Optimal induction of hepatitis C virus envelope-specific immunity by bicistronic plasmid DNA inoculation with the granulocyte-macrophage colony-stimulating factor gene

In this study, we have constructed various DNA vaccine vectors that carried hepatitis C virus (HCV) envelope genes without and with the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in several different ways. In Buffalo rats that received plasmids carrying the HCV envelope genes, which encode envelope proteins E1 and E2, both antibody and lymphoproliferative responses against these proteins were induced. These responses were greatly enhanced by the codelivery of the GM-CSF gene. In particular, inoculation with a bicistronic plasmid that independently expressed the GM-CSF gene and the envelope genes in the same construct generated the highest antibody titers and significantly increased lymphoproliferative responses against these proteins. Moreover, strong antibody responses to homologous and heterologous hypervariable region 1 peptides were elicited in the immunized rats.     

Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein

Immunization with irradiated sporozoites protects animals and humans against malaria, and the circumsporozoite protein is a target of this protective immunity. We now report that adjuvant-free intramuscular injection of mice with plasmid DNA encoding the Plasmodium yoelii circumsporozoite protein induced higher levels of antibodies and cytotoxic T lymphocytes against the P. yoelii circumsporozoite protein than did immunization with irradiated sporozoites. Mice immunized with this vaccine had an 86% reduction in liver-stage parasite burden after challenge with 5 x 10(5) sporozoites (> 10(5) median infectious doses). Eighteen (68%) of 28 mice that received two or three doses of vaccine were protected against challenge with 10(2) sporozoites, and the protection was dependent on CD8+ T cells. These studies demonstrate the utility of plasmid DNA immunization against a nonviral infection. By obviating the requirement for peptide synthesis, expression and purification of recombinant proteins, and adjuvants, this method of immunization provides an important alternative for rapid identification of protective B- and T-cell epitopes and for construction of vaccines to prevent malaria and other infectious diseases.     

Immunostimulatory CpG oligodeoxynucleotides enhance the immune response to vaccine strategies involving granulocyte-macrophage colony-stimulating factor

Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can activate various immune cell subsets and induce production of a number of cytokines. Prior studies have demonstrated that both CpG ODN and granulocyte-macrophage colony-stimulating factor (GM-CSF) can serve as potent vaccine adjuvants. We used the 38C13 murine lymphoma system to evaluate the immune response to a combination of these two adjuvants. Immunization using antigen, CpG ODN, and soluble GM-CSF enhanced production of antigen-specific antibody and shifted production towards the IgG2a isotype, suggesting an enhanced TH1 response. This effect was most pronounced after repeat immunizations with CpG ODN and antigen/GM-CSF fusion protein. A single immunization with CpG ODN and antigen/GM-CSF fusion protein 3 days before tumor inoculation prevented tumor growth. CpG ODN enhanced the production of interleukin-12 by bone marrow-derived dendritic cells and increased expression of major histocompatibility complex class I and class II molecules, particularly when cells were pulsed with antigen/GM-CSF fusion protein. We conclude that the use of CpG ODN in combination with strategies involving GM-CSF enhances the immune response to antigen and shifts the response towards a TH1 response and that this approach deserves further evaluation in tumor immunization approaches and other conditions in which an antigen-specific TH1 response is desirable.     

Protection against tuberculosis by a plasmid DNA vaccine

BACKGROUND: Incidence of smooth muscle tumors is increased in patients with human immunodeficiency virus infection and organ transplant recipients. Smooth muscle tumors in immunocompromised patients often occur in unusual locations and exhibit evidence of latent infection by clonal, presumably tumorigenic, Epstein-Barr virus (EBV). OBJECTIVE: To investigate the presence of EBV latent infection in smooth muscle tumors in patients with acquired immunodeficiency syndrome and in immunocompetent patients. DESIGN: Twenty-two extrauterine, extraintestinal smooth muscle and myofibroblastic tumors were reviewed pathologically, and clinical charts were screened. Tumors were malignant (15 patients), benign (6 patients), and borderline (1 patient). Tissue specimens were investigated for latent EBV infection by latent membrane protein immunocytochemistry and EBV-encoded RNA in situ hybridization. SETTING: University Hospital of the University of Nancy, France. PATIENTS: Patients were 18 adults and four children. Two adults had acquired immunodeficiency syndrome. Both had low-grade leiomyosarcomas located in adrenal gland. Moreover, in patient 1, leiomyosarcoma was multifocal in pericardium and lymph node; in lymph node, muscle tumor was adjacent to nodal and skin Kaposi's lesions. RESULTS: In both patients with acquired immunodeficiency syndrome and leiomyosarcoma, latent infection by EBV could be demonstrated in tumor cells, contrasting with absence of detectable EBV infection in adjacent non-neoplastic tissues and nearby Kaposi's lesions. Latent infection by EBV could not be demonstrated in smooth muscle and myofibroblastic tumors in immunocompetent patients. CONCLUSIONS: Latent EBV infection is associated with smooth muscle cell tumors in immunocompromised patients, but not in immunocompetent patients.     

Keratinocyte growth stimulation by granulocyte-macrophage colony-stimulating factor (GM-CSF)

A comprehensive review of ultrasonography of the knee is presented and includes the choice of equipment and best patient positioning as well as a review of the normal anatomy and major disorders involving the knee, menisci and ligaments.     

Selective expansion of alveolar macrophages in vivo by adenovirus-mediated transfer of the murine granulocyte-macrophage colony-stimulating factor cDNA

Based on the hypothesis that genetic modification of freshly isolated alveolar macrophages (AM) with the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA would induce AM to proliferate, this study focuses on the ability of adenoviral (Ad) vectors to transfer and efficiently express the murine (m) GM-CSF cDNA in murine AM with consequent expansion in the number of AM in vitro and in vivo. To demonstrate that an Ad vector can effectively transfer and express genes in AM, murine AM recovered by bronchoalveolar lavage from the lung of Balb/c mice were infected with an Ad vector coding for green fluorescent protein (GFP) in vitro and expressed GFP in a dose-dependent fashion. Infection of AM with an Ad vector containing an expression cassette coding for mGM-CSF led to GM-CSF expression and to AM proliferation in vitro. When AM infected with AdGFP were returned to the respiratory tract of syngeneic recipient mice, GFP-expressing cells could still be recovered by bronchoalveolar lavage 2 weeks later. In vitro infection of AM with AdmGM-CSF and subsequent transplantation of the genetically modified AM to the lungs of syngeneic recipients led to GM-CSF expression in vivo. Strikingly, the AM recovered by lavage 5 weeks after transplantation demonstrated an increased rate of proliferation, and the total number of alveolar macrophages was 1. 9-fold greater than controls. Importantly, the increase in the numbers of AM was selective (ie, other inflammatory cell numbers were unchanged), and there was no modification to the lung architecture. Thus, it is feasible to genetically modify AM with Ad vectors and to use this strategy to modify the behavior of AM in vivo. Based on the importance of AM in the primary defense of the respiratory epithelial surface, this strategy may be useful in enhancing pulmonary defenses in immunodeficiency states.     

Identification of functional domains in murine granulocyte-macrophage colony-stimulating factor using monoclonal antibodies to synthetic peptides

Granulocyte-macrophage (GM)-CSF is an important hematopoietic cytokine that regulates proliferation and differentiation of macrophages, neutrophils, and eosinophils. In this study, we generated mAb to five synthetic peptides that correspond to regions along the murine GM-CSF molecule. The ability of anti-peptide mAb to bind to and inhibit biologic activity of murine (m) GM-CSF was determined. mAb with the highest neutralization titers were derived from mice immunized with peptide II, which correspond to amino acids 27 to 38 of mGM-CSF. Immunochemical studies showed that peptide II specifically blocked binding of anti-peptide II mAb to GM-CSF. mAb to two other peptides in the N-terminal half corresponding to residues 7 to 17 and 47 to 58, respectively, of mGM-CSF also inhibited GM-CSF-dependent proliferation and differentiation of murine bone marrow precursors for macrophages and granulocytes. Anti-peptide mAb also inhibited growth of a murine hematopoietic cell line FDCP1 and a murine T cell line HT-2, which was shown to be dependent on GM-CSF for growth in vitro. Biologic activity of both natural and recombinant mGM-CSF was neutralized by anti-peptide mAb. These findings indicate that epitopes in the N-terminal region of mGM-CSF are important for biologic activity, and the epitope defined by peptide II (residues 27 to 38) lies within a particularly important functional domain of the mGM-CSF molecule.     

Seminal transforming growth factor beta1 stimulates granulocyte-macrophage colony-stimulating factor production and inflammatory cell recruitment in the murine uterus

Mating in rodents evokes an inflammatory-like reaction within the uterine endometrium, characterized by extensive infiltration and activation of macrophages, dendritic cells, and granulocytes. This response is initiated when seminal vesicle gland-derived factors in the ejaculate stimulate uterine epithelial cells to release proinflammatory cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF). Experiments in which seminal vesicle secretions were fractionated by Sephacryl S-400 chromatography and assayed in vitro for GM-CSF-stimulating activity revealed that the seminal moiety coeluted with transforming growth factor beta1 (TGFbeta1) in the 150-440-kDa range and was neutralized by anti-TGFbeta1 antibodies. Comparable amounts of recombinant TGFbeta1 stimulated GM-CSF release in cultures of uterine epithelial cells from estrous mice and, when instilled into the uterine lumen, caused an increase in GM-CSF content and an infiltration of leukocytes into the endometrium similar to the postmating response. These results show that seminal vesicular fluid contains TGFbeta1 at levels sufficient to be the primary causative agent in the postmating inflammatory cascade through induction of GM-CSF synthesis by uterine epithelial cells. Seminal TGFbeta1 is thus implicated as a key factor in initiation of the remodeling events and immunological changes that occur in the uterus during the preimplantation period of pregnancy.     

Particle-mediated gene transfer of granulocyte-macrophage colony-stimulating factor cDNA to tumor cells: implications for a clinically relevant tumor vaccine

The necessity for prolonged tissue culture manipulations limits the clinical application of many form of gene therapy in patients with malignancies. We hypothesized that granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a plasmid expression vector could be effectively introduced into resting tumor cells, without the need for tissue culture propagation prior to or following transfection, and that efficient expression of transgenic GM-CSF by the transfected tumor cells would confer an effective immune response against tumors. GM-CSF cDNA in expression vectors was coated onto gold particles and accelerated with a gene gun device into mouse and human tumor cells. Human tumor tissue transfected within 4 hr of surgery produced significant levels of transgenic human GM-CSF protein in vitro. Human GM-CSF was readily detectable in serum and at the injection site following subcutaneous implantation of these transfected tumor cells into nude mice. Transfected and irradiated murine B16 melanoma cells produced > or = 100 ng/ml murine GM-CSF/10(6) cells per 24 hr in vitro for at least 10 days. The antitumor efficacy of this nonviral approach was tested using irradiated B16 tumor cells that were transfected with mGM-CSF cDNA and injected into mice as tumor "vaccine". Subsequent challenge of these mice with nonirradiated, nontransfected B16 tumor cells showed that 58% of the animals wer protected from the tumor by the prior vaccine treatment. In contrast, only 2% of control animals were protected by prior treatment with irradiated B16 cells transfected with the vector containing the luciferase gene. These results suggest that particle-mediated transfection of fresh tumor explants with cytokine cDNA is an effective and clinically attractive approach for cancer therapy.     

Granulocyte colony-stimulating factor enhances protection by anti-K1 capsular IgM antibody in murine Escherichia coli sepsis

Combined prophylactic treatment with recombinant murine granulocyte colony-stimulating factor (G-CSF) and a suboptimal dose of anti-K1 capsular IgM monoclonal antibody (MAb) significantly enhanced survival in an experimental mouse Escherichia coli O7:K1 peritonitis model compared with untreated animals (67% vs. 11% survival; P < 0.001) and with either treatment alone (67 vs. 29% and 27% survival, respectively; P < 0.01), which suggests synergism between these agents. Enhanced survival by combined treatment was associated with increased neutrophil counts in blood and peritoneal lavage fluid, lower systemic and higher levels of local tumour necrosis factor (TNF) and lower bacterial counts in blood cultures. Mouse neutrophils treated with G-CSF but not infected with E. coli showed enhanced phagocytic and respiratory burst capacity, down-regulation of L-selectin receptors and enhanced expression of Fc RII-III receptors but not of complement receptors.     

Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine

To enhance the efficacy of DNA malaria vaccines, we evaluated the effect on protection of immunizing with various combinations of DNA, recombinant vaccinia virus, and a synthetic peptide. Immunization of BALB/c mice with a plasmid expressing Plasmodium yoelii (Py) circumsporozoite protein (CSP) induces H-2Kd-restricted CD8+ cytotoxic T lymphocyte (CTL) responses and CD8+ T cell- and interferon (IFN)-gamma-dependent protection of mice against challenge with Py sporozoites. Immunization with a multiple antigenic peptide, including the only reported H-2Kd-restricted CD8+ T cell epitope on the PyCSP (PyCSP CTL multiple antigenic peptide) and immunization with recombinant vaccinia expressing the PyCSP induced CTL but only modest to minimal protection. Mice were immunized with PyCSP DNA, PyCSP CTL multiple antigenic peptide, or recombinant vaccinia expressing PyCSP, were boosted 9 wk later with the same immunogen or one of the others, and were challenged. Only mice immunized with DNA and boosted with vaccinia PyCSP (D-V) (11/16: 69%) or DNA (D-D) (7/16: 44%) had greater protection (P < 0. 0007) than controls. D-V mice had significantly higher individual levels of antibodies and class I-restricted CTL activity than did D-D mice; IFN-gamma production by ELIspot also was higher in D-V than in D-D mice. In a second experiment, three different groups of D-V mice each had higher levels of protection than did D-D mice, and IFN-gamma production was significantly greater in D-V than in D-D mice. The observation that priming with PyCSP DNA and boosting with vaccinia-PyCSP is more immunogenic and protective than immunizing with PyCSP DNA alone supports consideration of a similar sequential immunization approach in humans.     

Augmentation of human macrophage candidacidal capacity by recombinant human myeloperoxidase and granulocyte-macrophage colony-stimulating factor

Phagocyte myeloperoxidase (MPO) is believed to be particularly important in defense against candida infection. We reported earlier that monocytes, rich in MPO, killed Candida albicans at a significantly higher rate and extent than did monocyte-derived macrophages, known to lack MPO, and that C. albicans is less resistant to MPO-dependent oxidants than less pathogenic Candida species. We hypothesized, therefore, that the capacity of macrophages to kill C. albicans might be improved in the presence of MPO. In this study, we evaluated the ability of recombinant human MPO (rhMPO) to augment the killing of C. albicans by resident macrophages and macrophages activated by recombinant human granulocyte-macrophage colony-stimulating factor. Addition of rhMPO (concentration range, 0.8 to 6.4 U/ml) to suspensions of resident and activated macrophages and opsonized C. albicans resulted in concentration-dependent and significant increases in candida killing. This enhancement was particularly pronounced with activated macrophages, whether C. albicans was opsonized or unopsonized and ingested through the macrophage mannose receptor. rhMPO did not affect the killing of C. albicans by monocytes, nor did it affect phagocytosis of opsonized or unopsonized C. albicans. These results indicate that exogenous rhMPO can augment the candidacidal capacity of both resident and activated macrophages, with a more profound effect on activated cells. We suggest that rhMPO may be effective in the treatment of invasive candidiasis.     

Up-regulation of neutral endopeptidase (CALLA) in human neutrophils by granulocyte-macrophage colony-stimulating factor

Neutral endopeptidase 24.11 (NEP/CALLA/CD10), an enzyme expressed on early lymphoid progenitors, neutrophils, and various other cell types, inactivates many biologically active peptides, including the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Inhibition of CD10/NEP on the surface of human neutrophils (PMNs) in vitro inhibits migration toward this chemotaxin, suggesting that enzymatic inactivation by NEP regulates the neutrophil response to fMLP. Because PMNs in inflammatory sites are exposed to various cytokines, we evaluated the effects of selected cytokines on CD10/NEP activity in vitro. Of five cytokines tested--interleukin-1 (IL-1), IL-6, and IL-8, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor (GM-CSF)--GM-CSF provided the most consistent increase in surface NEP activity. Low concentrations (10(-9)-10(-7) M) of GM-CSF increased NEP activity in a time- and concentration-dependent manner to more than 225% that of control (phosphate-buffered saline-treated) cells. Cytofluorometry of cells stained with a fluorescent antibody to CD10 indicated that GM-CSF increased expression of surface CD10/NEP antigen in a similar manner. The effect of GM-CSF on NEP activity was enhanced still further by simultaneous exposure to IL-1, suggesting that combinations of cytokines may direct and regulate the neutrophil response within an inflammatory site. Rapid upregulation of CD10/NEP underscores the importance of this enzyme for control of peptide mediators of inflammation.     

Critical role of Lyn kinase in inhibition of neutrophil apoptosis by granulocyte-macrophage colony-stimulating factor

The signal pathway for control of apoptosis in human neutrophils is currently unknown. In this study, we provide the first evidence that a Src family tyrosine kinase, Lyn, plays a key role in inhibition polymorphonuclear (PMN) cell death. Several nuclear proteins associated with apoptosis, i.e., p53, cdc2, and Rb, were absent from PMN. Bcl-2, known to inhibit apoptosis, was also not expressed. Programmed cell death that rapidly occurred in PMN could be arrested by granulocyte-macrophage CSF (GM-CSF), but this activation did not induce p53, cdc2, retinoblastoma, or Bcl-2 expression. Instead, GM-CSF produced a rapid activation of Lyn and Hck, but not Fgr, tyrosine phosphorylation within 1 min. Co-immunoprecipitation studies indicated that only Lyn, but not Hck, was physically coupled to GM-CSF receptor. By histologic assessment and evaluation of DNA fragmentation, only antisense Lyn, but not antisense Hck or antisense Fgr, could reverse the cell survival advantage provided by GM-CSF. Therefore, the physical coupling of Lyn to GM-CSF receptor and its early activation are required for inhibition or delay of apoptosis in PMN.     

Thrombocytopenia in dogs induced by granulocyte-macrophage colony-stimulating factor: increased destruction of circulating platelets

Administration of recombinant canine granulocyte-macrophage colony-stimulating factor (rcGM-CSF) to normal dogs in previous studies induced an increase in peripheral blood neutrophils and a dose-dependent decrease in platelet counts. In six dogs that received the highest tested dose of rcGM-CSF (50 micrograms/kg/d) for a minimum of 12 days, the mean nadir of the platelet count was 46,000/microL (range, 4,000 to 91,000/microL) on day 9 +/- 1.1 after starting therapy, compared with a mean baseline platelet count of 398,000/microL (range, 240,000 to 555,000/microL). In three dogs, survival of autologous 111In-labeled platelets was reduced from a mean of 4.9 days to 1.3 days during the administration of rcGM-CSF. Biodistribution studies with gamma camera imaging indicated that there was an increase in mean hepatic uptake during the administration of rcGM-CSF, from 15% to 44% of the total injected 111In-labeled platelets at 2 hours, whereas splenic uptake was not significantly changed. In contrast, in two evaluable dogs who were recipients of 111In-labeled platelets from matched allogeneic donors receiving rcGM-CSF, platelet survival was not reduced and no increased hepatic uptake was noted. A third dog became alloimmunized to the matched donor platelets and was not evaluable. Immunohistologic studies of liver and spleen were performed with monoclonal antibodies specific for canine gpIIb/IIIa and P-selectin in dogs treated with rcGM-CSF and compared with untreated controls. On treatment, a marked reduction of platelets in the red pulp of the spleen was evident, and in general, the presence of platelet antigen in the liver was unchanged. Therefore, platelets were not being sequestered, but destroyed in the liver and spleen. The platelet antigens, P-selectin and gpIIb/IIIa, were identified in association with Kupffer cells in the liver, but no difference in the number of distribution of these Kupffer cells was found between controls and rcGM-CSF-treated dogs. In the spleen during rcGM-CSF treatment, most platelet antigens were associated with large mononuclear cells in the marginal zone. During administration of rcGM-CSF, CD1c and CD11c expression was increased on Kupffer cells. Platelet P-selectin expression and binding of leukocytes to circulating platelets were unchanged from baseline studies with rcGM-CSF treatment. In conclusion, during the administration of rcGM-CSF to dogs, a local process in the liver and spleen is induced resulting in thrombocytopenia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Therapeutic use of granulocyte-macrophage colony-stimulating factor (GM-CSF). A review of recent experience

Recombinant cytokines are now available for clinical use. Several colony-stimulating factors (CFS) have been identified which induce activation, proliferation and maturation of myeloid lineage cells. Recent therapeutic trials with granulocyte-macrophage colony-stimulating factors (GM-CSF) in association with chemotherapy, bone marrow transplantation and leukemia treatment are reviewed. GM-CSF as primary treatment for myelodysplasia and other types of bone marrow failure is also of interest. Colony-stimulating factor therapy in AIDS may be useful in order to reduce myelodepression caused by antiviral treatment and chemotherapy for associated malignancies like Kaposi's sarcoma. However, the effect of neutrophil count increase on infection is far from clear, and the real benefit of GM-CSF in cancer therapy has yet to be demonstrated.     

Characterization of a cell-type-restricted negative regulatory activity of the human granulocyte-macrophage colony-stimulating factor gene

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of normal myeloid progenitor cells and can also stimulate the growth of acute myelogenous leukemia (AML) blasts. GM-CSF is not normally produced by resting cells but is expressed by a variety of activated cells including T lymphocytes, macrophages, and certain cytokine-stimulated fibroblasts and endothelial cells. Production of GM-CSF by cultured AML cells has been demonstrated, and GM-CSF expression by normal myeloid progenitors has been postulated to play a role in myelopoiesis. We have investigated the regulation of expression of GM-CSF in AML cell lines, and our results demonstrate the presence of a strong constitutive promoter element contained within 53 bp upstream of the cap site. We have also identified a negative regulatory element located immediately upstream of the positive regulatory element (within 69 bp of the cap site) that is active in AML cell lines but not T cells or K562 CML cells. Competition transfection and mobility shift studies demonstrate that this activity correlates with binding of a 45-kDa protein.     

In vitro increases in plasmid DNA supercoiling by hydrostatic pressure

Cells of the yeast strain Debaryomyces vanrijiae SBUG 770, grown with glucose, converted biphenyl to 4-hydroxybiphenyl as the major metabolite. In addition, 2-hydroxybiphenyl was formed in minor amounts. No further degradation of these substances was detected. However, these monohydroxylated derivatives were oxidised by alkane-grown cells in the presence of the co-metabolic substrate, tetradecane. Under these conditions 2-hydroxybiphenyl was oxidised to 2,5-dihydroxybiphenyl, and 4-hydroxybiphenyl was rapidly metabolised by formation of two major metabolites. One was identified as 3,4-dihydroxybiphenyl. Characterisation of the second product as 4-phenylmuconolactone points to a further metabolism of the initially formed dihydroxylated biphenyl via ortho-ring fission.     

MRL/lpr and MRL+/+ macrophage DNA synthesis in the absence and the presence of colony-stimulating factor-1 and granulocyte-macrophage colony-stimulating factor

Macrophage accumulation and proliferation as well as altered macrophage properties have been observed in autoimmune MRL mice. To determine whether there might be innate differences in the proliferative responses, we examined the DNA synthesis responses of peritoneal macrophages and macrophages derived in vitro from bone marrow precursors (bone marrow-derived macrophages (BMM)). Murine peritoneal exudate macrophages normally require the addition of macrophage CSF (CSF-1) to enter cell cycle in vitro. In contrast, we have found that many thioglycollate-induced adherent peritoneal macrophages, but not resident peritoneal macrophages, from both MRL/lpr and MRL+/+ mice atypically underwent DNA synthesis even in the absence of added CSF-1. They also responded very well to granulocyte-macrophage CSF. These findings may help to explain the appearance of increased macrophage numbers in MRL lesions. In contrast to a previous report, it was found that MRL/lpr and MRL+/+ BMM did not have an enhanced response to CSF-1 and that modulation of CSF-1 receptor expression was not more rapid in MRL BMM. We also found no evidence for abnormal CSF-1 internalization and degradation or for the lpr mutation to have any enhanced effect on BMM survival in the absence of CSF-1. TNF-alpha lowered the DNA synthesis response to CSF-1 of MRL/lpr BMM rather than enhanced it, as has been reported. Our data suggest that the enhanced accumulation of macrophages in the MRL/lpr kidney cannot be explained by a proposed model of enhanced responsiveness of MRL/lpr BMM to CSF-1, including a contribution by TNF-alpha.