Serum of lipopolysacharide-treated mice contains two types of colony-stimulating factor,separable by affinity chromatography

Serum from mice traated with bacterial lipopolysaccharide (LPS) was fractionated by Con A-Sepharose affinity chromatography, and assayed in vitro for colony-stimulating factor (CSF) using mouse bone marrow cells. The CSF failing to bind to concanavalin A-Sepharose (pool A) had similar biological properties to the unfractionated serum, i.e., it stimulated the formation of about equal numbers of granulocytic, mixed granulocyte-macrophage and macrophage colonies. The fraction eluted from the Con A-Sepharose column with α-methyl-D-glucopyranoside (pool B) had a steeper dose-response curve than either the unfractionated serum or the pool A CSF and most of the colonies were composed of macrophages. A mixture of the pool A and pool B CSFs stimulated colonies in a similar way as unfractionated serum and pool A. The apparent molecular weights of the two types of CSF were determined by two different gel-filtration procedures. Sephacryl S-200 gel-filtration suggested an apparent molecular weight of 85,000 for pool A CSF and 180,000 for pool B CSF. Gel-filtration on Sepharose CL-6B in the presence of guanidine hydrochloride (6M) yielded an apparent molecular weight of approximately 23,000 for pool A CSF and 33,000 for pool B CSF. The colony-forming cells (CFC) responding to pool B CSF were found to have a relatively high sedimentation velocity (peak sedimentation velocity 5.6–6.2 mm/hr) compared to the CFC responding to mouse-lung conditioned medium (MLCM) whose peak sedimentation velocity was between 4.0–4.5 mm/hour. The CFC responding to pool A CSF had an intermediate sedimentation velocity (peak 4.6–5.2 mm/hour). A time-course analysis of the morphology of clones or colonies in cultures stimulated with either MLCM or pool B CSF showed that the proporation of different colony types depends significantly on the incubation period and suggested that pool B CSF induced an early commitment of CFC towards macrophage differentiation.     

The role of colony-stimulating factor in granulopoiesis

The proliferation and maturation of granulocytic-monocytic stem cells appears to be controlled by a series of closely related glycoproteins termed “colony-stimulating factors” (CSFs). Recently, we devised a 6-step scheme for the purification of murine fibroblast (L-cell)-derived CSF. Ten liter pools of conditioned media were concentrated by ultrafiltration, precipitated by ethanol, and separated on DEAE cellulose, Con-A Sepharose, and Sephadex G 150. The CSF was separated from trace contaminants, including endotoxin, by density gradient centrifugation. The purified material was radioiodinated and used to define the serum half-life and in vivo distribution. Following IV injection there was a biphasic serum clearance with a t½ of 24–40 min and 2–2½ hours in the first and second phases. Approximately 25% of the tracer was excreted in the urine at 6 h; however, urinary radioactivity was due to low molecular weight peptides. Simultaneous studies by radioimmunoassay showed a similar rapid serum clearance of unlabeled CSF but virtually no urinary CSF activity. Thus, assays for urinary CSF may not provide useful measures of in vivo CSF activity. Further in vitro studies have defined the interaction of CSF with responsive cells in the marrow. Varying doses of CSF were incubated with 10⁷ marrow cells for intervals of 24–48 h. The major increment in cell-associated radioactivity occurred between 6 and 16 h. The reaction was saturable with 1–2 ng/ml CSF. Binding was prevented by cold CSF, but not by other proteins. Irradiation yielded only a minimal reduction in CSF binding. The interaction of CSF with marrow cells appeared to require new protein synthesis, as binding was completely inhibited by cycloheximide and puromycin. Irradiated mice injected with antibodies to CSF showed an inhibition of granulopoiesis by marrow cells in peritoneal diffusion chambers; however, granulopoiesis in the intact bone marrow was unaffected. Granulpoiesis in long-term marrow cultures was also unaffected by anti-CSF. These different responses may be due to accelerated clearance of injected CSF in nonirradiated mice or to extensive stromal interactions that modulate and perhaps control granulocytic differentiation in the intact bone marrow microenvironment.     

Molecular properties of a granulocyte/macrophage colony-stimulating factor obtained from the serum-free culture of Yoshida sarcoma cell Line YSSF-212T

A granulocyte/macrophage colony-stimulating factor (Peak-1 CSF) was partially purified from the medium of a serum-free culture of Yoshida sarcoma cells (Line YSSF-212T). Its elution position in gel-filtration chromatography corresponded to a molecular weight of about 22,000. The factor had an isoelectric point at pH 4.5 and a sedimentation coefficient of 2.3 S. The major part of its activity was not bound by Concanavalin A-Sepharose. Although CSF activity behaved as a single component in the gel-filtration and isoelectrofocussing procedures, subsequently it was resolved into two species by preparative discontinuous polyacrylamide gel-electrophoresis. This resolution indicates microheterogeneity of the CSF molecule. Oxidation with periodate readily inactivated L . P3-cell CSF, but the YSSF-cell CSF was fairly resistant. Moreover, titration with anti-L cell CSF serum showed a definite difference between L . P3-cell CSF and YSSF-cell CSF.     

Purification of two forms of colony-stimulating factor from mouse L-cell-conditioned medium

A modified procedure for the purification of the colony-stimulating factors (CSFs) in mouse L-cell-conditioned medium is used to isolate two forms of CSF, which are separable by reversed-phase high performance liquid chromatography with 300-A pore size supports. The specific biological activity of these CSFs (2 X 10(9) colonies/mg) was considerably higher than has been achieved by other methods. Even at high concentration (200 pM) both molecules stimulated predominantly more macrophage than granulocyte colonies; however, the less hydrophobic form appeared to stimulate the formation of more pure granulocytic colonies. Almost twice as much of the less hydrophobic CSF was recovered from L-cell-conditioned medium. Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both forms of L-cell CSF had apparent molecular masses of approximately 70,000 daltons. However, on reduction with 2-mercaptoethanol, while both forms generated a 39,000-dalton subunit, the less hydrophobic form also yielded a 32,000-dalton subunit. Storage of either form of L-cell CSF at pH 2.1, in the presence of acetonitrile or isopropanol, destroyed the biological activity. Electrophoretic analysis of the L-cell CSFs stored under these conditions indicated that this was associated with a spontaneous dissociation of the CSF dimer into the inactive subunits. There was some charge heterogeneity (pI 3.5-4.7) indicating different degrees of glycosylation. The unique N-terminal amino acid sequences of both forms of CSF were the same: (Lys-Glu-Val-Ser-Glu-His-X-Ser-His-Met-Ile-Gly-Asn). Thus, the polypeptide chains appear to be identical for the subunits of both forms of L-cell CSF.     

Isolation of colony stimulating factor from human milk

Human milk contains colony stimulating factor (CSF), a polypeptide growth factor, which stimulates in in vitro bone marrow culture proliferation and differentiation of colony forming granulocytic macrophage progenitor cells (CFU-GM) to form colonies. This activity was not found in either bovine milk or colostrum when assayed in human or mouse bone marrow cells. The human milk CSF activity is destroyed by treatment with proteases. However, neither 6M urea, 4M guanidine hydrochloride, 5 mM dithiothreitol, nor exposure to pH 2 will inactivate the milk derived CSF. Gel filtration and isoelectric focusing indicate that human milk CSF differs biochemically from the other CSFs isolated from various sources and has a molecular weight between 250,000 and 240,000 and an isoelectric point between 4.4 and 4.9.     

Stimulation of macrophage plasminogen activator activity by colony-stimulating factors

Colony-stimulating factors (CSFs) stimulate granulocyte-macrophage production from single hemopoietic progenitor cells. Various preparations of purified CSFs of two different subclasses have been shown here to stimulate a plasminogen-dependent fibrinolytic (plasminogen activator) activity from resident and starch-induced mouse peritoneal macrophages. Lymphocyte supernatants also stimulate macrophage plasminogen activator (PA) activitty. Since they contain colony stimulating activity, it is possible that one or more sublcasses of CSF in these supernatants is responsible for this effect. Since both colony-stimulating and macrophage growth activities have been detected at inflammatory sites, these findings could reflect a role for CSF in inflammatory processes.     

A granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by carrageenin-induced inflammatory cells of mice

Seven days after subcutaneous injection of 2% carrageenin solution in mice, the induced inflammatory tissue was isolated and treated with 0.1% trypsin. When the dispersed cells were incubated in a nutrient medium containing 5--20% calf serum, the cells grew adhering to the culture-dish surface and colony-stimulating factor (CSF) was accumulated in the medium gradually. Addition of lipopolysaccharide (Escherichia coli endotoxin) in the cell culture did not enhance the CSF production. It was shown by isoelectrofocusing that the majority of the produced CSF was an acidic molecule (pI = 3.8), while the treatment of this CSF with neuraminidase yielded a less acidic CSF species (pI = 5.1). Upon gel-filtration chromatography in the presence of 6 M guanidine HCl, the CSF exhibited an apparent molecular weight of 42,000. On the other hand, polyacrylamide gel-electrophoresis in the presence of 0.1% sodium dodecyl sulfate gave a molecular weight estimate of 65,000. Microscopic examination of the bone marrow cell culture showed that about one-third of the colonies were granulocytic. Addition of prostaglandin E₁(PGE₁) in the bone marrow cell culture significantly inhibited the action of the CSF, while prostaglandin D₂ was less inhibitory than PGE₁. The result suggests that the cells isolated from the inflammatory tissue are capable of producing CSF, which may have a role for proliferation and maturation of the mononuclear phagocytes at the site of inflammation.     

Purification of a human urinary colony-stimulating factor

Colony-stimulating factor (CSF), a protein required for the in vitro formation of colonies composed of granulocytes and/or macrophages, was isolated from the urine of anemic patients by using a seven-step procedure. The purified, homogeneous CSF had a specific activity of 1.9 X 10(8) U/absorbance unit at 280 nm (AU). This represents an overall purification of 25,330-fold and a total recovery of 3.8%. Upon iodination of the protein, the radioactivity migrated on sodium dodecyl sulfate (SDS) gel electrophoresis as a single peak with an apparent molecular weight of 46,000; reduction with mercaptoethanol caused dissociation to a single component of molecular weight 23,000. Only the dimer is active in stimulating colony formation. Urinary CSF stimulates formation of colonies comprising only macrophages in the mouse bone marrow cell culture assay. A neutralizing antibody raised against mouse L-cell CSF did not neutralize the activity of the urinary CSF but did bind it. This may indicate that the relative positions of antibody binding sites and the active sites are different in these two glycoproteins.     

T-cell hybridoma secreting hemopoietic regulatory molecules: granulocyte-macrophage and eosinophil colony-stimulating factors.

Murine spleen cells stimulated in vitro with pokeweed mitogen were fused with a HAT-sensitive AKR thymoma (BW5147) to produce T-cell hybridomas secreting hemopoietic colony-stimulating factors (CSFs). A stable cloned T-cell hybridoma has been isolated which expressed the H-2 antigens of both fusion parents, has a median chromosome number of 56 and secretes a factor(s) which stimulates the growth of granulocyte-macrophage and eosinophil colonies. The CSF-secreting hybridoma exhibited only the Thy 1.1 associated with the parent tumor, but no markers normally associated with normal T-cells or macrophages were detected. No CSF was secreted by the parent tumor line, but the hybridoma-conditioned medium, when used at 10% (v/v), contained sufficient CSF to stimulate 10–30 colonies per 10⁵ bone marrow cells. Lipopolysaccharide (1 μg/ml) stimulated the production of CSF by the hybridoma cells 3 fold. CSF production also increased when the cells were held at high density in serum-free medium. The colony-stimulating factor(s) secreted by the hybridoma exhibited similar molecular properties to those produced by pokeweed mitogen-stimulated spleen cells, and both the GM- and EO-CSFs had an apparent molecular weight by gel filtration of approximately 35,000.     

Purification of a colony-stimulating factor from cultured pancreatic carcinoma cells.

Serum-free conditioned medium prepared from an established line of human pancreatic carcinoma (MIA PaCa-2) provides a rich source of colony-stimulating factor (CSF). Two activities distinctly separable by isoelectrofocusing have been identified: a high molecular weight CSF exhibiting greater activity in mouse bone marrow and a low molecular weight CSF more active in human bone marrow. The high molecular weight CSF has been purified 1000-fold to apparent homogeneity by a two-step procedure including isoelectrofocusing and gel filtration chromatography. The purified CSF has a molecular weight of 50,000 and an isoelectric point of 3.7 to 4.6. It is a glycoprotein as shown by periodic acid-Schiff stain and exhibits greater activity in mouse marrow than in human marrow.     

Properties of colony-stimulating factors produced by macrophage cell lines and hybrid cells

Colony-stimulating factors (CSFs) produced by two simian virus 40(SV40) transformed macrophage cell lines (BAM1 and BAM3), and three hybrids (HM3-11, HM3-12, and HM3-14) derived from fusion between BAM3 and a Chinese hamster cell line (hs222-16) were examined. HM3-11 and HM3-14 produce two molecular species of CSF, which are not found in the conditioned media from cultures of BAM1 and BAM3 or lipopolysaccharide (LPS), phorbolmyristate-acetate (PMA), and zymosan-stimulated BAM3. HM3-12, which is classified into another group in terms of CSF secretion, does not produce these two CSFs. On the basis of various criteria, one of these CSF species (peak 1-CSF) was characterized as a macrophage-colony-stimulating factor (M-CSF). The other CSF (peak 2-CSF) induced a group of bone marrow cells in granulocytes and macrophages as well as growth of a mast cell line, IC2. This CSF has an apparent molecular weight of 18,000, estimated by SDS-polyacrylamide gel electrophoresis. Unlike interleukin 3 (IL3) from WEHI-3 cells, the growth factor activity of peak 2-CSF binds to DEAE-Sephacel. Thus, peak 2-CSF is similar to a granulocyte-macrophage colony-stimulating factor (GM-CSF) rather than to IL3. The anti L cell CSF serum does not inhibit the CSF activity in Chinese hamster fibroblast conditioned medium, and the IC2 cells do not respond to Chinese hamster lung conditioned medium (CHLCM), suggesting that peak 1- and peak 2-CSF are of mouse origin.     

Specific interaction of murine colony-stimulating factor with mononuclear phagocytic cells

L-cell colony-stimulating factor (CSF) is identical to macrophage growth factor and stimulates macrophage proliferation (Stanley et al., 1976, J. Exp. Med. 143: 631-647). The nature of the interaction of iodinated L-cell CSF (125I-CSF) with murine peritoneal exudate macrophages was studied. On incubation with 10 pM 125I-CSF at 0 degrees C, cellular binding of 125I-CSF reaches a stable maximum within 15 h. This is in contrast to the association behavior at higher temperatures. At 37 degrees C, cell-associated 125I-CSF levels reach, within 45 min, an unstable maximum which is up to 10-fold less than that occurring under the same conditions at 0 degrees C. At 0 degrees C, binding is saturated (approximately 5 X 10(4) sites/cell) at CSF concentrations of 1 nM. A comparison of binding and competition experiments indicates that iodinated L-cell CSF binds as effectively as L-cell CSF and that human urinary CSF and L-cell CSF equipotently compete for 125I-CSF binding. Specificity of the CSF-binding site is demonstrated by the failure of other known growth factors and hormones to compete for 125I- CSF binding. These studies and other findings suggest that 125I-CSF binding is restricted to macrophages and their precursors and to macrophage cell lines and that the binding site(s) is the receptor mediating the biological action of this CSF.     

Purification and characterization of human granulocyte colony-stimulating factor (G-CSF).

A colony-stimulating factor (CSF) has been purified to homogeneity from the serum-free medium conditioned by one of the human CSF-producing tumor cell lines, CHU-2. The molecule was a hydrophobic glycoprotein (mol. wt 19,000, pI = 6.1 as asialo form) with possible O-linked glycosides. Amino acid sequence determination of the molecule gave a single NH2-terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological activity was apparently specific for a neutrophilic granulocyte-lineage of both human and mouse bone marrow cells with a specific activity of 2.7 X 10(8) colonies/10(5) non-adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G-CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis.     

Identification of distinct receptors for two hemopoietic growth factors (granulocyte colony-stimulating factor and multipotential colony-stimulating factor) by chemical cross-linking

Granulocyte colony-stimulating factor (G-CSF) and multipotential colony-stimulating factor (multi-CSF or interleukin 3) are two members of a family of hemopoietic growth and differentiation factors. Using biologically active radioiodinated derivatives and chemical cross-linking (predominantly with the homobifunctional reagent disuccinimidyl suberate) followed by gel electrophoresis and autoradiography, receptors for these two factors have been identified. The G-CSF receptor was identified as a single subunit protein of Mr approximately 150,000 while two molecular species able to specifically cross-link to 125I-multi-CSF were identified of Mr approximately 75,000 and 60,000. For both CSFs specificity of formation of cross-linked species was demonstrated by showing that the homologous unlabeled CSF (but not other CSFs) competed for formation of the complexes with the appropriate dose-response relation, by showing that saturation occurred over the appropriate range of 125I-CSF concentration and by showing that the cellular specificity of CSF binding paralleled that for cross-linked complex formation. The formation of cross-linked complexes was dependent on the concentration and type of chemical cross-linker, especially for cross-linking of 125I-multi-CSF. Based on a number of criteria it is suggested that the two species cross-linked to 125I-multi-CSF do not represent receptors of different affinity but, rather, two noncovalently associated subunits of a receptor complex.     

Leishmania donovani amastigote components-induced colony-stimulating factors production

Increased hematopoiesis, driven by colony-stimulating factors (CSFs), is known to occur in infectious diseases. However, whether Leishmania donovani component(s) can directly induce the synthesis and secretion of CSFs is not known. We report that L. donovani amastigote antigens soluble in culture medium (LDAA; 0.01-10 mg/kg), injected intravenously in BALB/c mice, induced the production of serum CSFs; maximum induction (128>16 colonies) occurred at 1 mg/kg. In vitro also, LDAA (0.01-1 mg/ml) induced mouse peritoneal macrophages (M?s) to elaborate CSFs in the conditioned medium (CM); 0.1 mg/ml LDAA appeared optimal (68+/-9 colonies). Both in vivo and in vitro, the kinetics of CSF production were similar with peak response occurring 24 h after stimulation and return to background levels by 72 h. A predominant approximately 12 kDa LDAA protein (LDAA-12) also induced CSF production, both in serum and CM, in a dose-and time-dependent manner. Rabbit anti-LDAA-12 antibody significantly (p<0.05) reduced both the LDAA-and LDAA-12-induced CSF production, in vitro. Functionally, the LDAA-12-induced CSFs, both in the serum and CM, appeared to be similar as they supported the formation of granulocyte (G), M? (M) and GM colonies, in vitro, in similar proportion; GM colonies were maximum (>80%). Further, LDAA-12 induced significantly (p<0.05) high GM-CSF levels both in serum and CM (19+/-3 and 15+/-2 ng/ml, respectively), as compared to the controls. Neutralizing (100%) goat anti-mouse tumour necrosis factor-alpha (TNF-alpha) immunoglobulin G did not affect the LDAA-12-induced CSF production by M?s, indicating it to be TNF-alpha-independent. LDAA-12 induced de novo CSF production, as M?s co-treated with LDAA-12 and cycloheximide (50 microg/ml) did not elaborate CSFs. The CSF-inducing capability of LDAA-12 appeared to be heat (70 C; 1 h)-labile, destroyed by proteases (pronase E and trypsin) and was unaffected by sodium periodate treatment. In LDAA-12-treated mice, the splenic and femur colony forming unit-GM counts showed a maximum of 2.2- and 1.9-fold increase, respectively, as compared to the controls. These data are the first to directly demonstrate that L. donovani amastigote components can induce the production of CSFs that may play important role(s) in the pathogenesis of visceral leishmaniasis.     

Human macrophage colony-stimulating factor inhibits bone resorption by osteoclasts disaggregated from rat bone

Colony stimulating factors (CSFs) regulate the survival, proliferation and differentiation of haemopoietic progenitor cells, as well as the functional activity of mature cells. Because the osteoclast is derived from haemopoietic tissue, and because osteoblastic cells produce CSFs, we tested the effects of several CSFs on bone resorption by osteoclasts disaggregated from neonatal rat long bone. We found that recombinant macrophage (M)-CSF was a potent inhibitor of bone resorption, causing significant inhibition at concentrations similar to those required to support the growth of macrophage colonies in agar. Unlike other inhibitors of osteoclastic resorption, M-CSF did not alter cytoplasmic motility in time-lapse recordings, suggesting that M-CSF may inhibit osteoclasts through a different transduction mechanism. None of the remaining cytokines tested (granulocyte-macrophage CSF, interleukin 3, interleukin 6, or interferon γ) influenced bone resorption. M-CSF production may be a mechanism by which osteoblastic cells, which produce M-CSF, may regulate osteoclastic function. Alternatively, inhibition of osteoclastic resorption by a CSF that is responsible for amplification of the macrophage compartment may reflect a close lineage relationship between mononuclear phagocytes, in which M-CSF induces a diversion of lineage resources away from osteoclastic function.     

Leishmania donovani amastigote component-induced colony-stimulating factor production by macrophages: modulation by morphine

The neuroimmunomodulatory effects of opiates during microbial infections are now well known; however, not much is known during leishmaniasis. Here, we report the effects of morphine on purified approximately 12-kDa component of Leishmania donovani amastigote antigen (LDAA-12)-induced colony-stimulating factor (CSF) production by mouse peritoneal macrophages (PMs) in vitro. Low concentrations (1 x 10(-9) and 1 x 10(-11) M) of morphine significantly (P < 0.05) augmented the production of CSFs, whereas high concentrations (1 x 10(-3) and 1 x 10(-5) M) inhibited CSF production. Morphine exerted a similar concentration-dependent biphasic effect on the LDAA-12-induced elaboration of granulocyte (G)-macrophage (M)-CSF (GM-CSF) and M-CSF by PMs in their conditioned medium, as quantified by using enzyme-linked immunosorbent assay. Furthermore, selective agonists of mu-(DAGO) and delta-(DPDPE) opioid receptors also, respectively, augmented and inhibited the production of CSFs. Pretreatment of PMs with naloxone (1 x 10(-5) M) significantly (P < 0.05) blocked the augmenting effect of morphine. In contrast, at 1 x 10(-5) M, naloxone lacked any effect on the inhibitory effect of morphine; however, its 100-fold higher concentration partially blocked it. This study, apparently for the first time, demonstrates that morphine, via surface opioid receptors, biphasically modulates the LDAA-12-induced CSF production by PMs, in vitro. These results thus show the implications of opiate abuse on the outcome of therapeutic interventions in areas where both visceral leishmaniasis and drug abuse are rampant.     

Large-scale preparation and characterization of human colony-stimulating factor

In order to obtain human granulocytic colony-stimulating factor (G-CSF) in large quantities, a large-scale culture system of human G-CSF-producing cells has been established. The cell used for this system was T3M-1, which grew in a monolayered sheet in F-10 synthetic medium supplemented with 10% fetal bovine serum. T3M-1 cells grew in rolling bottles at the velocity of 0.5 r.p.m. with about 22 hr. of population doubling time. When the culture reached confluency, it was incubated in a serum-free medium supplemented with 1% bovine serum albumin. The conditioned medium was harvested every week, concentrated by Amicon PM-10 membrane, and loaded on a Sephadex G-75 column. The molecular weight of G-CSF was estimated at about 30,000. This G-CSF was stable over a pH range of 1.0 to 11.0 at 4°C for 21 hr. The CSF activity was destroyed by either trypsin or chymotrypsin, but resisted to RNase and DNase. A slight decrease in the activity was produced by treatment with neuramidase. G-CSF stimulated granulocytic colony formation of human and mouse marrow cells. By using the roller bottle culture system, we could obtain more than 100 liters of cultured medium in a month, which was able to form about 150,000,000 colonies of human bone marrow cells. The recovery of the human G-CSF activity from gel-filtration column was very high (91.7%), and a large increase of specific activity was obtainable (13.3-fold). This culture system is therefore expected to aid in the large-scale preparation of human G-CSF, thereby facilitating further studies on this granulopoietic factor.     

Induction of colony-stimulating factors by Plasmodium cynomolgi components

Plasmodium cynomolgi total parasite antigens soluble in culture medium (P.c.SA), when injected in monkeys (Macaca mulatta) intravenously, induced the synthesis and secretion of serum colony-stimulating factors (CSFs). In vitro cultured monkey splenic macrophages and blood monocytes, following incubation with P.c.SA, also elaborated CSFs: the splenic macrophages responded more. Peak CSFs levels, both in vivo and in vitro, were attained after 8 hours of P.c.SA stimulation, and thereafter declined to baseline values within 48 hours. CSFs, both in serum and in conditioned medium, induced the formation of macrophage, granulocyte and granulocyte-macrophage colonies in vitro, in the same proportion, indicating that committed progenitor cells responded to CSF from both sources in a similar way. Polymyxin B treatment had no effect on P.c.SA stimulated CSF elaboration by macrophages, suggesting an LPS-independent mechanism of CSF induction. CSF synthesis appeared to be de novo, as cycloheximide treatment of macrophages completely inhibited CSF production. These observations indicate that P. cynomolgi components can induce CSF synthesis.