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1.
Keratinocytes have been demonstrated to produce a number of cytokines, including growth factors such as the CSF IL-3. Circulating blood monocytes and some elicited macrophages retain a significant proliferative potential in response to colony-stimulating activity. Because a macrophage response is prominent in a variety of cutaneous immune reactions, we have studied the ability of conditioned media (CM) from a transformed murine keratinocyte cell line (PAM 212) and from normal murine keratinocytes to induce growth of peritoneal macrophages. CM from both normal and transformed keratinocyte cultures induces [3H]thymidine incorporation by thioglycollate-elicited, but not resident, peritoneal macrophages. IEF of PAM 212 CM reveals peaks of activity at pI 4.8 and less than or equal to 4.2. Analysis of CM by reversed-phase HPLC demonstrates active fractions that elute at 46 to 48% and 53 to 55% acetonitrile. The Mr of the 46 to 48% acetonitrile factor is 25 to 30 kDa by gel filtration HPLC. Polyclonal anti-granulocyte/macrophage (GM) CSF antibody blocks the induction of macrophage [3H]thymidine incorporation by factors with pI 4.8 and eluting at 46 to 48% acetonitrile but does not reduce the activity of crude CM or the factor eluting at 53 to 55% acetonitrile. Based on both physiochemical criteria and antibody neutralization, keratinocytes produce GM-CSF. Keratinocyte-derived factors, including GM-CSF, may play an important role in regulating cutaneous macrophage responses.  相似文献   

2.
The expression and modulation of IL-1 alpha in murine keratinocytes   总被引:6,自引:0,他引:6  
Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1. IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes. To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression. Our results indicated that as with the murine P388D1 monocyte cell line, PAM 212 keratinocytes constitutively express abundant amounts of IL-1 alpha mRNA. On exposure to LPS (100 micrograms/ml) for 8 h there was more than 10 times the increase in PAM 212 IL-1 alpha mRNA which was accompanied by a sixfold increase in supernatant IL-1 activity. Similarly UV irradiation had a significant effect on keratinocyte IL-1 alpha expression. High dose UV (300 mJ/cm2) inhibited PAM 212 IL-1 alpha expression at 4, 8, 24, 48 h post-UV whereas a lower dose of UV (100 mJ/cm2) inhibited UV at 4 and 8 h post-UV, but induced IL-1 expression at 24 and 48 h post-UV. The expression of IL-1 alpha varied with the differentiation state of the keratinocytes. Freshly removed newborn murine keratinocytes were found to constitutively express IL-1 alpha mRNA. Keratinocytes grown in low [Ca2+] tissue culture media (0.05 mM) for 6 days, functionally and phenotypically become undifferentiated and express increased quantities of IL-1 alpha mRNA, whereas cells grown in high [Ca2+] media (1.2 mM) for 6 days become terminally differentiated and IL-1 expression ceased. Keratinocytes cultured for 3 days in low [Ca2+] conditions expressed an intermediate level of IL-1 alpha. In contrast, little or no IL-1 beta mRNA was detected in either the PAM 212 cells or newborn murine keratinocytes. Thus LPS, UV, and cell differentiation state have a significant effect on expression of IL-1 alpha in murine keratinocytes.  相似文献   

3.
The influence of macrophage (M)-CSF on the production of inflammatory mediators has been examined in murine peritoneal macrophages. Cultures of macrophages treated with up to 30,000 U/ml of human rM-CSF or with 10,000 U/ml of L929-derived M-CSF did not reveal either PGE2, IL-1, or IL-6 secretion. In contrast, LPS, which served as a positive control, stimulated production of significant levels of PGE2, IL-1, and IL-6. Furthermore, Northern blot analysis of macrophage RNA revealed a strong induction of IL-1 alpha and IL-6 mRNA by LPS but not by M-CSF. Conditioned medium from macrophage cultures treated with purified L929 or human rM-CSF in combination with LPS exhibited a significant reduction of IL-1 bioactivity as compared with an LPS challenge alone. To investigate the mechanism involved in this M-CSF-dependent reduction of IL-1 bioactivity, we measured IL-1 alpha gene expression. The addition of M-CSF to LPS-treated macrophages did not affect IL-1 alpha mRNA levels suggesting that M-CSF may regulate production of an IL-1 inhibitor. This hypothesis was shown to be valid because removal of IL-1 alpha from conditioned medium of LPS plus M-CSF-treated cells allowed the detection of a nondialyzable factor that blocked IL-1-dependent thymocyte proliferation. The inhibitor appeared to be specific because it did not inhibit IL-2 and TNF bioactivities. Furthermore, this IL-1 inhibitor, which binds to cells and not to IL-1, competed with the binding of radioactive IL-1 alpha or beta to EL-4.6.1 cells. The results demonstrate that M-CSF alone does not induce proinflammatory mediators and PGE2 as was previously published. The data also suggest that M-CSF may play a role in the down-regulation of inflammatory responses.  相似文献   

4.
5.
GM-CSF and M-CSF (CSF-1) induce different phenotypic changes in macrophage lineage populations. The nature, extent, and generality of these differences were assessed by comparing the responses to these CSFs, either alone or in combination, in various human and murine macrophage lineage populations. The differences between the respective global gene expression profiles of macrophages, derived from human monocytes by GM-CSF or M-CSF, were compared with the differences between the respective profiles for macrophages, derived from murine bone marrow cells by each CSF. Only 17% of genes regulated differently by these CSFs were common across the species. Whether a particular change in relative gene expression is by direct action of a CSF can be confounded by endogenous mediators, such as type I IFN, IL-10, and activin A. Time-dependent differences in cytokine gene expression were noted in human monocytes treated with the CSFs; in this system, GM-CSF induced a more dramatic expression of IFN-regulated factor 4 (IRF4) than of IRF5, whereas M-CSF induced IRF5 but not IRF4. In the presence of both CSFs, some evidence of "competition" at the level of gene expression was observed. Care needs to be exercised when drawing definitive conclusions from a particular in vitro system about the roles of GM-CSF and M-CSF in macrophage lineage biology.  相似文献   

6.
We have previously reported that IL-3, a cytokine produced by both Th1 and Th2 type CD4+ T cells, displays macrophage-activating potential. IL-3, like IFN-gamma, readily induced functions related to Ag presentation (e.g., Ia and lymphocyte function-associated Ag-1 expression). However, in contrast to the response elicited by IFN-gamma, tumor cytotoxicity was not induced by IL-3. In this paper we have evaluated the capacity of IL-3 to regulate IL-1 expression. Our data demonstrate that although IL-3 alone was unable to induce the production of substantial IL-1 bioactivity in peritoneal exudate cells, it contributed synergistically to the induction of IL-1 bioactivity in the presence of suboptimal doses of LPS. It was of interest that IFN-gamma, which can also interact synergistically with LPS, was unable to complement the partial signals provided by IL-3 for the expression of IL-1 bioactivity, suggesting that IL-3 and IFN-gamma may be providing similar stimulatory signals in this respect. Our studies on the mechanism of synergy between IL-3 and LPS indicated that the effect of LPS did not appear to be mediated by the well-characterized LPS-inducible cytokines of macrophage origin (i.e., IL-1, alpha and beta, TNF-alpha, and IL-6). The best characterized function of IL-3 is its multicolony-stimulating activity as a CSF; in this context we also studied granulocyte-macrophage CSF and noted that it behaves similarly to IL-3 in that it can synergistically contribute to IL-1 induction. A similar, but more dramatic induction of IL-1 synthesis in response to IL-3 was demonstrated by the P388.D1 murine macrophage cell line. The kinetics and the molecular mechanism of the response of P388.D1 to IL-3 indicate several unique features of IL-3-induced IL-1 expression: 1) IL-3 itself induced IL-1 mRNA expression, which was unaccompanied by substantial production of bioactivity, either cell-associated or secreted into the culture supernatant; 2) IL-3 synergized with suboptimal doses of LPS to induce not only heightened IL-1 mRNA levels but bioactivity as well; and 3) IL-3, when combined with LPS, altered the kinetics of IL-1 message and bioactive protein production in response to LPS: IL-3 and LPS induced an early release (3 to 7 h poststimulation) of the IL-1 protein as well as a second peak of mRNA and bioactivity (at 12 to 36 h), which was not observed in response to either IL-3 or LPS alone.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

7.
8.
Mast cells and macrophages in normal C57/BL/6 mice   总被引:8,自引:2,他引:6  
Mast cells and macrophages have an important role in immunity and inflammation. Because mice are used extensively for experimental studies investigating immunological and inflammatory responses, we examined mast cell and macrophage distribution in normal murine tissues. Mast cells were abundant in the murine dermis, tongue, and skeletal muscle but were rarely found in the heart, lung, spleen, kidney, liver, and the bowel mucosa. In contrast, dogs exhibited large numbers of mast cells in the lung parenchyma, liver, and bowel. Some murine dermal mast cells had long cytoplasmic projections filled with granular content. Mouse mast cells demonstrated intense histamine immunoreactivity and were identified with histochemical enzymatic techniques for tryptase and chymase. Macrophages, identified using the monoclonal antibody F4/80, were abundant in the spleen, lung, liver, kidney, and bowel but relatively rare in the heart, tongue, and dermis. Using a nuclease protection assay we investigated mRNA expression of stem cell factor (SCF), a crucial survival factor for mast cells, and the macrophage growth factors macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Stem cell factor mRNA was highly expressed in the murine lung. Relatively low levels of SCF mRNA expression were found in the tongue and earlobe, which are tissues containing a high number of mast cells. Macrophage CSF and GM-CSF mRNA was highly expressed in the lung and spleen. The murine heart, an organ with a low macrophage content, expressed high levels of M-CSF but negligible levels of GM-CSF mRNA. Constitutive growth factor mRNA expression in murine tissues without significant populations of mast cells and macrophages may suggest an alternative role for these factors in tissue homeostasis.  相似文献   

9.
GM-CSF and M-CSF (CSF-1) can enhance macrophage lineage numbers as well as modulate their differentiation and function. Of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. What the critical actions are of these CSFs on macrophages during inflammatory reactions are unknown. To address this issue, adherent macrophages (GM-BMM and BMM) were first derived from murine bone marrow precursors by GM-CSF and M-CSF, respectively, and stimulated in vitro with LPS to measure secreted cytokine production, as well as NF-kappaB and AP-1 activities. GM-BMM preferentially produced TNF-alpha, IL-6, IL-12p70, and IL-23 whereas, conversely, BMM generated more IL-10 and CCL2; strikingly the latter population could not produce detectable IL-12p70 and IL-23. Following LPS stimulation, GM-BMM displayed rapid IkappaBalpha degradation, RelA nuclear translocation, and NF-kappaB DNA binding relative to BMM, as well as a faster and enhanced AP-1 activation. Each macrophage population was also pretreated with the other CSF before LPS stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and NF-kappaB activity. Thus, GM-CSF and M-CSF demonstrate, at the level of macrophage cytokine production, different and even competing responses with implications for their respective roles in inflammation, including a possible dampening or suppressive role for M-CSF in certain circumstances.  相似文献   

10.
Inhibition of inducible nitric oxide-synthase (iNOS) enzymatic activity during cutaneous wound repair leads to severely impaired tissue regeneration. To assess whether disturbed leukocyte infiltration might participate in impaired repair, we determined expressional kinetics of neutrophil-attracting macrophage inflammatory protein-2 (MIP-2), and monocyte-attracting macrophage chemoattractant protein-1 (MCP-1) using an excisional wound healing model in mice. MCP-1 was induced in epithelial keratinocytes upon wounding, and our data indicate that NO serves a negative regulatory role for MCP-1 expression in vivo, as clearly reduced numbers of wound margin keratinocytes associated with NO-deficient repair compensate for high MCP-1 expression levels observed during normal healing. MIP-2 expression was restricted to hair follicles which were not reduced in number during NO-deficient repair. In vitro studies confirmed a regulatory role of NO for keratinocyte-derived chemokine expression, as NO attenuated IL-1beta- and TNF-alpha-induced MCP-1 mRNA expression, whereas NO augmented IL-1beta-induced IL-8 (functional human homolog to murine MIP-2) mRNA expression in the human keratinocyte cell line HaCaT.  相似文献   

11.
12.
The effects of macrophage colony-stimulating factor (M-CSF or CSF-1) on the survival, proliferation, maturation and activation of human blood monocytes were examined. M-CSF (100-1,000 U/ml) doubled the number of monocytes surviving after eight days in culture and accelerated the usual increase in cell volume. Antiserum to M-CSF abolished both of these effects. There was no sizable increase in 3H-thymidine incorporation in monocytes over this time period. Of various factors tested, including gamma-interferon (gamma-IFN), interleukin (IL) 1 alpha, granulocyte CSF (G-CSF), platelet-derived growth factor (PDGF), and lipopolysaccharide (LPS), only granulocyte-macrophage CSF (GM-CSF) could also enhance survival and augment cell volume. While antiserum to human M-CSF eliminated the increase in survival induced by GM-CSF, it could not ablate the GM-CSF-stimulated increase in monocyte cell volume. Monocyte cell surface markers that increase with maturation (i.e., Fc gamma RIII) or with activation (i.e., Fc gamma RI) were unaffected by incubation with M-CSF.  相似文献   

13.
A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.  相似文献   

14.
Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, we examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. IL-10 mRNA expression was enhanced in UV-irradiated keratinocytes. The secretion of IL-10 by the irradiated keratinocytes was determined by Western blot analysis. A band reactive with anti-IL-10 mAb was found in supernatants from the UV-irradiated but not the mock-irradiated cells. IL-10 biologic activity was determined by the ability of the supernatants from the UV-irradiated keratinocytes to suppress IFN-gamma production by Ag-activated Th 1 cell clones. Anti-IL-10 mAb neutralized the ability of supernatants from UV-irradiated keratinocytes to suppress the induction of delayed-type hypersensitivity in vivo. Furthermore, injecting UV-irradiated mice with antibodies against IL-10 partially inhibited in vivo immunosuppression. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by UV-irradiated keratinocytes plays an essential role in the induction of systemic immunosuppression after total-body UV exposure.  相似文献   

15.
Epidermal epithelial cells (keratinocytes) produce and secrete a variety of immunologically active cytokines. We have previously reported that both transformed (PAM 212) and normal murine keratinocytes produce a soluble factor which induces proliferation of the T cell line, HT-2. In the present study we sought to compare keratinocyte-derived T cell growth factor (KTGF) with other T cell growth factors, characterize its physicochemical properties, and substantially purify KTGF from PAM 212 conditioned medium. KTGF from PAM 212 conditioned medium was not inhibited by antibodies which block the effect of interleukin 2 (IL 2) (S4B6) or B cell stimulatory factor 1 (BSF 1) (11B11). KTGF is heat-stable, has an isoelectric point of 4.8, and a relative molecular mass of 16 to 23 kilodaltons under nonreducing conditions. KTGF activity was enhanced at least 41,413-fold by sequential hydroxylapatite bulk preparation, desalting by reversed-phase chromatography, gel filtration high pressure liquid chromatography (HPLC), and reversed-phase HPLC. Keratinocytes produce a T cell growth factor with physicochemical properties distinct from IL 2 and BSF 1. KTGF may play a role in regulating the growth and differentiation of T cells in the epidermis.  相似文献   

16.
Peritoneal elicited macrophages (PEM) from mammary tumor-bearing mice have a decreased capacity to become cytotoxic against syngeneic, allogeneic, and xenogeneic target cells upon in vitro stimulation with LPS, as compared with PEM of normal mice. A regulatory mechanism other than PG release is suggested because the addition of both indomethacin and LPS to macrophage cultures from tumor-bearing mice caused no changes in their cytotoxic capability. Because tumor products have been implicated in the down-regulation of immune responses, we investigated whether pretreatment with supernatants from the tumor cell line DA-3, derived from the in vivo mammary adenocarcinoma D1-DMBA-3, affects the cytolytic capacity of macrophages. This treatment inhibits, in a dose-dependent fashion, the ability of stimulated normal PEM to kill target cells. Partial purification of DA-3 cell line supernatant showed that most of the inhibitory activity was exerted by factors with a molecular mass greater than 10 kDa and less than 30 kDa. However, slight inhibition could also be observed with fractions containing molecules less than 10 kDa. The data suggest that more than one factor released by the mammary tumor cells may be involved in the down-regulation of macrophage-mediated cytotoxicity. Because the DA-3 cells constitutively produce granulocyte-macrophage CSF (GM-CSF), which has a molecular mass of 27 kDa, we pretreated PEM from normal mice in vitro with rGM-CSF for 24 h. This resulted in a dose-dependent decrease in their capacity to kill tumor target cells upon LPS stimulation. Furthermore, PEM from normal mice injected with rGM-CSF for 25 days displayed a profound decrease in their cytolytic ability against DA-3 targets upon in vitro stimulation with increasing amounts of LPS. The pretreatment of PEM from normal mice with a combination of DA-3 cell supernatants and specific anti-GM-CSF partially neutralized the inhibitory effect of the DA-3 supernatant on macrophage tumoricidal capability. These results indicate that tumor-derived GM-CSF is an important factor involved in the decreased macrophage cytotoxicity during mammary adenocarcinoma progression.  相似文献   

17.
The development of culture conditions for growing normal human thymic epithelial (TE) cells free from contamination with other stromal cells has allowed us to identify and characterize TE cell-derived cytokines. In this study, we report that cultured human TE cells produced CSF that supported the growth of clonal hematopoietic progenitor cells in the light density fraction of human bone marrow cells. Thymic epithelial supernatants (TES) induced growth of granulocyte/macrophage colonies (CFU-GM), mixed granulocyte/erythrocyte/monocyte/megakaryocyte colonies (CFU-GEMM), and early burst-forming unit erythroid colonies (BFU-E). In addition, TES induced differentiation of the promyelocyte leukemic cell line HL-60 and stimulated growth of both granulocyte (CFU-G) and monocyte (CFU-M) colonies from murine bone marrow cells. Using anion exchange column chromatography, pluripotent CSF activities in TES were separated and shown to be distinct from an IL-1-like cytokine that has been shown as a TE cell-derived cytokine (TE-IL-1). Colony-stimulating activity supporting the growth of bone marrow CFU-GEMM, BFU-E, and CFU-GM co-eluted at 150 to 180 mM NaCl. A separate peak of CFU-GM-stimulating activity eluted early in the gradient at 20 mM NaCl. In Northern blot analysis of enriched RNA, synthetic oligonucleotide probes complementary to human G-CSF and M-CSF coding sequence each hybridized with a single RNA species of 1.7 and 4.4 kb, respectively. These data suggest that normal human TE cells synthesize G-CSF and M-CSF that promote differentiation of non-lymphoid hematopoietic cell precursors.  相似文献   

18.
The op/op mouse, in which the M-CSF gene is mutated, has greatly reduced numbers of macrophages and osteoclasts. We assessed the ability of M-CSF to induce osteoclast and macrophage formation in op/op hemopoietic cells in vitro. Osteoclast production was undetectable in op/op cell cultures, but was restored by M-CSF at concentrations approximately an order of magnitude higher than those that induced macrophages. In normal hemopoietic tissue M-CSF similarly increased macrophage numbers, but inhibited osteoclast formation. Despite cure of the macrophage defect, neither interleukin 3 nor granulocyte-macrophage CSF were able to induce osteoclastic differentiation in op/op cells. The results suggest that M-CSF induces osteoclastic differentiation but that macrophages, which are also induced by M-CSF, suppress osteoclast differentiation. Macrophages induced by other cytokines seem unable to contribute to osteoclast-formation.  相似文献   

19.
20.
We have conducted a systematic comparison of lysis of TNP-coated keratinocyte targets by TNP-specific antibody, by antibody plus complement, by antibody-dependent cellular cytotoxicity (ADCC), and by natural killing with the use of monocyte, lymphocyte, and neutrophil effectors. With chromium-release assays, human keratinocytes, HEp-2 cells (transformed human keratinocytes), PAM 212 cells (transformed mouse keratinocytes), and RSC (transformed rabbit keratinocytes) were all susceptible to monocyte- and lymphocyte-mediated ADCC (p less than 0.01 to p less than 0.02). All trypsinized keratinocyte targets were also susceptible to natural killing by monocyte or lymphocyte effectors (p = 0.05 to p less than 0.001). Antibody and antibody plus complement were poor mediators of keratinocyte lysis. If protein and complex lipid synthesis of keratinocytes were inhibited by 16-hr cycloheximide preincubation, then keratinocytes were susceptible to complement-mediated lysis, implying that the resistance of these cells to complement may be due to repair of transmembrane pores. Comparison of chromium-release assays with fluorescein diacetate dye uptake viability assays showed that human keratinocytes were still susceptible to monocyte and lymphocyte ADCC but not to antibody, antibody plus complement, or natural killing. The reproducible and uniform susceptibility of normal and transformed keratinocyte targets from three different species to monocyte and lymphocyte ADCC supports the hypothesis that this mechanism of cellular lysis may be important in antibody-associated diseases of epidermal cytotoxicity.  相似文献   

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