Is There a Role for Hematopoietic Growth Factors During Sepsis?

Sepsis is a complex syndrome characterized by simultaneous activation of pro- and anti-inflammatory processes. After an inflammatory phase, patients present signs of immunosuppression and possibly persistent inflammation. Hematopoietic growth factors (HGFs) are glycoproteins that cause immune cells to mature and/or proliferate. HGFs also have a profound effect on cell functions and behavior. HGFs play crucial role in sepsis pathophysiology and were tested in several clinical trials without success to date. This review summarizes the role played by HGFs during sepsis and their potential therapeutic role in the Management of sepsis-related immune disturbances.

[The comparative characteristics of antibacterial properties of the peptides of the active site of GM-CSF, and substances delivered from supernatants of hematopoietic progenitor CD34+45- cells].

The antibacterial activity of synthetic peptides of the active site of GM-CSF and supernatants of CD34+45- hematopoietic progenitor cells has been investigated GM-CSF peptides and cell supernatants were found to possess pronounced antibacterial activity, at that a combination of these substances has a more pronounced activity in comparison with the single substances. Possible mechanisms of the identified effects of synthetic peptides and substances from the supernatants of CD34+5- cells are discussed.

Cytopenias in HIV infection: mechanisms and alleviation of hematopoietic inhibition.

Hematopoietic abnormalities including anemia, cytopenias, and alterations of the stem cell plasticity in the bone marrow microenvironment commonly occur in HIV infected patients. These observations suggest that HIV-1 infection may affect processes important during early stages of hematopoiesis or stem cell differentiation. Hematopoietic abnormalities may be caused by altered stem cell differentiation possibly due to abnormal lineage specific expression of certain cellular genes such as cytokines relevant to hematopoiesis. These cytokines could affect regulatory signals important in hematopoiesis. However, in HIV infected individuals, it is not only the virus but also the highly active antiretroviral therapy (HAART) that both contribute to persistent hematopoietic suppression and ensuing cytopenias. Even if a lowering of HIV replication by HAART were to occur in infected individuals, prolonged HAART by itself and/or appearance of drug resistant mutants can contribute to hematopoietic suppression and resulting cytopenias. However, confounding factors such as opportunistic infections, immune mediated effects, or the consequences of prolonged physiological stress, which could contribute to decreased hematopoiesis in patients or other individuals, make the causative role of HIV in vivo, uncertain. The severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues (SCID-hu) is a small animal model which mimics HIV infection in humans, and is useful to determine the mechanisms of HIV-1 induced hematopoietic inhibition and development of drug therapies for interventions of stem cell differentiation. Further, SCID mouse serves as a useful small animal recipient of human progenitor cells and also allows us to study the differentiation of these cells in vivo. Results from our studies are expected to provide relief for HIV infected individuals from hematopoietic inhibition and ensuing cytopenias.

Therapeutically-induced autoantibodies in patients treated with recombinant hematopoietic growth factors: a brief summary.

Since recombinant gene technology was established to provide rare important regulatory proteins as recombinant molecules, cytokine therapies have widely developed and enormously contributed to the treatment of various diseases; nevertheless, it has been revealed that recombinant therapeutic molecules are not always perfect because of side-effects related to pharmacological functions of cytokines and/or potential antigenicity observed in some clinical cases. Although studies on the antigenicity of recombinant proteins have initiated, and observations in clinical studies have been accumulated over this decade, mechanisms of the autoantibody production are not clarified yet. Among various hematopoietic growth factors introduced into clinical trials, this report summarizes current issues of autoantibodies to primary regulators for terminal hematopoiesis.

Perinatal immunomodulation.

The fetus and the neonate are particularly vulnerable to injury caused directly by immunologic mechanisms or inflicted by infectious agents that take advantage of their relatively immature and inexperienced immune system. With increasing survival of high-risk neonates in the surfactant era, prevention/treatment of sepsis and chronic lung disease (CLD) has emerged as an area of priority in neonatal research. Considering the role of inflammatory mediators in the pathogenesis of sepsis and CLD, the clinical application of immunomodulator therapy to neonatology is perhaps more important at present than ever. Advances in molecular biology and immunology have led to development of newer immune modulator therapies that are directed towards specific cells or cytokines rather than resulting in a general suppression of the immune response. Failure of promising, newer immunomodulator therapies in sepsis trials in adults has, however, clearly documented the difficulties in diagnosing/correcting the imbalance between pro- and anti-inflammatory responses. As in the case of sepsis, development of a single magic bullet for prevention/management of a multi-factorial illness like CLD may be difficult, as prevention of prematurity - the single most important high-risk factor for CLD - is an unachievable goal at present. As new frontiers are being explored, older, well-established therapies like antenatal anti-D immunoglobulin prophylaxis continue to emphasize the tremendous potential of immunomodulator therapy in neonatology/perinatology. The current immunomodulators/immunotherapeutic agents with established/potential clinical applications in the perinatal period are reviewed.

Hematopoietic growth factors, dendritic cell biology, and vaccine therapy of cancer.

Hematopoietic growth factors have made it possible to collect, manufacture, and engineer dendritic cells ex vivo for clinical use and expand dendritic cell subsets in vivo when administered to patients. Dendritic cells are important vectors in the induction of an effective immune response against infection and neoplastic disease. Antigens alone, even those preprocessed to bind to antigen-presenting major histocompatibility complex class I and II molecules, are insufficient to regulate effective T-cell-mediated immunity. Activated dendritic cells are essential to this task. Studies of dendritic cell biology in the laboratory and preclinical studies have facilitated the implementation of clinical trials using dendritic cells in the treatment of melanoma and other cancers. Dendritic cell subset functional differences, effective tumor target antigen loading of dendritic cells for presentation to immune effector cells, dendritic cell maturation, the route of dendritic cell administration to humans, and immunologic monitoring are parameters that require vigorous study in the context of dendritic cell immunotherapy of cancer.

Lymphokine-dependent proliferation of T-lymphoid cells: regulated responsiveness and role in vivo.

The discovery of lymphokines stemmed from their ability to promote T-lymphocyte proliferation in vitro. Even after 20 years of intensive investigation, crucial aspects remain to be clarified about the role of specific lymphokines in T-cell proliferation and the biochemical mechanisms by which they play these roles, particularly in vivo. The present review focuses on conventional populations of TCR(alpha)beta T cells. Older findings and new insights into the function of specific lymphokines in T-lymphocyte proliferation in vivo are summarized along with unanswered questions raised by these observations. Vital contributions of lymphokines to clonal proliferation arise from two processes: the protection of cells against apoptosis and the activation of cell cycling. Findings are underscored indicating that the activity of a particular lymphokine depends on the subset of T cells (CD4 vs. CD8; naive vs. memory) to which it binds, and that point to potential pitfalls of extrapolating from tissue culture-adapted models to the regulation of T cells in vivo. After summaries of signaling mechanisms related to the proliferative activity of lymphokines, recent findings are highlighted suggesting that such signaling is a regulated and plastic process rather than one fixed schema of action.

Augmentation of the immune response with granulocyte-macrophage colony-stimulating factor and other hematopoietic growth factors.

Granulocyte-macrophage colony-stimulating factor is by far the most widely used hematopoietic growth factor to augment immune responses. At present, the best secured effect is as an adjuvant cytokine for vaccination. Granulocyte-macrophage colony-stimulating factor can be delivered as gene-transduced tumor cells, as plasmid DNA, or as the soluble free granulocyte-macrophage colony-stimulating factor protein. Granulocyte-macrophage colony-stimulating factor must be present at the same site as the vaccine component. Granulocyte-macrophage colony-stimulating factor may also augment the effect of therapeutic monoclonal antibodies by enhancing various effector functions such as antibody-dependent cellular cytotoxicity and amplifying an idiotypic network response (i.e., antitumor immunity). It may also be advantageous to combine granulocyte colony-stimulating factor with monoclonal antibodies (neutrophil and monocyte antibody-dependent cellular cytotoxicity) for tumor therapy. However, these growth factors might also induce immune suppression, which may hamper the contemplated effect of the growth factor. It is urgently warranted to better understand these dual effects on the immune system so that we can find optimal uses for the growth factors in various clinical settings.

Human dendritic cells require exogenous interleukin-12-inducing factors to direct the development of naive T-helper cells toward the Th1 phenotype.

Dendritic cells (DC) are important initiators of specific primary immune responses because they are the only APC that can efficiently activate naive Th cells. DC have the capacity to produce interleukin-12 (IL-12), a cytokine that plays a pivotal role in the development of Th1-mediated cellular immune responses. The present study focuses on the conditions under which human DC produce bioactive IL-12 p70 and, consequently, direct the development of naive T helper (Th) cells toward the Th1 phenotype. Bacteria or bacterial compounds such as Staphylococcus aureus Cowan strain I (SAC) or lipopolysaccharide (LPS) induced substantial IL-12 levels in DC, which could be further upregulated by interferon-gamma (IFN-gamma), whereas induction of IL-12 production via CD40 ligation required IFN-gamma as an obligatory, complementary signal. Also, activated naive Th cells were poor inducers of IL-12 production, unless exogenous IFN-gamma was present, whereas activated memory Th cells were effective inducers of IL-12 production and did not require exogenous IFN-gamma. Next, the cytokine profiles of matured Th cells that were primed by DC under different conditions were examined. DC promoted the development of naive Th cells into memory Th0 cells that produced both the type 1 cytokine IFN-gamma and the type 2 cytokine IL-4. In contrast, after activation with SAC, DC efficiently directed the development of Th1 cells through the release of IL-12. An APC-independent Th cell maturation model, using either recombinant IL-12 or supernatants of SAC-activated DC and neutralizing anti-IL-12 antibodies, confirmed that DC-derived IL-12 was the major Th1 skewing factor. Together, these data indicate that the contact between DC and naive Th cells during the initiation of specific immune responses does not result in the efficient induction of IL-12 production and that, consequently, exogenous IL-12-inducing factors are required to promote primary Th1-mediated cellular immune responses.

Allergen-induced increase in bone marrow progenitors in airway hyperresponsive dogs: regulation by a serum hemopoietic factor.

We have previously reported that bone marrow progenitors in dogs, specifically granulocyte-macrophage colony-forming units (GM-CFU), increase developing airway hyperresponsiveness after inhalation of the allergen Ascaris suum. In the present study, we evaluated whether this increased marrow hemopoietic activity can be stimulated by a factor in serum after allergen challenge. Serum samples taken from dogs prior to and 20 min, 2 h, and 24 h after Ascaris or diluent challenge were added to bone marrow cells aspirated prior to challenge, and GM-CFU measured. A second bone marrow aspirate was performed 24 h after challenge. Nonadherent mononuclear bone marrow cells were incubated for 8 days in the presence of the serum and recombinant canine hemopoietic cytokines (stem cell factor, granulocyte colony-stimulating factor, GM colony-stimulating factor). Eight dogs that developed (airway responders) and eight dogs that did not develop (airway nonresponders) allergen-induced airway hyperresponsiveness were studied. Allergen inhalation increased bone marrow GM-CFU in response to all three growth media in vitro for the airway responder (P < 0.05) but not airway nonresponder dogs. The 24-h serum, taken from the airway responder but not the airway nonresponder dogs, produced a similar increase in granulocyte progenitors when added to the bone marrow taken before allergen inhalation (P < 0.05). These findings demonstrate that bone marrow-derived granulocyte progenitors are upregulated by a factor that can be shown to be present in serum 24 h after allergen challenge in dogs that develop allergen-induced airway hyperresponsiveness. Whether in vivo stimulation of bone marrow inflammatory cell production is necessary for the development of allergen-induced airway hyperresponsiveness remains to be proven.

Stem cell factor contributes to intestinal mucosal mast cell hyperplasia in rats infected with Nippostrongylus brasiliensis or Trichinella spiralis, but anti-stem cell factor treatment decreases parasite egg production during N brasiliensis infection.

We assessed the effects of the c-kit ligand, stem cell factor (SCF), in the jejunal mucosal mast cell hyperplasia that occurs during infection with the intestinal nematodes, Nippostrongylus brasiliensis or Trichinella spiralis in rats. Compared with vehicle-treated rats, rats treated with SCF (25 micrograms/kg/d, intravenous [i.v.] for 14 days) during N brasiliensis infection exhibited significantly higher levels of the rat mucosal mast cell (MMC)-associated protease, rat mast cell protease II (RMCP II) in the jejunum and serum on day 8 of infection, but not on days 10 or 15 of infection. By contrast, in comparison to rats treated with normal sheep IgG, rats treated with a polyclonal sheep antirat SCF antibody exhibited markedly decreased numbers of jejunal MMCs, levels of jejunal RMCP II, and serum concentrations of RMCP II during infection with either nematode, particularly at the earlier intervals of infection (< or = day 10). Taken together, these findings indicate that SCF importantly contributes to MMC hyperplasia and/or survival during N brasiliensis or T spiralis infection in rats, but that levels of endogenous SCF are adequate to sustain near maximal MMC hyperplasia during infection with these nematodes. Notably, treatment of rats with SCF somewhat increased, and treatment with anti-SCF significantly decreased, parasite egg production during N brasiliensis infection. This finding raises the interesting possibility that certain activities of intestinal MMCs may contribute to parasite fecundity during infection with this nematode.

Murine spleen stromal cell line SPY3-2 maintains long-term hematopoiesis in vitro.

The hematopoietic microenvironment (HIM) of mouse spleen predominantly induces the differentiation of hematopoietic progenitors into erythroid lineage in vivo. However, the mechanisms of this phenomenon have not been fully explored because of the lack of an adequate in vitro system mimicking the spleen hematopoiesis. To reconstruct the HIM of mouse spleen in vitro, we established spleen stromal cell lines from a three-dimensional (3D) spleen primary culture in collagen gel matrix. Of these, SPY3-2 cells were negative for preadipocytic and endothelial markers, had a fibroblastoid morphology, and were not converted to adipocytes in the presence of 1 mumol/L hydrocortisone. They supported the maintenance and multilineal differentiation of hematopoietic progenitor cells for more than 8 weeks in vitro. The differentiated hematopoietic cells in the coculture medium were predominantly monocytes rather than granulocytes. Furthermore, erythropoiesis was predominantly induced in the presence of 2 U/mL erythropoietin and continued for more than 12 weeks. The number of burst-forming units-erythroid (BFU-E) was increased 10 times after 3 weeks of coculture, which was followed by pronounced production of erythroid cells in the coculture after week 4. SPY3-2 expressed high levels of c-kit ligand and low levels of granulocyte macrophage colony-stimulating factor and interleukin-3, and these molecules were all involved in this long-term erythropoiesis. Thus, the clonal SPY3-2 cell line will provide a novel HIM in vitro analogous to that of mouse spleen in vivo. These results suggest that 3D collagen gel culture may facilitate the establishment of functioning stromal cell lines of hematopoietic organ.

Immunological and biochemical characterization of biglycan-like haemopoietic factor.

Immunological and biochemical characteristics of a 100,000 MW biglycan-like haemopoietic factor, purified from thymic myoid cells 871207B, were studied to distinguish them from macrophage colony-stimulating factor (M-CSF), which they resemble in activity and biochemical properties. Rabbit antibody raised against a synthetic peptide fragment (J-1) designed from amino acid sequences specific to the 100,000 MW factor responded to 871207B cells, the conditioned medium of 871207B, and capillary-like structures in the thymus, but not to M-CSF producer L-929 cells or the conditioned medium of L-929 cells. In contrast, M-CSF epitope was detected in L-929 cells and the conditioned medium cells but not in 871207B cells or the conditioned medium, even after enzymatic digestion of glycosaminoglycan chains. Treatment of the 100,000 MW factor with chondroitinase ABC and AC produced a 50,000 MW component. Digestion of this product with N-glycanase resulted in a 40,000 MW protein component. These results suggest that the 100,000 MW factor is a proteoglycan consisting of a core protein with an apparent molecular mass of 40,000 MW, a 50,000 MW chondroitin sulphate chain and 10,000 MW N-linked oligosaccharide chains. A small amount of a 40,000 MW monocytic cell growth activity was also found in the 871207B cell-conditioned medium. An enzymatically obtained 40,000 MW factor, the conditioned medium 40,000 MW factor, and the 100,000 MW factor were specifically eluated from an anti-J-1 IgG-immobilized affinity column with monocytic cell growth activity, suggesting that the biological activity resides in the 40,000 MW core protein. The 100,000 MW factor induced the proliferation and differentiation of monocytic lineage cells from a variety of sources, such as bone marrow cells, peritoneal exudated cells and brain microglia cells.

Ultrastructural localization of stem cell factor in canine marrow-derived stromal cells.

Stromal cell lines derived from canine long-term bone marrow cultures (LTBMC) were characterized regarding the expression of growth factors and especially the localization of stem cell factor (SCF) (c-kit ligand). One cell line (DO64) was immortalized by transformation with a retroviral vector containing the open reading frames (ORFs) E6 and E7 of the human papilloma virus type 16 (HPV-16). Transfection did not change cellular characteristics but rendered the cell line more independent from culture conditions. The transformed line DO64 consisted mainly of fibroblast-like cells. In addition, some cells showed endothelial and some smooth-muscle cell features. Stromal cells expressed a broad spectrum of surface markers, including low levels of major histocompatibility-complex (MHC) class-II antigens. A new murine monoclonal antibody (MAb), RG7.6 (IgG1), specific for canine SCF, recognized the majority of fibroblast-like stromal cells. The staining pattern for SCF showed perinuclear and intracytoplasmic dense areas. Immunoelectron microscopy revealed the localization of SCF in secretory vesicles, the perivesicular cytoplasm, and bound to the cytoplasmatic membrane. RNA analysis showed that stromal cells transcribed, in addition to SCF, messages for granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte CSF (GM-CSF), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-beta). In summary, we have established and characterized canine marrow-derived stromal cell lines, and using the new MAb RG7.6, we have localized SCF to cytoplasmatic vesicles as well as the membrane of stromal cells.

Interactions between stem cell factor and c-Kit are required for intestinal immune system homeostasis.

Interactions between stem cell factor (SCF) and its receptor, c-Kit, are important for development of hematopoietic, melanocytes, and germ cells. T lymphocytes appeared normal in c-Kit (W/Wv) or SCF (SI/SId) mutant mice, except for those residing within the intestinal epithelium, the intraepithelial lymphocytes (IEL). Normally, IEL are composed of equal numbers of cells with alpha beta or gamma delta T cell receptors. In mutant mice, beginning at 6-8 weeks of age, the number of gamma delta IEL decreased, whereas alpha beta IEL increased. The latter was due largely to an increased CD4+ CD8+ TCR alpha beta subset, suggesting that these cells may be intermediates in the alpha beta IEL lineage. c-Kit or SCF was expressed by IEL or intestinal epithelial cells, respectively, indicating a potential for direct intercellular interaction. This possibility was supported by reconstitution studies that demonstrated that c-Kit mutations directly affected IEL. Thus, SCF-c-Kit interactions are important for homeostasis of the intestinal immune compartment.

Kit ligand/mast cell growth factor-independent differentiation of mast cells in myelodysplasia and chronic myeloid leukemic blast crisis.

Autonomous, factor-independent growth and differentiation of malignant cells in preleukemic and leukemic disease states is a well-recognized phenomenon and is often associated with a poor prognosis. Mast cells are distinct hematopoietic cells and express a unique profile of antigens. Growth and differentiation of normal mast cells is dependent on mast cell growth factor (MGF), the ligand of the c-kit protooncogene product. In this study, we screened for mast cell-lineage involvement in 52 patients suffering from myeloid leukemias, myelodysplastic syndromes (MDS), systemic mastocytosis, or other diseases by probing for mast cell-related molecules (c-kit, tryptase, histamine, and MGF) and by analyzing kit ligand/MGF-independent growth of mast cells in long-term suspension culture. Of the 52 patients tested, 2 patients with refractory anemia with excess of blast cells in transformation and 1 patient suffering from chronic myeloid leukemia blast crisis (CML-BC) were diagnosed as mastocytic disease. These patients were characterized by complex chromosomal abnormalities, splenomegaly, high percentages of circulating metachromatic cells (5% to 25%), high levels of cellular tryptase (> 10 ng/10(5) peripheral blood mononuclear cells/mL) and a tryptase/histamine (ng:ng) ratio greater than 1. The metachromatic cells expressed the mast-cell-related surface antigen c-kit, but not basophil-related antigens (CD11b, CDw17). Furthermore, in these 3 patients, spontaneous, MGF-independent growth of mast cells along with spontaneous synthesis of tryptase was demonstrable in long-term culture. No autocrine production, paracrine production, or overproduction of MGF was found. The spontaneous growth of mast cells could neither be abbrogated by addition of monoclonal antibodies (MoAbs) to c-kit nor by MoAbs against MGF (< 5% inhibition), whereas factor (MGF)-dependent differentiation of mast cells in these patients could be abbrogated by MoAbs to c-kit or MoAbs to MGF (> 70% inhibition, P < .001). In addition, serum MGF levels in these patients were within the normal range and MGF could not be detected in cell-free culture supernatants. All 3 patients showed rapid progression of disease and had a survival time of less than 1 year. In conclusion, we describe a unique form of transformation in MDS and CML-BC characterized by mast cell lineage involvement and factor-independent differentiation of mast cells. This form of leukemic transformation has to be delineated from chronic myeloid leukemia with basophilia or basophil crisis, from primary mast cell leukemia, and from monocytic leukemias and myelodysplastic disorders associated with basophilia.

Overview of the role of hematopoietic growth factors in bone marrow transplant recovery and bone marrow transplant failure.

Morbidity of bone marrow transplantation has been reduced by the prophylactic use of myeloid stimulating growth factors. Trials are current exploring the usage of hematopoietic growth factors in a variety of other potential clinical applications in bone marrow transplantations. These include stimulation of multipotent progenitor cells, enhancement of monocyte macrophage function, enhancement of lymphocyte function, and alteration of inflammatory mediators which are causal of nonhematologic toxicities of bone marrow transplantation. Hematopoietic growth factors, factors which induce production of stimulatory cytokines and/or factors which alter inflammatory pathways, may be effective in improving the tolerability of patients undergoing bone marrow transplant.

IL-12 protects bone marrow from and sensitizes intestinal tract to ionizing radiation.

IL-12, a potent stimulator of hemopoietic progenitor cells, was evaluated as a potential protector against 60Co-gamma radiation-induced lethal hemopoietic syndrome in mice. Administration of IL-12 before lethal irradiation of genetically distinct strains of mice, B6D2F1 and C3H/HeJ, protected a significant fraction of both strains of mice from death. Radioprotection was associated with a fivefold increase in the number of bone marrow cells at 6 days after irradiation. Even at supralethal doses of radiation (1200 cGy), the number of c-kit+ bone marrow cells 3 days after irradiation was twofold greater in IL-12-treated mice than in saline-treated mice. However, mice that received IL-12 and 1200 cGy (B6D2F1) or 900 cGy (C3H/HeJ) died of the gastrointestinal syndrome, as was evident by gross necroscopy and histologic evaluation, within 4 to 6 days after irradiation. Induction of the gastrointestinal syndrome in mice not treated with IL-12 required radiation doses of 1500 cGy or greater in both strains. Thus, at doses of radiation at which IL-12 still protects c-kit+ hemopoietic cells, it sensitizes the intestinal tract to damage. Radioprotection with IL-12 was abrogated by anti-IL-1R or anti-stem cell factor Ab. Anti-IFN-gamma Ab did not affect IL-12-induced hemopoietic radioprotection, but abrogated sensitization of the intestinal tract by IL-12. The sensitizing effect of IL-12 may be related to its ability to prime mice to subsequent inflammatory challenge, as demonstrated by an almost 100-fold increase in circulating TNF and IL-6 levels in normal B6D2F1 mice challenged with IL-12 and LPS. This priming effect of IL-12 also was abrogated by anti-IFN-gamma Ab.