Matrix immobilization enhances the tissue repair activity of growth factor gene therapy vectors.

Although growth factor proteins display potent tissue repair activities, difficulty in sustaining localized therapeutic concentrations limits their therapeutic activity. We reasoned that enhanced histogenesis might be achieved by combining growth factor genes with biocompatible matrices capable of immobilizing vectors at delivery sites. When delivered to subcutaneously implanted sponges, a platelet-derived growth factor B-encoding adenovirus (AdPDGF-B) formulated in a collagen matrix enhanced granulation tissue deposition 3- to 4-fold (p < or = 0.0002), whereas vectors encoding fibroblast growth factor 2 or vascular endothelial growth factor promoted primarily angiogenic responses. By day 8 posttreatment of ischemic excisional wounds, collagen-formulated AdPDGF-B enhanced granulation tissue and epithelial areas up to 13- and 6-fold (p < 0.009), respectively, and wound closure up to 2-fold (p < 0.05). At longer times, complete healing without excessive scar formation was achieved. Collagen matrices were shown to retain both vector and transgene products within delivery sites, enabling the transduction and stimulation of infiltrating repair cells. Quantitative PCR and RT-PCR demonstrated both vector DNA and transgene mRNA within wound beds as late as 28 days posttreatment. By contrast, aqueous formulations allowed vector seepage from application sites, leading to PDGF-induced hyperplasia in surrounding tissues but not wound beds. Finally, repeated applications of PDGF-BB protein were required for neotissue induction approaching equivalence to a single application of collagen-immobilized AdPDGF-B, confirming the utility of this gene transfer approach. Overall, these studies demonstrate that immobilizing matrices enable the controlled delivery and activity of tissue promoting genes for the effective regeneration of injured tissues.

Fibroblast growth factor 2-retargeted adenoviral vectors exhibit a modified biolocalization pattern and display reduced toxicity relative to native adenoviral vectors.

Targeted vectors provide a number of advantages for systemic and local gene delivery strategies. Several groups have investigated the utility of using various ligands to alter the tropism of adenovirus (Ad) vectors. We have previously demonstrated that fibroblast growth factor (FGF) ligands can specifically target DNA transfection and Ad transduction through high-affinity FGF receptors (FGFRs). FGFRs are overexpressed in abnormally proliferating tissues, such as malignancies. The present studies explore the effects of retargeting with FGF2 on the tissue localization pattern and the systemic toxicity of Ad in mice. Results of semiquantitative PCR analyses indicate that the distribution of FGF2-Ad vector genome sequences after intravenous administration in mice is altered. Markedly lower amounts (10- to 20-fold) of FGF2-Ad localize to the liver when compared with native Ad. This decrease in liver deposition translates into a significant reduction in subsequent toxicity as measured by serum transaminases and histopathology in mice injected with FGF2-AdHSV-thymidine kinase with and without ganciclovir administration. In an intraperitoneal model of ovarian cancer, FGF2-Ad generates increased transgene expression in tumor tissue when compared with Ad. Taken together, these results indicate that the retargeting of Ad with FGF2 results in a more efficient vector system for systemic and regional gene therapy applications, with concomitant lower levels of systemic toxicity.

Fibroblast growth factor 2 retargeted adenovirus has redirected cellular tropism: evidence for reduced toxicity and enhanced antitumor activity in mice.

Adenovirus (Ad) have been used as vectors to deliver genes to a wide variety of tissues. Despite achieving high expression levels in vivo, Ad vectors display normal tissue toxicity, transient expression, and antivector immune responses that limit therapeutic potential. To circumvent these problems, several retargeting strategies to abrogate native tropism and redirect Ad uptake through defined receptors have been attempted. Despite success in cell culture, in vivo results have generally not shown sufficient selectivity for target tissues. We have previously identified (C. K. Goldman et al., Cancer Res., 57: 1447-1451, 1997) the fibroblast growth factor (FGF) ligand and receptor families as conferring sufficient specificity and binding affinity to be useful for targeting DNA in vivo. In the present studies, we retargeted Ad using basic FGF (FGF2) as a targeting ligand. Cellular uptake is redirected through high-affinity FGF receptors (FGFRs) and not the more ubiquitous lower-affinity Ad receptors. Initial in vitro experiments demonstrated a 10- to 100-fold increase in gene expression in numerous FGFR positive (FGFR+) cell lines using FGF2-Ad when compared with Ad. To determine whether increased selectivity could be detected in vivo, FGF2-Ad was administered i.v. to normal mice. FGF2-Ad demonstrates markedly decreased hepatic toxicity and liver transgene expression compared with Ad treatment. Importantly, FGF2-Ad encoding the herpes simplex virus thymidine kinase (TK) gene transduces Ad-resistant FGFR+ tumor cells both ex vivo and in vivo, which results in substantially enhanced survival (180-260%) when the prodrug ganciclovir is administered. Because FGFRs are up-regulated on many types of malignant or injured cells, this broadly useful method to redirect native Ad tropism and to increase the potency of gene expression may offer significant therapeutic advantages.

Prevalent expression of fibroblast growth factor (FGF) receptors and FGF2 in human tumor cell lines.

Basic fibroblast growth factor (FGF2) has potent mitogenic and angiogenic activities that have been implicated in tumor development and malignant progression. The biological effects of FGF2 and other members of the FGF ligand family are mediated by 4 transmembrane tyrosine kinase receptors (FGFRs). To better understand the roles of FGFRs in cancer, the expression of FGF2 and each of the 4 FGFRs was assessed by RNase protection analysis of 60 human tumor cell lines, representing 9 tumor types. Expression of at least one FGFR isoform was detected in 90% and FGF2 mRNA in 35% of the cell lines. Our comprehensive analysis of FGF2 and FGFR expression in human tumor cell lines provides evidence that FGF signaling pathways are active in a majority of human tumor cell lines, and lends support to the development of anti-tumor strategies that target FGFRs.

Targeting tumor cells via EGF receptors: selective toxicity of an HBEGF-toxin fusion protein.

Over-expression of the epidermal growth factor receptor (EGFR) is a hallmark of numerous solid tumors, thus providing a means of selectively targeting therapeutic agents. Heparin-binding epidermal growth factor (HBEGF) binds to EGFRs with high affinity and to heparan sulfate proteoglycans, resulting in increased mitogenic potential compared to other EGF family members. We have investigated the feasibility of using HBEGF to selectively deliver a cytotoxic protein into EGFR-expressing tumor cells. Recombinant fusion proteins consisting of mature human HBEGF fused to the plant ribosome-inactivating protein saporin (SAP) were expressed in Escherichia coli. Purified HBEGF-SAP chimeras inhibited protein synthesis in a cell-free assay and competed with EGF for binding to receptors on intact cells. A construct with a 22-amino-acid flexible linker (L22) between the HBEGF and SAP moieties exhibited an affinity for the EGFR that was comparable to that of HBEGF. The sensitivity to HBEGF-L22-SAP was determined for a variety of human tumor cell lines, including the 60 cell lines comprising the National Cancer Institute Anticancer Drug Screen. HBEGF-L22-SAP was cytotoxic in vitro to a variety of EGFR-bearing cell lines and inhibited growth of EGFR-over-expressing human breast carcinoma cells in vivo. In contrast, the fusion protein had no effect on small-cell lung carcinoma cells, which are EGFR-deficient. Our results demonstrate that fusion proteins composed of HBEGF and SAP exhibit targeting specificity and cytotoxicity that may be of therapeutic value in treating a variety of EGFR-bearing malignancies.

Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality.

Keratinocyte growth factor (KGF) stimulates the proliferation and differentiation of epithelial cells including those of the gastrointestinal tract. Although chemotherapeutics and radiation exposure kill rapidly proliferating tumor cells, rapidly dividing normal cells of the host's gastrointestinal tract are also frequently damaged, leading to the clinical condition broadly termed "mucositis." In this report, recombinant human KGF used as a pretreatment in several mouse models of chemotherapy and/or radiation-induced gastrointestinal injury significantly improved mouse survival. Using multiple-dose 5-fluorouracil, methotrexate, and radiation in combination and total body radiation alone models, KGF increased survival by 55% or greater. In the models that used chemotherapy with or without radiation, KGF significantly ameliorated weight loss after injury and accelerated weight gain during recovery. The basis of these systemic benefits appears to be due in part to the trophic effects of the growth factor on the intestinal epithelium because KGF pretreatment caused an increase in measures of mucosal thickness (villus height and crypt depth) that persisted during the course of 5-fluorouracil chemotherapy. Treatment with KGF also afforded a 3.5-fold improvement in crypt survival in the small intestine, suggesting that KGF also has a direct effect on the crypt stem cells. These data indicate that KGF may be therapeutically useful to lessen the intestinal side effects of current cancer therapy regimens.

Keratinocyte growth factor ameliorates mucosal injury in an experimental model of colitis in rats.

BACKGROUND & AIMS: Keratinocyte growth factor (KGF) is known to enhance tissue repair in the skin; however, its role in the gastrointestinal tract is largely unknown. The aim of this study was to evaluate the effects of exogenous KGF in an experimental model of colitis in rats. METHODS: KGF was administered before or after induction of colitis with 2,4,6-trinitrobenzenesulfonic acid/ethanol. In the first two study groups, KGF (5 mg/kg) was administered intraperitoneally 24 hours and 1 hour before induction of colitis; animals were killed 8 hours (n=10) and 1 week (n=10) after injury. In subsequent study groups, KGF or vehicle treatment was begun 24 hours after the induction of colitis at doses of 5 (n=20), 1 (n=10), and 0.1 (n=10) mg/kg intraperitoneally and continued once daily for 1 week. Colonic tissue samples were evaluated macroscopically and microscopically for mucosal injury and assayed for myeloperoxidase activity. RESULTS: Administration of KGF after but not before induction of colitis significantly ameliorated tissue damage. Macroscopic necrosis and microscopic ulcerations were reduced by 40%-50% at KGF doses of 1 and 5 mg/kg. CONCLUSIONS: Exogenous KGF has a key role in mucosal healing in an experimental model of colitis in rats.

Impaired tumor growth in colony-stimulating factor 1 (CSF-1)-deficient, macrophage-deficient op/op mouse: evidence for a role of CSF-1-dependent macrophages in formation of tumor stroma.

Macrophages have been suggested to play a major role in the immune response to cancer. They have also been suggested to stimulate the formation of tumor stroma and to promote tumor vascularization. The availability of the op/op mouse, which has no endogenous colony-stimulating factor 1 (CSF-1) and which possesses a profound macrophage deficiency, provides a new model to verify these notions. Subcutaneous growth of transplantable Lewis lung cancer (LLC) is markedly impaired in the op/op mice compared with normal littermates. Treatment of tumor-bearing op/op mice with human recombinant CSF-1 corrects this impairment. Histological analysis of tumors grown in op/op and normal mice revealed marked differences. Tumors grown in op/op mice display a decreased mitotic index and pronounced necrosis, particularly hemorrhagic. Moreover, particularly in the op/op tumors, peculiar sinusoid-like abortive vessels (not filled with blood) have been observed. These tumors, in contrast to tumors grown in normal mice, are almost deprived of regular arteries and veins. In contrast to tumors grown in normal mice, they exhibit almost no Sirius red-stained collagenous fibers and Gomori silver-stained reticular fibers. Our data suggest that the CSF-1-dependent macrophage subpopulation missing in op/op mice plays a primary role in supporting tumor stroma formation and tumor vascularization in murine LLC tumors.

Keratinocyte growth factor causes cystic dilation of the mammary glands of mice. Interactions of keratinocyte growth factor, estrogen, and progesterone in vivo.

Keratinocyte growth factor (KGF) is a paracrine mediator of epithelial cell proliferation that has been reported to induce marked proliferation of mammary epithelium in rats. In this study, systemic administration of KGF into naive and oophorectomized mice causes mammary gland proliferation, as evidenced histologically by the appearance of cysts lined by a single layer of epithelium and by hyperplastic epithelium. Whole mount preparations of the mammary glands reveal that the histologically noted cysts are actually ducts that are dilated along much of their length. The histology of the mammary glands of KGF-treated mice is similar to the histology of fibrocystic disease in the human female breast. The response in mice differs significantly from the appearance of the mammary glands in KGF-treated rats in which ductal epithelial proliferation is most prominent. Estrogen and progesterone when administered in combination but not alone cause the development of numerous endbuds in the mouse mammary gland. KGF in estrogen- and progesterone-pretreated mice causes the growth of dilated ducts, hyperplastic epithelium within ducts and endbuds, and a fibrous metamorphosis of periductal adipose tissue. The mammary epithelial hyperplasia caused by KGF is rapidly reversible in both mice and rats after cessation of KGF treatment. The spectrum of KGF-, estrogen-, and progesterone-induced mammary histopathology in mice provides a model for the study of fibrocystic and hyperplastic breast disease.

Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract.

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, was identified as a specific keratinocyte mitogen after isolation from a lung fibroblast line. Recently, recombinant (r)KGF was found to influence proliferation and differentiation patterns of multiple epithelial cell lineages within skin, lung, and the reproductive tract. In the present study, we designed experiments to identify additional target tissues, and focused on the rat gastrointestinal (GI) system, since a putative receptor, K-sam, was originally identified in a gastric carcinoma. Expression of KGF receptor and KGF mRNA was detected within the entire GI tract, suggesting the gut both synthesized and responded to KGF. Therefore, rKGF was administered to adult rats and was found to induce markedly increased proliferation of epithelial cells from the foregut to the colon, and of hepatocytes, one day after systemic treatment. Daily treatment resulted in the marked selective induction of mucin-producing cell lineages throughout the GI tract in a dose-dependent fashion. Other cell lineages were either unaffected (e.g., Paneth cells), or relatively decreased (e.g., parietal cells, enterocytes) in rKGF-treated rats. The direct effect of rKGF was confirmed by demonstrating markedly increased carcinoembryonic antigen production in a human colon carcinoma cell line, LIM1899. Serum levels of albumin were specifically and significantly elevated after daily treatment. These results demonstrate rKGF can induce epithelial cell activation throughout the GI tract and liver. Further, endogenous KGF may be a normal paracrine mediator of growth within the gut.

Keratinocyte growth factor induces pancreatic ductal epithelial proliferation.

Keratinocyte growth factor (KGF) causes a proliferation of pancreatic ductal epithelial cells in adult rats after daily systemic administration for 1 to 2 weeks. Even before the proliferation of intralobular ducts is histologically evident, KGF also induces proliferating cell nuclear antigen expression within the ductal epithelium of intercalated, intralobular, and interlobular ducts. KGF also causes incorporation of 5-bromodeoxyuridine in ductal epithelial cells. Epithelial cell proliferation is histologically most prominent at the level of the intralobular ducts adjacent to and within the islets of Langerhans. Pancreatic ductal proliferation is not histologically apparent in rats sacrificed 7 to 10 days after the cessation of KGF administration. The pancreatic hormones insulin, glucagon, somatostatin, and pancreatic polypeptide are normally distributed within islets that demonstrate intrainsular ductal proliferation. The proliferating ductal epithelium does not show endocrine differentiation as evidenced by the lack of immunoreactivity for pancreatic hormones. KGF is a potent in vivo mitogen for pancreatic ductal epithelial cells.

Antifungal activity of recombinant human macrophage colony-stimulating factor in models of acute and chronic candidiasis in the rat.

Models of acute and chronic candidiasis were developed in Fischer 344 rats to evaluate the therapeutic efficacy of recombinant human macrophage colony-stimulating factor (rhM-CSF) alone and in combination with the antifungal agent fluconazole. In the acute model, rats were challenged by intravenous injection with 2 x 10(6) Candida albicans, approximately 4 times the LD50. Daily subcutaneous (sc) bolus injections of rhM-CSF for 10 days plus a single sc bolus dose of 0.3 mg/kg of fluconazole improved the median survival time from 5 days (32% survival) with fluconazole alone to > 30 days (88% survival) in the rhM-CSF- and fluconazole-treated rats. In the chronic model, daily sc bolus injections of rhM-CSF for 10 days plus a single sc bolus dose of 1.0 mg/kg of fluconazole decreased the median titer of C. albicans cultured from the kidneys by 10-fold at 15 and 30 days after infection. These studies showed that rhM-CSF treatment improved the therapeutic outcome in both the acute and chronic rat model of candidiasis when used with fluconazole, a standard fungistatic agent.

Effect of macrophage-modulating agents on in vivo growth of transplantable Lewis lung cancer in mice.

C57Bl/6 mice, bearing transplantable Lewis lung cancer (non-metastatic subline) implanted either subcutaneously or intraperitoneally were treated with macrophage colony stimulating factor (M-CSF, 10(6) units per mouse, per day for 19 days), Escherichia coli lipopolysaccharide or both. Lipopolysaccharide (5 micrograms per mouse) administered daily once a day for up to 30 days impaired both subcutaneous and intraperitoneal tumor growth and prolonged survival of tumor bearing mice. Macrophage colony stimulating factor, administered daily, inhibited only subcutaneous tumor growth, both when administered alone and in combination with with lipopolysaccharide, and had no effect on intraperitoneal tumor. Moreover, it did not prolong survival of tumor bearing mice, when administered alone, and nullified the effects of lipopolysaccharide when administered concomitantly. These data suggest that macrophage colony stimulating factor, at least in this tumor model and in this dose schedule, offers little benefit. In contrast, the present data confirm earlier suggestions on therapeutic usefulness of bacterial lipopolysaccharide in neoplastic disease, which makes this compound an interesting candidate for future clinical trials.

Antitumor protection from the murine T-cell leukemia/lymphoma EL4 by the continuous subcutaneous coadministration of recombinant macrophage-colony stimulating factor and interleukin-2.

Combined continuous s.c. coadministration of macrophage-colony stimulating factor (M-CSF) plus interleukin-2 (IL-2) by osmotic pump protected mice given i.v. injections of a lethal dose of EL4 T-cell leukemia/lymphoma. Antitumor protection was significantly greater than that afforded by treatment with either cytokine alone. Since neither IL-2 receptors nor M-CSF receptors were expressed on EL4, the antitumor effect was likely attributed to murine effector cells. To determine how M-CSF+IL-2 provided this effect, we performed immunophenotypic and functional analyses as well as in vivo depletion studies of putative antitumor effector cells. Splenic phenotyping experiments revealed that the highest levels of macrophages and natural killer cells were observed in mice given the cytokine combination rather than either M-CSF or IL-2 alone. In vivo depletion of natural killer cells ablated the antitumor protective effect of M-CSF and IL-2. T-cells were also important for M-CSF+IL-2 efficacy, since adult thymectomy/T-cell depletion significantly inhibited the ability of cytokine coadministration to protect against EL4. Coadministration of the 2 cytokines significantly elevated in vivo levels of CD3+CD4+, CD3+CD8+, CD3+NK1.1+ T-cells, and CD3+CD25+ (activated) T-cells, and elevated anti-EL4 cytotoxic T-cell activity measured in vitro. Although WBC counts and fluorescence-activated cell sorter studies showed that M-CSF+IL-2 treatment significantly elevated neutrophils, s.c. delivery of granulocyte-colony stimulating factor at doses sufficient to induce neutrophilia was unable to confer anti-EL4 protection. These studies indicate that macrophages, T-cells, and natural killer cells are all important in the M-CSF+IL-2 anti-EL4 response. The superior antitumor effect of this cytokine combination along with the ability of M-CSF to diminish the toxicity of IL-2 in this model suggests that further investigations into the clinical potential of this combination treatment are warranted.

The skeletal effects of colony-stimulating factor-1 in toothless (osteopetrotic) rats: persistent metaphyseal sclerosis and the failure to restore subepiphyseal osteoclasts.

Toothless (tl), one of four osteopetrotic mutations in the rat, is characterized by few osteoclasts, undetectable bone resorption, and failure of correction by bone marrow transplantation. We recently reported that CSF-1 treatment improves these skeletal problems but that metaphyseal sclerosis persists. In the present study we demonstrate that optimal reduction of the skeletal sclerosis in tl rats following CSF-1 treatment has lower and upper dosage thresholds and that skeletal sclerosis returns after CSF-1 withdrawal. Osteoclasts increase significantly in tl rats after CSF-1 treatment, but compared to untreated normal littermates, histochemical staining for characteristic enzymes and osteoclast number is reduced and no osteoclasts appear in the subepiphyseal areas of long bones. These data are interpreted to mean that there are dosage limits to the beneficial skeletal effects of CSF-1, that persistent sclerosis is related to the failure to restore subepiphyseal osteoclasts, that osteoclast or progenitor populations may be deficient or differ in their responses to CSF-1, and that the defect in tl rats is not merely lack of a circulating, biologically active form of CSF-1.

Macrophage colony-stimulating factor induces indirect angiogenesis in vivo.

The cytokine macrophage colony-stimulating factor was implanted in the rabbit cornea over a wide dose range (1 ng to 100 microg) to assay its angiogenic activity in vivo. Neovascularization occurred in a dose-dependent manner, and maximum angiogenesis occurred only with 100 microg. Histologic analysis revealed that the corneas were free of inflammation at the lower doses, but had slight inflammation at 50 and 100 microg. Nonspecific esterase staining of frozen sections and transmission electron microscopy revealed that the inflammatory cells were predominantly macrophages, with very few neutrophils present. This association of capillary formation with inflammation suggests an indirect mechanism of angiogenesis. The lack of neutrophils within the inflammatory cell infiltrate demonstrates that indirect angiogenesis can proceed without the local presence of neutrophils. This distinguishes macrophage colony-stimulating factor from other indirect-acting angiogenesis factors that have been identified to date.

The effects of colony-stimulating factor-1 on tooth eruption in the toothless (osteopetrotic) rat in relation to the critical periods for bone resorption during tooth eruption.

The toothless (tl) rat is an osteopetrotic mutation characterized by a generalized skeletal sclerosis, reduced bone resorption, few osteoclasts and a total absence of erupted teeth. This mutation is not cured by bone marrow transplants from normal littermates. It is known that the skeletal defects in tl rats are greatly improved after treatment with colony-stimulating factor-1 (CSF-1). This investigation concerns the effects of CSF-1 on the development and eruption of the dentition of tl rats. Untreated tl rats had no erupted teeth by 56 days after birth, and the roots of incisors and molars were severely distorted by compression against bone. The apex of the mandibular incisor did not extend past the first molar and continued growth of its apical end produced odontoma-like masses consisting of distorted dentine and enamel matrices. In addition, few osteoclasts were seen on alveolar bone surfaces surrounding the developing teeth. Mutants given CSF-1 were characterized by delayed eruption of all molars and sometimes incisors. The incidence of incisor eruption was related inversely to the age at which CSF-1 treatment began. Molars of treated tl rats had well-developed roots similar to those in normal rats. Treated mutants had numerous osteoclasts in alveolar bone and well-developed haemopoietic marrow spaces in the mandible. Histochemical staining for both tartrate-resistant acid phosphatase and tartrate-resistant acid ATPase was reduced or negligible in osteoclasts of untreated tl rats, heavy in normal osteoclasts and of intermediate intensity in CSF-1-treated mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of recombinant human macrophage colony-stimulating factor in irradiated murine recipients of T-cell-depleted allogeneic or non-depleted syngeneic bone marrow transplants.

Recombinant macrophage colony-stimulating factor (rM-CSF), which reacts exclusively with cells of monocyte lineage, was evaluated in the murine bone marrow (BM) transplant setting for in vivo effects on recipient survival, hematologic recovery, and engraftment. Two types of fully allogeneic donors were selected based on the expression (BALB/c), or lack of expression (DBA/1), of hybrid hematopoietic histocompatibility (Hh1) antigens. These antigens are established targets for monocyte and/or natural killer (NK) cell-mediated graft rejection. Irradiated C57BL/6 mice were used as recipients for all experiments. Recipients of T-cell-depleted (TCD) BALB/c BM and a 14-day continuous subcutaneous infusion of 16.8 micrograms/d rM-CSF (n = 30) showed a significant decrease in donor cell engraftment as compared with recipients of donor BM administered pumps delivering saline. These mice administered rM-CSF also displayed significantly reduced levels of circulating leukocytes (predominantly lymphocytes) on day 14 posttransplant (compared with saline controls). Neither engraftment effects nor leukocyte effects were observed when C57BL/6 recipients were administered Hh1 nonexpressing TCD DBA/1 BM cells (n = 30), suggesting that the monocyte/macrophage population is important in long-term alloengraftment in certain donor-recipient strain combinations in which donor Hh1 antigens can serve as target antigens for host effector cells, but are not important in strain combinations in which they are not recognized. Circulating tumor necrosis factor alpha (TNF alpha) levels measured at two time periods during rM-CSF infusion were not elevated. Thus, the reduction in alloengraftment is not likely to be directly related to TNF alpha. However, in vivo elimination of NK cells in the BALB/c into C57BL/6 model prevented the impairment of engraftment mediated by rM-CSF. Thus, rM-CSF-mediated inhibition of alloengraftment is contingent on the presence of host NK cells with antidonor reactivity. Survival was unaffected when rM-CSF was administered in either allogeneic BM transplant model, but was significantly reduced when rM-CSF was administered to C57BL/6 recipients of syngeneic BM transplants. These data are the first analyzing the effects of rM-CSF in murine allogeneic BM transplantation and extend our previous studies using the BALB/c into C57BL/6 model in which in vivo infusions of recombinant granulocyte-macrophage CSF, but not recombinant granulocyte-CSF, lead to decreases in alloengraftment. These data show that rM-CSF-induced stimulation of monocytes may increase BM graft rejection in instances in which NK cells are involved in the rejection process. These data may have future clinical implications for the use of rM-CSF in allogeneic BM transplantation.

Administration of colony stimulating factor-1 corrects some macrophage, dental, and skeletal defects in an osteopetrotic mutation (toothless, tl) in the rat.

The toothless (tl/tl) mutation in the rat results in a paucity of osteoclasts and osteopetrosis that cannot be corrected by bone marrow transplantation. In the present study we demonstrate that tl/tl rats also have profound deficiencies of femoral, peritoneal, and pleural cavity macrophages. Furthermore, the macrophage colony stimulating activity of post-endotoxin sera from tl/tl rats is substantially reduced, suggesting that, as in the case of the op mutation in mice, the basis of the tl mutation is a deficiency of the macrophage growth factor, colony stimulating factor-1 (CSF-1). Consistent with this suggestion, treatment of tl/tl rats from birth for up to six weeks with CSF-1 reduced the osteopetrosis, increased body weight, and permitted tooth eruption. In addition, CSF-1 treatment induced large numbers of osteoclasts in tl/tl bones and macrophages in the peritoneal cavity and bone marrow. Persistence of metaphyseal sclerosis, however, indicated that the disease was not totally corrected by this treatment. These studies indicate that the basis of the tl mutation is most likely another CSF-1 deficiency, and further emphasize the role of this growth factor in osteoclast differentiation.

Circulating macrophage colony-stimulating factor is not reduced in malignant osteopetrosis.

Malignant osteopetrosis is a disorder characterized by a deficiency in osteoclast number or function. In one animal model of osteopetrosis, the op/op mouse, macrophage colony-stimulating factor (M-CSF) is absent, and the administration of M-CSF corrects the defects. We evaluated the serum of 13 patients with malignant osteopetrosis by an M-CSF radioimmunoassay to determine if a quantitative M-CSF deficiency existed in these patients. All patients had M-CSF present in levels equal to or higher than control serum. In addition, serum from 6 osteopetrotic patients was tested in a bioassay to determine if the M-CSF present is biologically active, and in all cases there was demonstrable activity in these samples. We provide evidence that deficiency of circulating M-CSF is unlikely to be a major contributor to the etiologic basis for the majority of children with malignant osteopetrosis.