Human Macrophage Colony-stimulating Factor Research Articles

Immunoreactive macrophage colony-stimulating factor is increased in atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits after recombinant human macrophage colony-stimulating factor therapy.

Infiltration of monocytes into arteries is an early event in the pathogenesis of atherosclerosis. This recruitment is interpreted as enhancing lesion development, but it could also be a host response limiting lipid accumulation. The ability of macrophages to limit cholesterol uptake, however, can be reduced by the impaired mobility and metabolic activity associated with foam cell development. As lesions enlarge, foam cells die and become the nidus for the necrotic core. Treatments to improve viability might improve foam cell function and promote regression. Macrophage colony-stimulating factor (M-CSF) is vital to monocyte/macrophage differentiation, proliferation, and activation. We found that foam cells of Watanabe heritable hyperlipidemic (WHHL) rabbits had faint staining for M-CSF. Treatment of rabbits with recombinant human M-CSF (rhM-CSF) increased M-CSF staining, which correlated with reduced cholesterol content of these foam cells.

Protective effect of mucosal administration of recombinant human macrophage colony-stimulating factor (rhM-CSF) on mucosal infection of Sendai virus in mice

We investigated the protection confered by the mucosal administration of recombinant human macrophage colony-stimulating factor (rhM-CSF) against mucosal infection of Sendai virus in mice. In an experimental infection model using Sendai virus, an intranasal (i.n.) administration of rhM-CSF (20 μg per mouse) 2 days before infection induced significant protection against a lethal infection of this virus. Also, its antiviral activity was dependent upon the dose of rhM-CSF. However, a subcutaneous (s.c.) administration of rhM-CSF with an effective dose (20 μg per mouse) i.n. did not confer protection. In a time course analysis of virus growth in the lungs, mice given rhM-CSF i.n. significantly inhibited the early period of infection, compared with the untreated mice. Moreover, the level of interferon-γ (IFN-γ) in lung wash fluids from the rhM-CSF-treated mice was higher than that of the untreated mice. These results suggested that the mucosal (i.n.), but not the systemic (s.c.) administration of rhM-CSF augments host resistance against mucosal infection with Sendai virus, and that its prophylactic activity is related to growth inhibition of the virus and enhanced IFN-γ secretion in the lungs.

Synthesis and secretion of macrophage colony stimulating factor by mature human monocytes and human monocytic THP-1 cells induced by human serum albumin derivatives modified with methylglyoxal and glucose-derived advanced glycation endproducts

Human serum albumin minimally-modified by methylglyoxal (MG min-HSA) stimulated the synthesis and secretion of macrophage-colony stimulating factor (M-CSF) by mature human monocytes in vitro. Human serum albumin minimally-modified by glucose-derived advanced glycation endproducts (AGE min-HSA) and human serum albumin highly-modified by glycose-derived advanced glycation endproducts (AGE-HSA) stimulated much lower secretion of M-CSF from human monocytes than did MG min-HSA. MG min-HSA and AGE-HSA but not AGE min-HSA also stimulated the growth of human monocytic THP-1 cells in vitro which was inhibited by polyclonal antibodies to human M-CSF. For MG min-HSA, the median growth stimulatory concentration EC 50 value was 0.24 ± 0.07 μM and the maximal increase in cell growth was 36% of control cell growth ( n = 24). Similar induction of secretion of M-CSF from monocytes in vivo may contribute to atherosclerosis in macro- and micro-angiopathy, particularly in the development of diabetic complications.

Recombinant Human Macrophage Colony-Stimulating Factor in Nonhuman Primates: Selective Expansion of a CD16+ Monocyte Subset With Phenotypic Similarity to Primate Natural Killer Cells

The CD16 receptor (Fc gamma R-III) is found on many tissue macrophages (M phi s), but its expression on circulating monocytes is restricted to a small, phenotypically distinct subset. The number of these CD16+ monocytes may be markedly increased in response to sepsis, human immunodeficiency virus infection, or metastatic malignancy. We have recently shown that the CD16+ monocyte population is selectively expanded by administration of recombinant human macrophage colony-stimulating factor (rhM-CSF). In the current study, we used the highly rhM-CSF-responsive cynomolgus primate model to further characterize this novel monocyte population. Animals treated with rhM-CSF underwent a progressive and essentially complete conversion to the CD16+ monocyte phenotype, with up to a 50-fold increase in the number of CD16+ cells. This increase was paralleled by the emergence of a population of circulating cells that morphologically resembled large granular lymphocytes (LGLs). However, quantitatively, this population corresponded closely to the number of CD16+ monocytes, and fluorescence-activated cell sorting (FACS) confirmed that they were the same. In addition to their LGL-like morphology, many rhM-CSF-induced CD16+ monocytes showed a pattern of size, granularity, and quantitative cell surface marker expression that closely resembled the pretreatment LGL/natural killer (NK) cell population but that did not resemble the pretreatment monocyte population. However, rhM-CSF-induced CD16+ monocytes could be distinguished from LGL/ NK cells by fact that they all expressed cell surface receptors for rhM-CSF, and many of them showed reduced but detectable phagocytic and respiratory burst activity. Studies of human subjects treated with rhM-CSF also showed an analogous population of "LGL-appearing" CD16+ mononuclear cells. Thus, our studies reveal a previously unsuspected ability of cells in the monocyte lineage to adopt a phenotype similar to that of LGL/NK cells. The extent of this phenotypic convergence suggests that the two lineages retain access to elements of a similar developmental pathway.

Structural Requirements for the Ectodomain Cleavage of Human Cell Surface Macrophage Colony-stimulating Factor

One form of human macrophage colony-stimulating factor (CSF-1(256), M-CSFalpha) is a member of a restricted set of cell surface transmembrane proteins, which is selected to undergo proteolytic ectodomain cleavage. To determine the substrate requirements for this cleavage, we have constructed a series of mutations in the cytoplasmic tail, transmembrane domain, and juxtamembrane region of CSF-1(256) and stably expressed the mutated genes in NIH 3T3 cells. Our results demonstrate that membrane association of the CSF-1 precursor is required for cleavage of its growth factor ectodomain and furthermore that the juxtamembrane region Pro161-Gln162-Leu163-Gln164-Glu165 (PQLQE) (residues 161-165 of the ectodomain) is an essential determinant of cell surface CSF-1(256) cleavage and that the cleavage site is partially sequence-specific. Furthermore, a mechanism of steric hindrance, which likely involves interference with protease accessibility, is postulated to explain the observed decreases in the cleavage efficiency in certain CSF-1 mutants. Finally, our results strongly suggest that the CSF-1 ectodomain is cleaved at or very near the cell surface by a membrane-associated proteolytic system.

Role of cytokines in leukemic type growth of myelodysplastic CD34+ cells

The clonal growth of progenitor cells from myelodysplastic syndromes (MDS) can be subdivided into four growth patterns: (1) normal, (2) no growth or low plating efficiency, (3) low colony and high cluster number, and (4) normal or high colony number with a large number of clusters. The former two (1 and 2) can be referred to as nonleukemic patterns and latter two (3 and 4) as leukemic. In a search for a role for cytokines in leukemic-type growth of MDS progenitor cells, marrow CD34+ cells were purified up to 94% for 8 normal individuals and 88% for 12 MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). The purified CD34+ cells were cultured for 14 days with various combinations of cytokines, including recombinant human macrophage colony-stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte-macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3), and stem cell factor (SCF; a ligand for c-kit) in serum-free medium. The clonal growth of MDS CD34+ cells supported by a combination of all of the above cytokines was subdivided into the two patterns of leukemic or nonleukemic, and then the role of individual or combined cytokines in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. Evidence we obtained showed that SCF plays a central role in the leukemic-type growth of MDS CD34+ cells and that G-CSF, GM-CSF; and/or IL-3 synergize with SCF to increase undifferentiated blast cell colonies and clusters over that seen in normal CD34+ cells. SCF is present in either normal or MDS plasma at a level of nanograms per milliliter, and this physiologic concentration of SCF can stimulate progenitor cells. This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blast cells. This depends, at least in part, on cytokines such as G-CSF, GM-CSF, IL-3, and SCF.

Role of cytokines in leukemic type growth of myelodysplastic CD34+ cells

The clonal growth of progenitor cells from myelodysplastic syndromes (MDS) can be subdivided into four growth patterns: (1) normal, (2) no growth or low plating efficiency, (3) low colony and high cluster number, and (4) normal or high colony number with a large number of clusters. The former two (1 and 2) can be referred to as nonleukemic patterns and latter two (3 and 4) as leukemic. In a search for a role for cytokines in leukemic-type growth of MDS progenitor cells, marrow CD34+ cells were purified up to 94% for 8 normal individuals and 88% for 12 MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). The purified CD34+ cells were cultured for 14 days with various combinations of cytokines, including recombinant human macrophage colony-stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte-macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3), and stem cell factor (SCF; a ligand for c-kit) in serum-free medium. The clonal growth of MDS CD34+ cells supported by a combination of all of the above cytokines was subdivided into the two patterns of leukemic or nonleukemic, and then the role of individual or combined cytokines in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. Evidence we obtained showed that SCF plays a central role in the leukemic-type growth of MDS CD34+ cells and that G-CSF, GM-CSF; and/or IL-3 synergize with SCF to increase undifferentiated blast cell colonies and clusters over that seen in normal CD34+ cells. SCF is present in either normal or MDS plasma at a level of nanograms per milliliter, and this physiologic concentration of SCF can stimulate progenitor cells. This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blast cells. This depends, at least in part, on cytokines such as G-CSF, GM-CSF, IL-3, and SCF.

Recombinant Human Macrophage-Colony Stimulating Factor Suppresses the Mouse Mixed Lymphocyte Reaction

We examined the effect of recombinant human macrophage-colony stimulating factor (rhM-CSF) on mouse macrophage accessory functionsin vitro.The addition of rhM-CSF to an allogenic mixed lymphocyte culture (MLC) consisting of mouse spleen cells suppressed the proliferation of lymphocytes in a concentration-dependent manner. However, rhM-CSF did not suppress the mixed lymphocyte reaction (MLR) on the MLC that contained only purified T and dendritic cells. This suggested that the suppressive effect of rhM-CSF on the MLR occurs via spleen macrophages. Suppression of the MLR by rhM-CSF could not be neutralized by the addition of synthetic inhibitors of prostaglandin E2(indomethacin) and nitric oxide (NG-monomethyl-L-arginine). An antibody specific to mouse interleukin-10 (IL-10), which can neutralize the inhibitory effect of recombinant IL-10in vitro,did not prevent rhM-CSF-induced suppression of the MLR. Even higher concentrations of IL-1 could not overcome the inhibitory effect of rhM-CSF. Moreover, culture supernatants of macrophages stimulated with rhM-CSF had a suppressive effect on the MLR. The concentrations of transforming growth factor β (TGF-β) and IL-10 in the suppressive culture supernatants were lower than those in the supernatants of nonstimulated macrophages. The supernatants suppressed the proliferation of the LBRM 33 TG6 mouse T cell line, but it did not affect the proliferation of other cell lines (L1210, L5178Y-R, and MC/9). These results suggest that rhM-CSF modulates a macrophage accessory function by stimulating macrophages to secrete a suppressive factor that causes suppression of the T cell responsein vitro.

Inhibition of apoptosis in polymorphonuclear neutrophils from burn patients.

Normal human polymorphonuclear neutrophils (PMN) undergo rapid apoptosis during in vitro culture. In contrast, apoptosis is inhibited in PMN from patients with severe burns. This inhibition is not an inherent property of the cells but is caused by thermolabile factors present in the plasma. Endotoxin and the proinflammatory cytokines interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) do not appear to be directly responsible. The ability of burn plasma to inhibit apoptosis was reduced by neutralizing antibodies to human granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF levels could not be detected in the burn plasma. However, the incubation of burn-derived or normal leukocyte populations consisting primarily of PMN in burn plasma induced the production of GM-CSF. The results suggest that activation of GM-CSF synthesis by factor(s) in burn plasma may play a role in regulating inflammation by the inhibition of apoptosis.

Effect of recombinant human macrophage-colony stimulating factor on marrow, splenic, and peripheral hematopoietic progenitor cells in mice.

The effects of human macrophage colony-stimulating factor (M-CSF) on marrow, splenic, and peripheral progenitor cells (CFU-M, CFU-GM, and CFU-G) were investigated in mice administered recombinant human M-CSF (8-4,000 micrograms/kg). Single injection of 4,000 micrograms/kg of M-CSF resulted in a decrease in the number of marrow progenitor cells (CFU-M, CFU-GM, and CFU-G) on day 2 followed by a gradual increase, returning to the original level on day 4 or 5. In contrast, each type of splenic progenitors tested for started to increase markedly on day 2, reaching a level 4- to 15-fold higher than that of the basal value on day 3 or 4. Peripheral CFU-M, CFU-GM, and CFU-G also increased on day 2. In addition, administration of 800 micrograms/kg of M-CSF in mice caused a decrease in marrow CFU-G, as well as an increase in splenic CFU-G. The present results indicate that treatments of mice with pharmacological concentrations of human M-CSF affect the number of progenitor cells not only of monocyte/macrophage lineage but also of granulocyte lineage. Also, the coincidence between decrease of marrow progenitor cells and increase of splenic and peripheral progenitor cells suggests that the progenitor cells are released from bone marrow to peripheral blood and reseeded to the spleen by the action of M-CSF.

Selective biochemical modification of functional residues in recombinant human macrophage colony-stimulating factor beta (rhM-CSF beta): identification by mass spectrometry.

A rapid method combining classical chemical modification with mass spectrometry was developed to identify amino acids in the recombinant human macrophage colony-stimulating factor (rhM-CSF) protein of potential import to the ligand-receptor interaction. Diethyl pyrocarbonate modification of rhM-CSF beta (under nondenaturing conditions) results in a time- and concentration-dependent loss in receptor binding and biological activity. Peptide mapping of the reaction products by mass spectrometry showed that, with low DEP:M-CSF ratios (< 50:1), there was selective modification of histidine residues, whereas at higher ratios (> 50:1), Tyr and Lys residues were also modified. The loss in rhM-CSF beta activity was directly correlated with the extent of carbethoxylation of His9 and His15, as determined by matrix-assisted laser desorption/ionization mass spectrometric molecular weight determinations (MALDIMS). For these residues mono-modification was observed. By contrast, C-terminal histidine residues His176 and His210 showed bis-modifications, the extent of which had no correlation to losses in biological activity. These data suggest the importance of residues in the A-helix (His9 and His15) to ligand-receptor binding.

M-CSF併用cytosine arabinoside少量療法が著効した骨髄異形成症候群の急性白血病移行例：白血病細胞のM-CSFによる増殖と細胞内蛋白チロシンリン酸化

M-CSF併用cytosine arabinoside少量療法が著効した骨髄異形成症候群の急性白血病移行例：白血病細胞のM-CSFによる増殖と細胞内蛋白チロシンリン酸化

Transcriptional activation of the macrophage-colony stimulating factor gene by minimally modified LDL. Involvement of nuclear factor-kappa B.

Minimally modified LDL (MM-LDL), obtained by mild iron oxidation or prolonged storage at 4 degrees C, has been shown to induce the expression of macrophage-colony stimulating factor (M-CSF) in cultured aortic endothelial cells. To examine whether other cell types also respond to MM-LDL, we investigated its effect on the expression of M-CSF mRNA in mouse L-cells and human aortic smooth muscle cells. Both L-cells and human aortic smooth muscle cells showed increased levels of M-CSF mRNA in response to 10 to 200 micrograms/mL MM-LDL in a dose-dependent manner. This allowed us to use mouse L-cells as a model to study the mechanism involved in MM-LDL-mediated increase in M-CSF mRNA. Nuclear runon assays showed that M-CSF gene transcription was activated by MM-LDL. In the present study, we identified specific elements that conferred MM-LDL-mediated transcriptional activation of the human M-CSF gene. Chimeric constructs containing sequential deletions in the 5'-promoter region of the M-CSF gene linked to a reporter chloramphenicol acetyltransferase (CAT) gene were transfected into mouse L-cells. The human M-CSF promoter region extending upstream from the transcription start site to nucleotide -406 showed maximum induction of CAT activity by MM-LDL. Induction of CAT activity was drastically reduced, with a deletion plasmid lacking the promoter region -406 to -344. A functional nuclear factor (NF)-kappa B binding site present in this critical region was required for MM-LDL-mediated induction of CAT activity since an internal deletion construct lacking this element showed significant loss of transcriptional activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Macrophage Colony-Stimulating Factor on Antifungal Activity of Mononuclear Phagocytes against Aspergillus fumigatus

The effects of recombinant human macrophage colony-stimulating factor (M-CSF) on antifungal activity of human monocytes (MNC), MNC-derived macrophages (MDM), and rabbit pulmonary alveolar macrophages (PAM) against Aspergillus fumigatus were studied. MNC-induced hyphal damage was augmented by incubation with M-CSF (P = .027); PAM-induced hyphal damage was moderately enhanced by M-CSF (P = .046). Phagocytosis of Aspergillus conidia by MDM and PAM was strongly enhanced by M-CSF (P < .01). MNC pretreated with M-CSF exhibited enhanced superoxide anion production in response to PMA (P = .026). This effect was not associated with increased levels of mRNA transcripts of the components of NADPH oxidase, the enzyme responsible for superoxide anion production. M-CSF augments antifungal activity of mononuclear phagocytes against both conidia and hyphae of Aspergillus fumigatus partly by enhancement of oxidation-dependent mechanisms and may have an important immunomodulatory role in prevention and treatment of invasive aspergillosis in leukopenic patients.

Characterization of disulfide linkages and disulfide bond scrambling in recombinant human macrophage colony stimulating factor by fast-atom bombardment mass spectrometry of enzymatic digests

Fast-atom bombardment mass spectrometry was used to study disulfide bonding patterns in heat-denatured human recombinant macrophage colony stimulating factor (rhM-CSF). The heat-denaturated protein was studied by analysis of the pattern of peptides in the proteolytic digests. Native rhM-CSF is a homodimer with intramolecular disulfide linkages between Cys7–Cys90, Cys48–Cys139, and Cys102–Cys146 and intermolecular linkages between Cys31-Cys31, and the pairs Cys157 and Cys159. Brief heating for 1 min leads to partial disulfide bond scrambling. In addition to the native disulfide bonds between Cys7–Cys90, Cys48–Cys139, and Cys31-Cys31, nonnative disulfide bonds were detected between Cys48–Cys90 and Cys48–Cys102. When heated for 5 min the disulfide bonds of rhM-CSF are completely scrambled and lead to nonnative intramolecular disulfide bonds between Cys48–Cys102 and Cys90–Cys102 and one intermolecular disulfide bond between Cys102–Cys102.

Characterization of disulfide linkages and disulfide bond scrambling in recombinant human macrophage colony stimulating factor by fast-atom bombardment mass spectrometry of enzymatic digests

Fast-atom bombardment mass spectrometry was used to study disulfide bonding patterns in heat-denatured human recombinant macrophage colony stimulating factor (rhM-CSF). The heat-denaturated protein was studied by analysis of the pattern of peptides in the proteolytic digests. Native rhM-CSF is a homodimer with intramolecular disulfide linkages between Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146 and intermolecular linkages between Cys31-Cys31, and the pairs Cys157 and Cys159. Brief heating for 1 min leads to partial disulfide bond scrambling. In addition to the native disulfide bonds between Cys7-Cys90, Cys48-Cys139, and Cys31-Cys31, nonnative disulfide bonds were detected between Cys48-Cys90 and Cys48-Cys102. When heated for 5 min the disulfide bonds of rhM-CSF are completely scrambled and lead to nonnative intramolecular disulfide bonds between Cys48-Cys102 and Cys90-Cys102 and one intermolecular disulfide bond between Cys102-Cys102.

Macrophage colony-stimulating factor enhancement of antibody-dependent cellular cytotoxicity against human colon carcinoma cells

Antibody-dependent cellular cytotoxicity (ADCC) has been suggested to be an important defense mechanism against tumors. The effects of recombinant human macrophage colony-stimulating factor (rhM-CSF) on ADCC activity of human monocytes were investigated. Human peripheral monocytes were pre-incubated for 72 h with rhM-CSF at various concentrations (50, 100, 200, 400 U/ml) and then used as effector cells in a 24-h 111-Indium release assay. Human carcinoma cell lines LS-174T, CBS, and KLE were used as targets to react with anti-carcinoma monoclonal antibodies (mAbs: murine D612, murine CC49, and chimeric CC49). A significant increase in ADCC activity was observed after monocytes were incubated in 100–400 U/ml of human rhM-CSF. Variation in ADCC activity of monocytes among donors was observed. The enhancement of ADCC activity was blocked by the addition of a neutralizing antibody to rhM-CSF. Less D612 mAb was required for the rhM-CSF-treated monocytes to mediate an equivalent level of ADCC activity as compared to the untreated monocytes. Because of the low levels of rhM-CSF required in these studies to enhance ADCC, treatment of monocytes alone with comparable levels of rhM-CSF did not enhance antibody-independent cytotoxicity. Moreover, it is demonstrated here that recombinant human interleuken-4 (rhIL-4) and rhM-CSF can have a synergistic effect of monocyte-mediated ADCC on human tumor cells. These results thus indicate that rhM-CSF augments ADCC of human peripheral blood monocytes using mAbs to human carcinomas, suggesting a potential role for rhM-CSF in cancer immunotherapy.

Effects of macrophage colony-stimulating factor on folliculogenesis in gonadotrophin-primed immature rats.

The effect of macrophage colony-stimulating factor (M-CSF) on folliculogenesis and ovulation was studied. Folliculogenesis and ovulation were induced in immature female rats with a s.c. injection of equine chorionic gonadotrophin (eCG), followed 48 h later by human chorionic gonadotrophin (hCG). The ovulation rate was measured after the following treatments. (1) Graded doses of human M-CSF (1-300 x 10(3) iu per rat) were administered i.p. daily for 3 consecutive days. (2) M-CSF (100 x 10(3) iu) was administered i.p. at designated times between 96 h before and 10 h after hCG injection. (3) Rabbit anti-human M-CSF polyclonal antibody (5 micrograms) was administered into the left ovarian bursa at designated times between 49 h before and 10 h after hCG injection. In addition, the effect of M-CSF on ovarian macrophages was investigated using immunohistochemistry with mouse anti-rat macrophage monoclonal antibody, TRPM-3. The treatment with M-CSF (> 30 x 10(3) iu per rat) significantly increased the ovulation compared with controls in a dose-dependent manner. This stimulatory effect of M-CSF was observed when it was administered between 96 h and 49 h before hCG injection. The ovarian intrabursal administration of anti-M-CSF antibody significantly inhibited the number of ovulated ova from the treated ovaries compared with either those from control rats or from the contralateral untreated ovaries between 24 h before and 3 h after hCG injection. The immunohistochemistry revealed that M-CSF increased the number of ovarian macrophages in growing follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage Colony-Stimulating Factor Complementary DNA: a Candidate for Gene Therapy in Metastatic Melanoma

At present, there is no highly effective treatment for metastatic melanoma. Innovative approaches aimed at inducing a more effective immune response against tumors have shown promising results in animal models. One approach involves the genetic modification of tumor cells so that they produce cytokines that stimulate an immune response. The aim of this study was to determine the effectiveness of cytokine gene therapy for metastatic melanoma in a murine melanoma model. B16F10 murine melanoma cells, which readily metastasize to the lungs, were transduced with a retroviral vector containing genes encoding neomycin resistance and human macrophage colony-stimulating factor (M-CSF). The presence of M-CSF messenger RNA in transduced cells was examined by coupled reverse transcription and polymerase chain reaction. Concentrations of soluble M-CSF in cell culture supernatants were determined by enzyme-linked immunosorbent assays (ELISAs). A clonal cell line, designated N+/CSF+, that expressed and secreted M-CSF was identified. Another clonal cell line, designated N+/CSF-, did not secrete M-CSF at levels detectable by ELISA. B16F10, N+/CSF-, and N+/CSF+ cells, individually or in combination, were injected intravenously or subcutaneously into C57BL/6 mice; we then evaluated the tumorigenicity and metastatic behavior of the cells, as well as the immune responses and survival of the mice. The immune responses assayed were the cytotoxic T lymphocyte (CTL) and peritoneal exudate cell (PEC) tumoricidal activities. Injection of B16F10 cells into the tail vein of C57BL/6 mice led to the establishment of lung metastases by week 2 and death by week 8. Injection of the N+/CSF+ or N+/CSF- cells led to the establishment of lung metastases that were detected at 2 and 3 weeks, respectively; however, these metastatic lesions were eliminated, and the animals had survival rates similar to those of the noninjected control mice. Injection of mice with a mixture of B16F10 and N+/CSF- cells resulted in the development of metastatic disease and 0% survival at 8 weeks, whereas mice that had been given an injection of a mixture of B16F10 and N+/CSF+ cells had an 80% survival rate at 8 weeks and survived at least two times longer (P = .007). The CTL and PEC tumoricidal activities in animals given an injection of N+/CSF+ cells suggested that monocytes and lymphocytes were responsible for the observed antitumor response. These findings suggest that the expression of M-CSF by genetically modified melanoma cells caused an effective antitumor immune response in host C57BL/6 mice and, thus, prolonged survival over that observed in the control mice.

CD16+ Monocytes in Patients With Cancer: Spontaneous Elevation and Pharmacologic Induction by Recombinant Human Macrophage Colony-Stimulating Factor

The small subset of circulating monocytes that express the maturation-associated CD16 antigen has recently been reported to be elevated in patients with bacterial sepsis. We now show that this novel CD16+ monocyte population is also spontaneously expanded in patients with cancer. We studied 14 patients with metastatic gastrointestinal carcinoma enrolled ina clinical trial of recombinant human macrophage colony-stimulating factor (rhMCSF) plus monoclonal antibody D612. We found that before any cytokine treatment, 12 of 14 patients constitutively displayed significant elevations in both the percentage and the absolute number of CD16+ monocytes, as compared with both normal subjects and ill patients with elevated monocyte counts but without malignancy. CD16+ monocytes accounted for 46% +/- 22% of total monocytes in the patients with cancer versus 5% +/- 3% for controls (P < .01). The increase was not attributable to infection or intercurrent illness and appeared to be associated with the underlying malignancy itself. A similar spontaneous elevation of CD16+ monocytes was observed in 35 of 44 additional patients diagnosed with a variety of other solid tumors. When patients with gastrointestinal carcinoma were treated with rhMCSF, there was a marked further increase in the percentage of CD16+ monocytes (to 83% +/- 11%), as well as in the absolute number of CD16+ cells and the level of CD16 antigen expression. In every case, the patients with cancer showed a greater CD16+ monocyte response than the maximal response obtained in normal volunteer subjects treated witha similar regimen of rhMCSF (n = 5, P < .001), suggesting that the presence of malignancy primed patients for enhanced responsiveness to rhMCSF. We hypothesize that spontaneous expansion of the CD16+ monocyte population may represent a novel biologic marker for a widespread and previously unsuspected host immune response to malignancy.