The AMPK pathway triggers autophagy during CSF1-induced microglial activation and may be implicated in inducing neuropathic pain.

The development and maintenance of neuropathic pain is now given far more attention in the clinic work. Increasing evidence has shown that colony-stimulating factor 1 (CSF1) is involved in microglial activation and may further induce pain. Here, we observed the signaling events that link the CSF1-induced microglial activated and consequences for pain processing. For the in vitro study, flow cytometry showed the microglial activity was markedly increased after CSF1 stimulation. Western blot showed the increased expression of p-PRKAA1/PRKAA1, p-AMPK/AMPK, p-ULK1/ULK1, p-S6k/S6k and LC3-II/LC3-I. QRT-PCR showed the IL-1, TNF-α and BDNF were simultaneously upregulated in the activated microglia cells, whereas the specific AMPK inhibitor compound C exhibited reverse effects in microglia. Using immunofluorescence staining and electron microscopy, we found CSF1 decreased microglial p62 expression and induced the number of autophagosomes, whereas compound C significantly exhibited the reverse effects. For the in vivo study, compared with the control and AMPK-siRNA transfection, the mice under CSF1 intrathecal injection increased CSF1 receptor and LC3 expressed in the activated spinal microglia. More importantly, qRT-PCR showed CSF1 intrathecal injection substantially upregulated BDNF and c-Fos mRNA expression as well as the ensuing neuropathic pain. Our findings demonstrated that CSF1 induced a significant upregulation of microglial activation via the AMPK signaling pathway and resulted in an increasing microglial autophagic level. An increasing CSF1 level in the central nervous system can mimic and cause pain syndromes by up-regulation of AMPK-depended autophagy, thus offering a new target for the therapy of neuropathic pain.

Macrophage colony-stimulating factor increases hepatic macrophage content, liver growth, and lipid accumulation in neonatal rats.

Signaling via the colony-stimulating factor 1 receptor (CSF1R) controls the survival, differentiation, and proliferation of macrophages. Mutations in CSF1 or CSF1R in mice and rats have pleiotropic effects on postnatal somatic growth. We tested the possible application of pig CSF1-Fc fusion protein as a therapy for low birth weight (LBW) at term, using a model based on maternal dexamethasone treatment in rats. Neonatal CSF1-Fc treatment did not alter somatic growth and did not increase the blood monocyte count. Instead, there was a substantial increase in the size of liver in both control and LBW rats, and the treatment greatly exacerbated lipid droplet accumulation seen in the dexamethasone LBW model. These effects were reversed upon cessation of treatment. Transcriptional profiling of the livers supported histochemical evidence of a large increase in macrophages with a resident Kupffer cell phenotype and revealed increased expression of many genes implicated in lipid droplet formation. There was no further increase in hepatocyte proliferation over the already high rates in neonatal liver. In conclusion, treatment of neonatal rats with CSF1-Fc caused an increase in liver size and hepatic lipid accumulation, due to Kupffer cell expansion and/or activation rather than hepatocyte proliferation. Increased liver macrophage numbers and expression of endocytic receptors could mitigate defective clearance functions in neonates. NEW & NOTEWORTHY This study is based on extensive studies in mice and pigs of the role of CSF1/CSF1R in macrophage development and postnatal growth. We extended the study to neonatal rats as a possible therapy for low birth weight. Unlike our previous studies in mice and pigs, there was no increase in hepatocyte proliferation and no increase in monocyte numbers. Instead, neonatal rats treated with CSF1 displayed reversible hepatic steatosis and Kupffer cell expansion.

The colony-stimulating factors and collagen-induced arthritis: exacerbation of disease by M-CSF and G-CSF and requirement for endogenous M-CSF.

There is increasing evidence that the colony-stimulating factors (CSFs) may play a part in chronic inflammatory autoimmune diseases, such as rheumatoid arthritis (RA). We examined the involvement of macrophage CSF (M-CSF or CSF-1) and granulocyte CSF (G-CSF) in collagen-induced arthritis (CIA), a murine model of RA. Daily injections of M-CSF or G-CSF, 20-24 days postprimary immunization with type II collagen, exacerbated disease symptoms in suboptimally immunized DBA/1 mice. Support for the involvement of endogenous M-CSF in CIA was obtained by studies in which neutralizing monoclonal antibody reduced the severity of established CIA and also by studies showing the resistance of M-CSF-deficient op/op mice to CIA induction. These studies show that M-CSF and G-CSF can be proinflammatory in CIA and provide evidence that macrophage- and granulocyte-lineage cells can exacerbate CIA. Our results also show that M-CSF-dependent cells are essential for CIA development, suggesting M-CSF may be a suitable target for therapeutic intervention in RA.

Exacerbation of acute inflammatory arthritis by the colony-stimulating factors CSF-1 and granulocyte macrophage (GM)-CSF: evidence of macrophage infiltration and local proliferation.

CSF-1 and GM-CSF have been implicated in the pathogenesis of rheumatoid arthritis. We report the effects of CSF-1 and GM-CSF in the development of an acute methylated bovine serum albumin (mBSA)-induced murine arthritis model. Examination of histopathological features revealed that the systemic administration of CSF-1 or GM-CSF following mBSA administration into the knee resulted in the exacerbation of arthritis. This included synovial hyperplasia and joint inflammation, most evident at 7 and 14 days post-mBSA administration, and the appearance of erosive pannus tissue. The exacerbation by CSF-1 and GM-CSF was not sustained but declined in incidence and severity by 21 days post-mBSA administration, similar to the effects of IL-1beta in this model, reported here and previously. Macrophages expressing Mac-2 and F4/80 were a prominent feature of the pathology observed, particularly the infiltration of Mac-2+ macrophages seen in all mice administered CSF-1, GM-CSF or IL-1beta. Present in inflamed knees was a locally dividing population of cells which included Mac-2+ and F4/80+ macrophages. These studies demonstrate that CSF-1 and GM-CSF can exacerbate and prolong the histopathology of acute inflammatory arthritis and lend support to monocytes/macrophages being a driving influence in the pathogenesis of inflammatory arthritis.

Macrophage colony-stimulating factor increases bone resorption by osteoclasts disaggregated from human fetal long bones.

Macrophage colony-stimulating factor (M-CSF) is known to play an important role in human and murine osteoclast formation. Although M-CSF has been shown to inhibit isolated neonatal rat osteoclasts from resorbing bone, its action on the mature human osteoclast has not been described. We now report that M-CSF increases osteoclastic bone resorption in a dose-responsive manner. Bone resorption by mature human fetal osteoclasts, including pit area, depth, and volume, was increased in the presence of M-CSF compared with vehicle. The number of osteoclasts in the cultures was similar after 2 and 18 h in the presence of M-CSF, whereas there was a significant reduction in osteoclast number, whether assessed as the number of tartrate-resistant acid phosphatase (TRAP)-positive or vitronectin receptor-positive cells after 18 h in M-CSF-free cultures. The number of nuclei per osteoclast after 2 or 18 h in M-CSF was also similar and there was no difference in the number of vitronectin receptor-positive mononucleate cells at 2 and 18 h. This suggests that the increased bone resorption is likely to be accounted for by enhanced osteoclast survival in M-CSF compared with controls rather than by formation of new osteoclasts.

Transient thrombocytopenia produced by administration of macrophage colony-stimulating factor: investigations of the mechanism.

Administration of macrophage colony-stimulating factor (M-CSF) to mice (2 to 8 mg/kg/d x 5d) produced dose-dependent thrombocytopenia, which reached its nadir on days 4 to 5, followed by rapid recovery. Surprisingly, when administration of M-CSF was prolonged, the thrombocytopenia completely resolved, despite continued treatment. Splenectomy did not prevent the thrombocytopenia. Readministration of M-CSF after various intervals continued to produce the thrombocytopenic effect, even after 35 days. Measurements of Meg-CFC and megakaryocyte ploidy during the periods of M-CSF treatment and recovery of normal platelet levels showed no evidence of bone marrow suppression. Platelet survival was markedly decreased after 5 days of M-CSF (at the platelet count nadir) and after 9 days of continued M-CSF treatment, when the platelet count had returned to normal. Platelets from M-CSF-treated donors demonstrated normal survival when transfused into normal recipients. We concluded that thrombocytopenia produced by M-CSF was not due to suppression of thrombopoiesis, but to increased activity of the monocyte/macrophage system, which caused shortened platelet survival, and that subsequently, increased platelet production compensated for ongoing platelet destruction and resulted in normal platelet levels.

Macrophage-colony forming cells (M-CFC), with different sensitivities to colony stimulating factors, from peritoneal exudates and tissues of chronically inflamed mice.

OBJECTIVE AND DESIGN: To determine whether nonadherent macrophage precursors are present within the inflamed peritoneal cavity in mice, we analysed the mononuclear cell populations from different peritoneal tissues. OBJECTS: A group of 90 female mice BDF1 (C57BL/ 6 x DBA/2) was used for the study. Mononuclear cells were harvested from the peripheral blood, bone marrow, peritoneal exudate, omentum, mesentery, parietal peritoneum and diaphragm. TREATMENT: Mice were injected intraperitoneally with 0.2 ml of Freund's incomplete adjuvant. Animals were sacrificed at 6, 13, 16, 21 and 30 days. Three to six animals were examined for each time period. METHODS: Progenitor cell assay was performed in 1 ml of semi-solid agarose (0.3% Seakem GTG) DMEM which was supplied either with recombinant colony stimulating factors or with mesothelial cell-conditioned medium. RESULTS: Nonadherent macrophage-colony forming cells were present in all peritoneal compartments (35-140 precursor cells/5 x 10(4) mononuclear cells). Granulocyte/ macrophage-colony forming cells were found in the inflamed omentum. Combined simultaneous treatment with GM-CSF and M-CSF blocked the proliferation of the exudate and mesentery-derived macrophage precursors, but not other peritoneal tissue-derived macrophage precursors. Sequential stimulation with GM-CSF and M-CSF did not inhibit macrophage colony formation. CONCLUSIONS: GM-CSF can possibly influence the proliferative response induced by M-CSF. Nonadherent macrophage precursors recovered from different tissue compartments seem to differ in their sensitivity to growth regulation.

Functions of the M-CSF receptor on osteoclasts.

Macrophage colony-stimulating factor (M-CSF) receptor has been previously reported to be present in osteoclasts both at mRNA and protein levels. However, the biochemical interactions between M-CSF and its receptor on osteoclasts are less well characterized than in mononuclear phagocytes. In this study, we show that (1) 125I-labeled M-CSF ligand specifically binds to the M-CSF receptor on osteoclasts by autoradiography; (2) binding of M-CSF to the receptor stimulates protein tyrosine phosphorylation in osteoclasts by immunostaining; (3) oxygen-derived free radicals produced by calvarial osteoclasts are increased by M-CSF stimulation (1.37 +/- 0.08, n = 10, P < 0.01); and (4) bone resorption in calvarial explants is enhanced by M-CSF (1.153 +/- 0.09, n = 10, p < 0.001). Thus, our data provide multiple lines of evidences that mouse calvarial osteoclasts are activated by M-CSF. These data suggest that under the conditions present in the calvarial model, M-CSF activates osteoclastic bone resorption.

Hematopoietic recovery following high-dose combined alkylating-agent chemotherapy and autologous bone marrow support in patients in phase-I clinical trials of colony-stimulating factors: G-CSF, GM-CSF, IL-1, IL-2, M-CSF.

Hematopoietic recovery in 115 patients with metastatic breast cancer or metastatic melanoma, enrolled in phase-I studies of recombinant growth factors while undergoing treatment with high-dose chemotherapy with autologous bone marrow support, was examined with assays of bone marrow progenitor cells and peripheral blood progenitor cells, and by evaluation of peripheral blood counts. Groups of patients receiving hematopoietic cytokine support [with interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), or monocyte CSF (M-CSF)] post marrow infusion were compared with contemporaneous control patients not receiving growth factor support. Patients receiving GM-CSF demonstrated statistically significant increases in the growth of granulocyte/macrophage colony-forming units (CFU-GM) in the bone marrow and peripheral blood compared with control patients. The effect of GM-CSF was dose dependent in the early period post marrow infusion (day +6) with bone marrow CFU-GM colonies at doses 8-16 micrograms/kg/day 34 times those measured in controls. Significant increases in bone marrow multipotential progenitor cells (CFU-GEMM) were seen in patients receiving GM-CSF day +21 post marrow infusion. Patients receiving IL-1 demonstrated significant increases in bone marrow CFU-GM at day +21, maximal at dosages of 24-32 ng/kg/day. There were no significant increases in burst forming unit-erythroid (BFU-E) among any study group. Patients receiving G-CSF had significantly increased absolute neutrophil counts (ANC) and total white blood cell counts (WBC) by day +11 post transplant compared with control patients. Patients receiving GM-CSF demonstrated significantly increased WBC (greater than 2000/mm3) at day +11 and ANC greater than 500/mm3 at day +16. Optimal dose of G-CSF and GM-CSF to stimulate neutrophil recovery post transplant was 4-8 micrograms/kg/day and 8-16 micrograms/kg/day, respectively. Platelet recovery did not differ among the six study groups. These data demonstrate accelerated myeloid recovery after high-dose chemotherapy and autologous bone marrow support in patients receiving either G-CSF or GM-CSF. Moreover, GM-CSF and IL-1 stimulate myelopoiesis at the level of bone marrow CFU-GM, while G-CSF causes earlier neutrophil recovery peripherally.

Long-term follow-up of patients with invasive fungal disease who received adjunctive therapy with recombinant human macrophage colony-stimulating factor.

Mortality of bone marrow transplant (BMT) patients who develop invasive fungal infection is greater than 80%. Long-term follow-up of 46 consecutive BMT patients who received recombinant human macrophage colony-stimulating factor (rhM-CSF) as adjunctive therapy with standard antifungal treatment who were entered into phase I/II trials at The Fred Hutchinson Cancer Research Center is reported. rhM-CSF (100 micrograms/m2 to 2,000 micrograms/m2; Chiron/Cetus Corporation, Emeryville, CA) was administered from day 0 to 28 after determination of progressive fungal disease. Results of long-term follow-up of fungal infection, relapse, and survival were compared with 58 similar historical controls. Multivariable analysis of the patients who received rhM-CSF showed two factors that significantly correlated with poor survival: Karnofsky score < or = 20% and Aspergillus infection. Overall, survival of patients who received rhM-CSF was greater than that of historical patients (27% v 5%) and was entirely because of a 50% survival rate in patients with Candida infection and Karnofsky scores greater than 20%. Prospective, randomized, controlled trials to determine efficiency of rhM-CSF are indicated and should be directed at patients with invasive candidiasis.

A phase I trial of recombinant human macrophage colony-stimulating factor by rapid intravenous infusion in patients with refractory malignancy.

Twenty patients with advanced cancer for which there was no effective standard therapy or whose disease was refractory to standard therapy were treated with recombinant macrophage colony-stimulating factor (rM-CSF). The rM-CSF was administered by intravenous bolus infusion for 5 consecutive days every other week for 2 treatment weeks. The doses administered ranged from 30 to 33,000 micrograms/m2/day. There was no intrapatient dose escalation. There were minimal to no systemic side effects seen, except for acute dyspnea noted in three patients. The dyspnea was felt to be related to the rate of infusion and did not recur in one patient given additional rM-CSF at a slower infusion rate. The major hematologic effect seen was a mild decrease in platelet count, which began to recover while the patients continued to receive the rM-CSF. The clearance of rM-CSF was dose dependent. Lower doses resulted in a saturable mechanism felt to represent cellular uptake. Clearance at higher doses demonstrated both a first-order mechanism at high serum rM-CSF concentrations, representing renal clearance, as well as a saturable mechanism at low serum concentrations. The maximum mean serum half-life was reached at dose levels of greater than or equal to 3,690 micrograms/m2 and was in the range of 234-258 min. By this route of administration, rises in absolute monocyte count were slight and seen only at doses of greater than or equal to 450 micrograms/m2 during the second therapy week. The maximum tolerated dose was not reached in this study because of lack of availability of rM-CSF.