, and CD95, suggesting that reduced transduction of costimulatory signals is involved. Although HLA class I signals inducing cell aggregation required engagement of positions within the TCR binding region, growth inhibitory signals could be generated through positions both within and adjacent to this domain. Taken together, engagement of specific positions within the TCR binding domain of the class I alpha 2 helix results in active cellular responses. Thus, this region may be directly involved in signal transduction following CTL recognition of target cells.

RG1, a new murine monoclonal antibody recognizing a "supertypic" determinant on HLA-A molecules.

Monomorphic and polymorphic anti-HLA monoclonal antibodies (mAb) are valuable reagents for assessment of the structural and functional importance of different class I determinants. We have generated a new mAb, RG1, reacting with an epitope variably expressed on normal and leukemic hematopoietic cells of different lineages. Immunoprecipitation of the RG1 antigen disclosed a bimolecular complex characteristic of class I proteins. The RG1 epitope was expressed on an HLA-A2 transfected cell line but not on cells transfected with HLA-E, -F or -G molecules. MAb reactivity with reference B-lymphoblastoid cell lines and HLA typing of RG1 reactive and unreactive cells demonstrated that the epitope was expressed in conjunction with defined HLA-A molecules. Cells expressing HLA-A2, -A24(9) and -A68(28) proteins were brightly stained with RG1 whereas mAb binding to HLA-A1, -A11 and a split of A3 molecules was significantly lower. In contrast, the RG1 epitope was apparently not expressed on HLA-A23(9), -A25(10), -A26(10), -A29(19), -A30(19), -A31(19), -A32(19), -A33(19) and some HLA-A3 molecules. Based on class I alpha sequence data, these results suggest that the RG1 epitope is localized to a region of the alpha 2 helix accessible to the T cell receptor for antigen on cytotoxic T lymphocytes. Lys in position 144 and His in position 151 are apparently critical for RG1 binding.

Transforming growth factor beta modulates C3 and factor B biosynthesis and complement receptor 3 expression in cultured human monocytes.

Complement biosynthesis in monocytes is stimulated by different pathogens and modulated by a variety of cytokines, but little is known about the possible effect of transforming growth factor beta (TGF-beta) on this monocyte function. We therefore studied the effect of TGF-beta 1 and TGF-beta 2 on constitutive, lipopolysaccharide (LPS)- and Candida albicans-induced monocyte biosynthesis of complement components C3 and factor B. Under all three conditions, both forms of TGF-beta (20 ng/ml) induced a two- to fourfold increase in C3 concentration in monocyte supernatants harvested after 2 or 5 days of cell culture, an effect that was abrogated by cycloheximide. In contrast, constitutive and pathogen-induced production of factor B was suppressed by TGF-beta. The effects of TGF-beta on complement production were neutralized by a monoclonal anti-TGF-beta antibody. Moreover, TGF-beta suppressed the pathogen-induced release of granulocyte-macrophage colony-stimulating factor and down-regulated the expression of complement receptor 3 (CD11b/CD18), while the expression of CD11a/CD18, a related beta 2 integrin, was unaffected. These novel effects of TGF-beta emphasize the immunomodulatory significance of this cytokine.

Interleukin-1 (IL-1) directly and indirectly promotes hematopoietic cell growth through type I IL-1 receptor.

Interleukin-1 (IL-1) has been shown to stimulate hematopoietic progenitor cell growth both in vitro and in vivo. Although IL-1 alone lacks the ability to promote hematopoietic progenitor growth in vitro, it is a potent synergistic factor in combination with other colony-stimulating factors (CSFs). Because it was unknown whether type I (p80), type II (p68), or other IL-1-binding proteins mediated the synergistic effects of IL-1 on purified progenitor cells, we used the difference in immunoreactivity between type I and type II IL-1 receptor (IL-1R) to better assess the role of these receptors in hematopoietic progenitor growth. Therefore, the synergistic effects of IL-1 alpha on IL-3-, CSF-1-, and granulocyte macrophage (GM)-CSF-induced progenitor growth, both in CFU-c and single-cell assays, were determined in the presence of monoclonal antibodies (MoAbs) 35F5 and 4E2 that block the binding of IL-1 alpha to type I and type II IL-1R, respectively. The synergistic effect of IL-1 alpha on IL-3 responsive Lin- and Lin(-)-Thy-1+ progenitors was indirectly mediated and could be inhibited by MoAb 35F5. In contrast, IL-1 alpha directly synergized with CSF-1 and GM-CSF to promote progenitor cell growth. The direct synergistic effect of IL-1 alpha on CSF-1-induced progenitor growth was observed in all progenitor populations examined (Lin-, Lin-Thy-1+, and Lin-Thy-1-) and was inhibited by MoAb 35F5. However, the direct synergistic effect of IL-1 alpha on GM-CSF-responsive progenitors. Lin- and Lin-Thy-1+, was partially inhibited by MoAb 35F5. In contrast, the MoAb antitype II IL-1R (MoAb 4E2) could not inhibit the direct synergistic effects of IL-1 alpha on CSF-1- or GM-CSF-induced progenitor growth. Thus, IL-1 alpha directly and indirectly stimulates the growth and differentiation of purified progenitors through the type I IL-1R but not the type II IL-1R.

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.

Granulocyte-macrophage colony-stimulating factor, but not macrophage colony-stimulating factor, suppresses basal and lipopolysaccharide-stimulated complement factor production in human monocytes.

Monocyte/macrophage contribution of C biosynthesis is important, particularly during inflammation. Since granulocyte-macrophage CSF (GM-CSF) and macrophage-CSF (M-CSF) exert a variety of stimulatory effects on monocyte/macrophage functions in vitro, we studied their impact on the biosynthesis of the C components C3 and factor B by human monocytes in culture. GM-CSF at doses of 10 ng/ml and higher inhibited the basal C3 synthesis. This effect was most pronounced when the cytokine was added to freshly isolated monocytes. No effect was found on the basal production of factor B. Furthermore, GM-CSF abrogated the LPS-stimulated production of both C3 and factor B. These suppressive effects were neutralized by a polyclonal anti-GM-CSF antibody. Moreover, when anti-GM-CSF was added to unstimulated or LPS-stimulated cells, their C3 production increased. This indicates that both spontaneous and LPS-triggered release of monocyte-produced GM-CSF has an autocrine function in regulating monocyte C3 biosynthesis. GM-CSF also down-modulated the expression of CD14 at an early stage of cell culture. This might be the mechanism through which the LPS-effects are suppressed because CD14 has been shown to be a LPS receptor. Contrary to this, M-CSF at doses of 100 U/ml and higher stimulated the synthesis of C3, whereas the basal production of factor B and the LPS-stimulated production of C3 and factor B were unaffected. Granulocyte-CSF (G-CSF) did not influence monocyte C biosynthesis, and neither anti-M-CSF nor anti-G-CSF influenced the LPS-induced C3 production. The effects of GM-CSF and M-CSF on C biosynthesis may be important in regulating the availability of C components during an inflammatory response, and these observations may also have implications for the clinical use of CSF.

Increased granulopoiesis after sequential administration of transforming growth factor-beta 1 and granulocyte-macrophage colony-stimulating factor.

Transforming growth factor-beta 1 (TGF-beta 1) is an inhibitor of the growth and differentiation of immature hematopoietic progenitors in vitro; however, we have demonstrated that TGF-beta 1 can promote granulopoiesis in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. We therefore examined the effect of the combined administration of TGF-beta 1 and GM-CSF in vivo. First, TGF-beta 1 enhanced the specific binding of GM-CSF (2.0-fold) on bone marrow cells, reaching a maximum 40 hours after injection, while the specific binding of interleukin-3 (IL-3) was unaffected. Using GM-CSF-specific binding to determine the optimal regimen for cytokine administration in vivo, we found that the administration of TGF-beta 1 and GM-CSF in sequence increased myelopoiesis. Total numbers of colony-forming units-granulocyte/macrophage (CFU-GM) and myeloblasts per femur were increased above the level obtained with the simultaneous injection of TGF-beta 1 plus GM-CSF, GM-CSF alone or TGF-beta 1 alone. Further, the sequential administration of TGF-beta 1 and GM-CSF resulted in enhanced numbers of mature granulocytes in both the bone marrow and peripheral blood. In contrast, the sequential combination of TGF-beta 1 and GM-CSF did not enhance the numbers or increase the recovery of erythroid cells in the bone marrow. These results show that TGF-beta 1 in vivo as in vitro has a multifunctional effect on bone marrow progenitors, and by using an optimal combination of TGF-beta 1 and GM-CSF in vivo, one can selectively increase both the central and peripheral granulopoietic compartments.

In vivo effect of interleukin-1 alpha on hematopoiesis: role of colony-stimulating factor receptor modulation.

To determine the mechanism(s) by which interleukin-1 (IL-1) promotes granulopoiesis in vivo, we examined the effect of in vivo administration of IL-1 alpha on colony-stimulating factor (CSF) receptor expression on bone marrow cells (BMCs) and whether this directly correlated with progenitor cell responsiveness. Administration of IL-1 alpha to mice induced the upregulation of both granulocyte-macrophage-CSF (GM-CSF) and IL-3 receptors, which reached a maximum 24 hours after IL-1 alpha injection on unfractionated BMCs. This upregulation was more pronounced on the progenitor-enriched cell population (lineage-negative [Lin(-)]). The enhanced GM-CSF and IL-3 receptor expression directly correlated with enhanced IL-3- or GM-CSF-induced growth of colony-forming unit-culture (CFU-c) or CFU-mixture (CFU-Mix; colonies containing macrophages, granulocytes, and erythroid cells). In addition, the absolute number of high proliferative potential-colony-forming cells (HPP-CFC) was increased fivefold. In contrast, granulocyte-CSF (G-CSF)-specific binding on unfractionated BMCs was rapidly (4 hours) reduced after IL-1 alpha administration and returned to control levels by 24 hours. This reduction correlated with IL-1 alpha-induced margination of mature granulocytes (RBC-8C5hi cells), which express high levels of G-CSF receptors. IL-1 alpha treatment did not affect G-CSF receptor expression on Lin- cells. Pretreatment of mice with anti-type I IL-1 receptor antibody blocked the IL-1 alpha-induced upregulation of GM-CSF and IL-3 receptor expression on BMCs. Taken together, as one possible mechanism, IL-1 alpha in vivo may stimulate the expression of functional GM-CSF and IL-3 receptors on BMCs indirectly, and, in concert with the induction of circulating CSF levels, may account for the ability of IL-1 alpha to stimulate hematopoiesis in vivo.

Transforming growth factor-beta: a bidirectional regulator of hematopoietic cell growth.

It is now apparent that the transforming growth factor-beta (TGF-beta) family of proteins has potent hematopoietic regulatory properties ranging from effects on the growth and differentiation of primitive stem cells to the differentiated functions of mature cells. Although most reports have described the inhibitory activities of TGF-beta on hematopoiesis, recent evidence supports the concept that TGF-beta can have both inhibitory and stimulatory actions on these systems. These differences depend on the differentiation state of the target cell and the other cytokines interacting with the cell. Furthermore, TGF-beta has direct bidirectional effects on cell surface expression of many cytokine receptors suggesting that it is part of the mechanism of action of TGF-beta. The major biological effect of TGF-beta on hematopoietic cell growth is the reversible inhibition of entry into the cell cycle. Importantly, the effect of in vivo administration of TGF-beta has mimicked the in vitro effects. Ultimately, well designed clinical trials will determine whether the exciting potential of TGF-beta can be used to treat or prevent myelotoxicity and other bone marrow dysfunctions.