Moving beyond GK-12.

The National Science Foundation GK-12 program has made more than 300 awards to universities, supported thousands of graduate student trainees, and impacted thousands of K-12 students and teachers. The goals of the current study were to determine the number of sustained GK-12 programs that follow the original GK-12 structure of placing graduate students into classrooms and to propose models for universities with current funding or universities interested in starting a program. Results from surveys, literature reviews, and Internet searches of programs funded between 1999 and 2008 indicated that 19 of 188 funded sites had sustained in-classroom programs. Three distinct models emerged from an analysis of these programs: a full-stipend model, in which graduate fellows worked with partner teachers in a K-12 classroom for 2 d/wk; a supplemental stipend model in which fellows worked with teachers for 1 d/wk; and a service-learning model, in which in-classroom activity was integrated into university academic coursework. Based on these results, potential models for sustainability and replication are suggested, including establishment of formal collaborations between sustained GK-12 programs and universities interested in starting in-classroom programs; development of a new Teaching Experience for Fellows program; and integration of supplemental fellow stipends into grant broader-impact sections.

Transcription of a single mannose receptor gene by macrophage and retinal pigment epithelium.

To determine if the macrophage mannose receptor transcript is present in mouse, rat, pig, and human retinal pigment epithelium (RPE), primary cultures and/or freshly dissected retinal pigment epithelium from four different species were used to isolate total RNA. RT-PCR was used to amplify segments of the macrophage mannose receptor from each sample. Amplified products were sequenced and compared with known sequences of the macrophage mannose receptor. Macrophage mannose receptor transcripts were identified in all RPE samples. Comparison between sequences identified in RPE with macrophage sequences from the same species revealed 100% identity. Sequence homology between the different species was 74% or greater. These data are consistent with the transcription of a single mannose receptor gene by these two phagocytic cell types.

Lung surfactant and reactive oxygen-nitrogen species: antimicrobial activity and host-pathogen interactions.

Surfactant protein (SP) A and SP-D are members of the collectin superfamily. They are widely distributed within the lung, are capable of antigen recognition, and can discern self versus nonself. SPs recognize bacteria, fungi, and viruses by binding mannose and N-acetylglucosamine residues on microbial cell walls. SP-A has been shown to stimulate the respiratory burst as well as nitric oxide synthase expression by alveolar macrophages. Although nitric oxide (NO.) is a well-recognized microbicidal product of macrophages, the mechanism(s) by which NO. contributes to host defense remains undefined. The purpose of this symposium was to present current research pertaining to the specific role of SPs and reactive oxygen-nitrogen species in innate immunity. The symposium focused on the mechanisms of NO*-mediated toxicity for bacterial, human, and animal models of SP-A- and NO.-mediated pathogen killing, microbial defense mechanisms against reactive oxygen-nitrogen species, specific examples and signaling pathways involved in the SP-A-mediated killing of pulmonary pathogens, the structure and binding of SP-A and SP-D to bacterial targets, and the immunoregulatory functions of SP-A.

The role of surfactant-associated protein A in pulmonary host defense.

Resident alveolar macrophages play a key role in the initial defense against inhaled pathogens. Surface molecules bind opsonized as well as nonopsonized microbes and mediate their internalization by the macrophage. The recent discovery that specific C-type lectins can bind to the surface of a wide range of pathogens has led to the hypothesis that these lectins are involved in the initial phases of microbe recognition by the macrophage. Studies in our laboratory focus on the role of the lung-specific lectin surfactant associated protein A (SP-A) in host defense against pulmonary pathogens. SP-A contains a carbohdyrate recognition domain that appears to bind specifically to exposed carbohydrate residues on the surface of microorganisms. This lectin-microorganism interaction leads to entry of specific pathogens into macrophages and activation of intracellular pathways, resulting in the production of antimicrobial mediators such as nitric oxide. Many studies, including those involving SP-A-deficient mice, underscore the importance of this protein in pulmonary innate immunity. However, the intramacrophage mechanisms underlying the effects of SP-A are still unclear. This article describes our current knowledge of SP-A and its interactions with immune cells and pathogens with a focus on recent findings from our laboratory regarding SP-A interactions with mycobacteria.

HIV-1 Tat represses transcription from the mannose receptor promoter.

The mannose receptor is expressed on mature macrophages and immature dendritic cells, and functions to mediate phagocytosis of pathogens and capture of Ags for delivery to MHC class II-containing intracellular compartments. It has been previously reported that HIV-1-infected macrophages have reduced functions associated with the mannose receptor, including impaired Pneumocystis carinii phagocytosis and mannosylated albumin uptake. Several HIV-1-derived proteins including the Tat protein have been shown to transcriptionally repress host cell genes. The present study was undertaken to define the role of the HIV-1-derived protein Tat in HIV-mediated mannose receptor down-regulation. Cotransfection of the human macrophage cell line U937 with a Tat expression vector and a mannose receptor promoter-luciferase reporter construct resulted in down-regulation of mannose receptor promoter activity. This repression was targeted to the basal promoter. Expression of either one- or two-exon Tat resulted in decreased promoter activity. The addition of the transactivation response element (TAR) sequence enhanced the Tat-mediated repression. Down-regulation was also seen when transfected cells were treated with exogenously added Tat protein. These results are consistent with a mechanism whereby Tat reduces mannose receptor promoter activity by interfering with the host transcriptional initiation machinery, potentially resulting in decreased levels of surface mannose receptor available for Ag or pathogen capture.

Surfactant protein-A enhances uptake of respiratory syncytial virus by monocytes and U937 macrophages.

Surfactant protein (SP)-A is a known opsonin for a variety of pulmonary pathogens. SP-A enhances ingestion of these pathogens by interaction with an SP-A receptor (SP-AR) found on phagocytic cells such as peripheral blood monocytes (PBMC) and alveolar macrophages. Respiratory syncytial virus (RSV) is the most important respiratory pathogen in children. Recent studies have indicated that SP-A levels may be decreased in RSV bronchiolitis and pneumonia. In this study we examined the role of SP-A in uptake of RSV by both PBMC and U937 macrophages, a human macrophage cell line known to express SP-ARs. In addition, we studied the effect of SP-A- mediated uptake of RSV on production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 by these cells because incomplete immunity to recurrent RSV infection has been partially attributed to abnormal cytokine responses by macrophages. SP-A enhanced binding and uptake of fluorescently labeled RSV (RSV-FITC) by PBMC in a dose-dependent manner, with a maximal effect seen with 10 to 15 microg/ml SP-A as measured by both percent fluorescent monocytes and linear mean fluorescence (lmf) of individual cells. SP-A also enhanced uptake of RSV-FITC by U937 macrophages, with a maximal effect seen with 20 microg/ml SP-A as measured by both percent fluorescent monocytes and lmf. With respect to TNF-alpha levels, RSV alone slightly enhanced TNF-alpha production by PBMC and decreased TNF-alpha production by U937 macrophages measured at 12 h after addition of RSV. SP-A-mediated uptake of RSV significantly enhanced TNF-alpha production by PBMC and reversed the RSV-induced depression of TNF-alpha by U937 macrophages. RSV significantly enhanced IL-10 production by both cell types, which was reversed by SP-A-mediated uptake. These findings suggest that SP-A is an important opsonin for RSV and that SP-A-mediated uptake of RSV may alter some of the unusual cytokine responses that are postulated to be involved in incomplete immunity to recurrent infection.

Surfactant protein A enhances mycobacterial killing by rat macrophages through a nitric oxide-dependent pathway.

Surfactant-associated protein A (SP-A) is involved in surfactant homeostasis and host defense in the lung. We have previously demonstrated that SP-A specifically binds to and enhances the ingestion of bacillus Calmette-Guerin (BCG) organisms by macrophages. In the current study, we investigated the effect of SP-A on the generation of inflammatory mediators induced by BCG and the subsequent fate of ingested BCG organisms. Rat macrophages were incubated with BCG in the presence and absence of SP-A. Noningested BCG organisms were removed, and the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide were measured at varying times. TNF-alpha and nitric oxide production induced by BCG were enhanced by SP-A. In addition, SP-A enhanced the BCG-induced increase in the level of inducible nitric oxide synthase protein. Addition of antibodies directed against SPR210, a specific macrophage SP-A receptor, inhibited the SP-A-enhanced mediator production. BCG in the absence of SP-A showed increased growth over a 5-day period, whereas inclusion of SP-A dramatically inhibited BCG growth. Inhibition of nitric oxide production blocked BCG killing in the presence and absence of SP-A. These results demonstrate that ingestion of SP-A-BCG complexes by rat macrophages leads to production of inflammatory mediators and increased mycobacterial killing.

PU.1 and USF are required for macrophage-specific mannose receptor promoter activity.

In the current study we report the isolation of 854 base pairs of the rat mannose receptor promoter. Analysis of the sequence revealed one Sp1 site, three PU.1 sites, and a potential TATA box (TTTAAA) 33 base pairs 5' of the transcriptional start site. The tissue specificity of the promoter was determined using transient transfections. The promoter was most active in the mature macrophage cell line NR8383 although the promoter also showed activity in the monocytic cell line RAW. No activity was observed in pre-monocytic cell lines or epithelial cell lines. Mutation of the TTTAAA sequence to TTGGAA resulted in a 50% decrease in activity in transient transfection assays suggesting that the promoter contains a functional TATA box. Using electrophoretic mobility shift assays and mutagenesis we established that the transcription factors Sp1, PU.1, and USF bound to the mannose receptor promoter, but only PU.1 and USF contributed to activation. Transient transfections using a dominant negative construct of USF resulted in a 50% decrease in mannose receptor promoter activity, further establishing the role of USF in activating the rat mannose receptor promoter. Comparison of the rat, mouse, and human sequence demonstrated that some binding sites are not conserved. Gel shifts were performed to investigate differences in protein binding between species. USF bound to the rat and human promoter but not to the mouse promoter, suggesting that different mechanisms are involved in regulation of mannose receptor expression in these species. From these results we conclude that, similar to other myeloid promoters, transcription of the rat mannose receptor is regulated by binding of PU.1 and a ubiquitous factor at an adjacent site. However, unlike other myeloid promoters, we have identified USF as the ubiquitous factor, and demonstrated that the promoter contains a functional TATA box.

Surfactant protein A inhibits T cell proliferation via its collagen-like tail and a 210-kDa receptor.

Investigation of possible mechanisms to describe the hyporesponsiveness of pulmonary leukocytes has led to the study of pulmonary surfactant and its constituents as immune suppressive agents. Pulmonary surfactant is a phospholipid-protein mixture that reduces surface tension in the lung and prevents collapse of the alveoli. The most abundant protein in this mixture is a hydrophilic molecule termed surfactant-associated protein A (SP-A). Previously, we showed that bovine (b) SP-A can inhibit human T lymphocyte proliferation and interleukin-2 production in vitro. Results presented in this investigation showed that different sources of human SP-A and bSP-A as well as recombinant rat SP-A inhibited human T lymphocyte proliferation in a dose-dependent manner. A structurally similar collagenous protein, C1q, did not block the in vitro inhibitory action of SP-A. The addition of large concentrations of mannan to SP-A-treated cultures also did not disrupt inhibition, suggesting that the effect is not mediated by the carbohydrate recognition domain of SP-A. Use of recombinant mutant SP-As revealed that a 36-amino acid Arg-Gly-Asp (RGD) motif-containing span of the collagen-like domain was responsible for the inhibition of T cell proliferation. A polyclonal antiserum directed against an SP-A receptor (SP-R210) completely blocked the inhibition of T cell proliferation by SP-A. These results emphasize a potential role for SP-A in dampening lymphocyte responses to exogenous stimuli. The data also provide further support for the concept that SP-A maintains a balance between the clearance of inhaled pathogens and protection against collateral immune-mediated damage.

Characterization of a rat alveolar macrophage cell line that expresses a functional mannose receptor.

The mannose receptor is a single polypeptide transmembrane glycoprotein expressed on the surface of macrophages that binds and internalizes soluble and particulate ligands. Physiological ligands for this receptor are pathogens, such as mycobacteria, and extracellular acid hydrolases and peroxidases. Expression of the mannose receptor is tightly linked to the functional state of the macrophage: the receptor appears during differentiation, is increased by macrophage deactivating agents, and is reduced in the presence of macrophage activating agents. Studies of the mechanisms underlying these regulatory processes have been hampered by the lack of a stable cell line that expresses a functional and appropriately regulated mannose receptor. In this study we describe expression and modulation of the mannose receptor by the rat alveolar cell line NR8383. Similar amounts of the mannose receptor ligand horseradish peroxidase were internalized by both NR8383 cells and alveolar macrophages. In addition, NR8383 cells expressed immunoreactive mannose receptor protein and mannose receptor mRNA as detected by Northern analysis. Regulation studies showed that mannose receptor expression was regulated at the levels of activity, protein, and mRNA in NR8383 cells similarly to regulation in primary rat macrophages. In addition, NR8383 cells could be successfully transfected with a luciferase reporter gene, providing the transfectable, mannose receptor-positive macrophage cell line. These results support the hypothesis that NR8383 cells potentially represent the best current macrophage cell line for studying various aspects of macrophage function, and are particularly critical in studies of regulation of the mannose receptor, a key receptor in host defense and immune regulation.

Ingestion of Candida albicans down-regulates mannose receptor expression on rat macrophages.

The frequency of infection and death due to various Candida species has increased steadily during the past decade, with mucocutaneous candidal infections as a common problem in the immunocompromised host. Mononuclear phagocytes are important in phagocytosis of this organism. In areas where there are low levels of opsonins, the macrophage-specific mannose receptor plays a dominant role in mediating Candida albicans ingestion. Following receptor-mediated infection, the host macrophage produces inflammatory cytokines and mediators that lead to ultimate killing of the invading Candida. Infection of macrophages by pathogens often leads to altered function that might effect their subsequent host defense properties. For example, function of both the complement receptor type 3 and the mannose receptor are down-regulated following exposure to pathogens or pathogen-derived products. In the current study, we have examined the down-regulation of mannose receptor expression following Candida infection and have investigated possible mechanisms that might be involved. Mannose receptor activity was decreased following 24 h postinfection with Candida. Both tumor necrosis factor and nitric oxide were produced during the infection, and inhibition of the these mediators partially blocked the effect on the receptor. Infection with Candida also inhibited the ability of dexamethasone to up-regulate mannose receptor expression. Finally, mannose receptor protein turnover was accelerated in Candida-infected macrophages. We conclude that Candida down-regulates one of the receptors involved in its internalization through a combination of production of modulatory molecules and enhanced receptor degradation. These results support the hypothesis that pathogens that infect macrophages have the ability to alter the phagocytic pathways available for subsequent host defense.

SP-A enhances uptake of bacillus Calmette-Guérin by macrophages through a specific SP-A receptor.

Surfactant-associated protein A (SP-A) is a C-type lectin that is involved in surfactant metabolism as well as host defense functions in the lung. We have recently identified a receptor on macrophages [specific 210-kDa SP-A receptor (SPR210)] that binds SP-A. In the current study we have investigated the role of SP-A in mediating uptake of bacillus Calmette-Guérin (BCG) by rat macrophages and human monocytes and have examined the role of the macrophage SPR210 in this process. 125I-labeled SP-A bound BCG in a Ca(2+)-, carbohydrate-, and dose-dependent manner. To examine association of SP-A-BCG complexes with macrophages, BCG were opsonized with SP-A and were incubated with rat bone marrow-derived macrophages (RBMM), rat alveolar macrophages (RAM), or human monocytes at a 1-to-1 ratio for 4 h. The cells were washed, fixed in formalin, and stained with auramine-rhodamine. Cell-associated organisms were enumerated by fluorescent microscopy. The percentage of cells with one or more associated BCG was increased by SP-A from 27% of RBMM with BCG alone to 54% with SP-A-BCG complexes; 1-16% in RAM; and 39-67% in human monocytes. This enhanced uptake was dependent on the dose of SP-A, with maximal increases seen with 10 micrograms/ml. Electron microscopic analysis supported the conclusion that organisms were ingested by and not simply bound to the macrophages. Inclusion of SPR210 antibodies blocked association of SP-A-BCG complexes, suggesting a role for SPR210 in mediating the interaction of SP-A-BCG with the macrophages. This was further supported by the finding that modulation of SPR210 activity resulted in altered SP-A-BCG uptake. These results demonstrate that SP-A binds to BCG and that uptake of these SP-A-BCG complexes is mediated in part by the SPR210 on rat macrophages and human monocytes.

Purification of a cell-surface receptor for surfactant protein A.

In the present report we have characterized the binding of surfactant protein A (SP-A) to bone marrow-derived macrophages, U937 cells, alveolar macrophages, and type II epithelial cells. The binding of SP-A to all cell types is Ca2+-dependent and trypsin-sensitive, but type II cells express distinct Ca2+-independent binding sites. The binding of SP-A to macrophages is independent of known cell surface carbohydrate-specific receptors and of glycoconjugate binding sites on the surface of the cells and is distinct from binding to C1q receptors. Based on ligand blot analysis, both type II cells and macrophages express a 210-kDa SP-A-binding protein. The 210-kDa protein was purified to apparent homogeneity from U937 macrophage membranes using affinity chromatography with noncovalently immobilized surfactant protein A, and was purified from rat lung by differential detergent and salt extraction of isolated rat lung membranes. Polyclonal antibodies against the rat lung SP-A-binding protein inhibit binding of SP-A to both type II cells and macrophages, indicating that the 210-kDa protein is expressed on the cell surface. The polyclonal antibodies also block the SP-A-mediated inhibition of phospholipid secretion by type II cells, indicating that the 210-kDa protein is a functional cell-surface receptor on type II cells. In a separate report we have determined that antibodies to the SP-A receptor block the SP-A-mediated uptake of Mycobacterium bovis, indicating that the macrophage SP-A receptor is involved in SP-A-mediated clearance of pathogens.

Differential regulation of the mannose and SP-A receptors on macrophages.

Two carbohydrate-dependent mechanisms exist on alveolar macrophages to clear mannose-containing pathogens: receptor-mediated entry of non-opsonized microorganisms via the mannose receptor and receptor recognition of pathogens opsonized with surfactant-associated protein A (SP-A). A number of studies have demonstrated that mannose receptor expression is tightly linked to the functional state of the macrophage. In the present study, we investigated regulation of binding of SP-A to its receptor on macrophages by the same agents that regulate mannose-receptor expression. Phorbol 12-myristate 13-acetate, lipopolysaccharide (LPS), and interferon-gamma treatment of rat marrow-derived macrophages increased SP-A binding by 163, 296, and 337%, respectively, over untreated controls. Mannose-receptor activity was reduced to 75, 60, and 25% of control levels by these agents. Dexamethasone increased mannose receptor activity to 225%, while decreasing SP-A binding to 44% of controls. Addition of granulocyte macrophage-colony stimulating factor (GM-CSF) to human monocytes on day 0 dramatically increased mannose-receptor activity on day 5 over the non-serum control. SP-A binding was highest to freshly isolated monocytes and decreased to < 10% after differentiation in the presence of GM-CSF. After intraperitoneal injection of dexamethasone, rat alveolar macrophages isolated at 24 h expressed increased mannose-receptor activity and decreased SP-A binding. LPS injection resulted in increased SP-A binding and decreased mannose-receptor activity. In every instance, SP-A binding was inversely regulated with respect to mannose-receptor expression. We therefore speculate that the mannose receptor is a first-line host-defense receptor that is turned off during inflammation. SP-A in the alveolar space can then act as a lung-specific opsonin and mediate clearance of pathogens via the upregulated SP-A receptor.

Characterization of rod outer segment plasma membrane proteins which bind to the mannose receptor.

Previous work by our laboratory has demonstrated that rod outer segment (ROS) phagocytosis can be mediated by mannose-receptor dependent activity. This study was designed to probe for potential ligands on the ROS surface which could interact with the mannose receptor during the phagocytic cycle. Solubilized ROS plasma membranes were passed over a mannose receptor-Sepharose column in the presence of CaCl2. Proteins specifically bound to the column were eluted using methyl-D-mannoside and EDTA and characterized by gel electrophoresis, lectin blots, and immunoblots. Silver stained gels of ROS plasma membrane proteins eluted from the mannose-receptor column demonstrated six bands: a major band at 36 kD, identified by monospecific antibodies as rhodopsin, and bands of Mr = 39 kD, 67 kD, 76 kD, 97 kD and 100 kD. Lectin blots of the eluted fractions confirmed that all six proteins in these fractions could bind concanavalin A. In summary, these results showed that rhodopsin and several other mannose-containing glycoproteins on ROS plasma membranes were bound to a mannose receptor column, and thus could serve as ligands for mannose receptor-mediated ROS phagocytosis.

Activation of human THP-1 cells and rat bone marrow-derived macrophages by Helicobacter pylori lipopolysaccharide.

The mechanism by which Helicobacter pylori, which has little or no invasive activity, induces gastric-tissue inflammation and injury has not been well characterized. We have previously demonstrated that water-extracted proteins of H. pylori are capable of activating human monocytes by a lipopolysaccharide (LPS)-independent mechanism. We have now compared activation of macrophages by purified LPS from H. pylori and from Escherichia coli. LPS was prepared by phenol-water extraction from H. pylori 88-23 and from E. coli O55. THP-1, a human promyelomonocytic cell line, and macrophages derived from rat bone marrow each were incubated with the LPS preparations, and cell culture supernatants were assayed for production of tumor necrosis factor alpha (TNF-alpha), prostaglandin E2 (PGE2), and nitric oxide. THP-1 cells showed maximal activation by the LPS molecules after cell differentiation was induced by phorbol 12-myristate 13-acetate. Maximal TNF-alpha and PGE2 production occurred by 6 and 18 h, respectively, in both types of cells. In contrast, NO was produced by rat bone marrow-derived macrophages only and was maximal at 18 h. The minimum concentration of purified LPS required to induce TNF-alpha, PGE2, and NO responses in both types of cells was 2,000- to 30,000-fold higher for H. pylori than for E. coli. Purified LPS from three other H. pylori strains with different polysaccharide side chain lengths showed a similarly low level of activity, and polymyxin B treatment markedly reduced activity as well, suggesting that activation was a lipid A phenomenon. These results indicate the low biological activity of H. pylori LPS in mediating macrophage activation.

Nitric oxide inactivates xanthine dehydrogenase and xanthine oxidase in interferon-gamma-stimulated macrophages.

Interferon-gamma (IFN-gamma) has been reported to up-regulate transcription of the xanthine dehydrogenase (XDH) gene and to regulate XDH and xanthine oxidase (XO) activity in endothelial cells and liver tissue. Macrophages are a source of XDH/XO activity at inflammatory sites and are functionally regulated by IFN-gamma. We studied the effect of IFN-gamma on XDH and XO in rat bone marrow macrophages, rat alveolar macrophages, and murine RAW cells. Instead of an induction of enzyme activity, XDH/XO activity was almost totally lost after incubation with 100 to 1,000 U/ml of IFN-gamma for 24 h in all three cell types. The loss of cell-associated XDH/XO activity was not correlated with the appearance of XDH/XO activity in the media. In addition, the loss of XDH/XO activity could not be accounted for by transcriptional repression, since there was an increase in steady-state levels of XDH mRNA. To determine whether XDH/XO activity might be lost through nitric oxide-mediated inactivation of XDH/XO, we compared the time course and dose response for XDH/XO inactivation with that of nitric oxide production and found them similar. Treatment with the nitric oxide inhibitor N-monomethyl arginine appeared to totally block inactivation of XDH/XO by IFN-gamma. We conclude that upon stimulation with IFN-gamma, inducible nitric oxide in macrophages leads to post-transcriptional inhibition of XDH/XO, possibly minimizing the potential for tissue injury from XO released from macrophages into the inflammatory milieu. Inactivation of XDH may represent yet another "protective" role for nitric oxide at sites of inflammation.

Dexamethasone blocks the interferon-gamma-mediated downregulation of the macrophage mannose receptor.

The macrophage mannose receptor is highly susceptible to modulation by a variety of proinflammatory and antiinflammatory agents. Previous studies have demonstrated that mannose receptor activity is dramatically increased in rat bone marrow-derived macrophages by dexamethasone treatment and decreased following incubation with interferon-gamma (IFN-gamma). Regulation by both agents occurs at least in part by alteration of mannose receptor mRNA levels. In the present study, we have investigated the ability of dexamethasone to block mannose receptor downregulation in rat marrow macrophages by IFN-gamma. Incubation of rat macrophages with IFN-gamma resulted in downregulation of mannose receptor activity, receptor synthesis, and mRNA levels, with no change in turnover rate of the receptor. IFN-gamma appeared to act at least partially through the generation of nitric oxide: inclusion of the nitric oxide inhibitor N-monomethyl arginine inhibited the IFN-gamma-induced effects on the mannose receptor by approximately 50%. When cells were coincubated with dexamethasone plus IFN-gamma, dexamethasone blocked the decrease in mannose receptor activity, synthesis, and mRNA. Additionally, dexamethasone inhibited nitric oxide production in response to IFN-gamma. These results suggest that mannose receptor expression is downregulated by IFN-gamma by at least two mechanisms: a decrease in mannose receptor mRNA levels and inhibition involving the production of nitric oxide. Dexamethasone has the capability of blocking the effect of IFN-gamma on mannose receptor expression through inhibition of IFN-gamma-mediated downregulation of mannose receptor transcription and/or inhibition of IFN-gamma-mediated induction of nitric oxide production.

Regulation of tumor necrosis factor-alpha receptors on macrophages: differences between primary macrophages and transformed macrophage cell lines.

Macrophages are a pivotal cell in the production of a variety of cytokines. In addition, macrophages express receptors on their surface which allows them to act as target cells for cytokines. The regulation of both cytokine production and cytokine receptor expression in macrophages may play a key role in modulating the processes of inflammation, injury, and repair. In this report we have studied the regulation of macrophage receptors for tumor necrosis factor-alpha (TNF alpha) on rat bone marrow-derived macrophages, rat alveolar macrophages, human monocyte-derived macrophages, and the human monocyte-like cell line, U937, by a variety of immunomodulatory and inflammatory agents. U937 cells and rat and human macrophages bound TNF alpha in a saturable process, with an affinity in each cell type of approximately 1 nM. Following incubation with phorbol myristate acetate for 60 min, TNF alpha binding to all cells was decreased. Interleukin-1 treatment increased TNF alpha binding to human and rat macrophages. Interferon-gamma treatment decreased receptor activity in both human and rat macrophages, but increased TNF alpha binding to U937 cells. Treatment of human and rat macrophages with endotoxin resulted in a rapid loss of TNF alpha binding, while endotoxin treatment increased receptor expression in U937 cells. In all cases, receptor regulation was the result of a change in receptor number. Finally, following intraperitoneal injection of endotoxin, TNF alpha receptor expression was down-regulated on alveolar lavage cells from rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxin induces the expression of macrophage inflammatory protein 1 alpha mRNA by rat alveolar and bone marrow-derived macrophages.

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is a newly described cytokine that is present in large amounts in the culture supernatant of an endotoxin-stimulated murine macrophage-like cell line (RAW 264.7). There is increasing information that suggests that this cytokine mediates acute neutrophilic inflammation, although the mechanism of mediation is unknown. Data examining the production and regulation of MIP-1 alpha by primary rat macrophages are lacking, and MIP-1 alpha has not been studied previously in an animal model of endotoxin-induced neutrophilic alveolitis. In this study, we performed Northern analysis of steady-state rat MIP-1 alpha mRNA using an oligonucleotide probe complementary to amino acids 4-13 of murine MIP-1 alpha. Our data demonstrate that rat alveolar and bone marrow-derived macrophages can be induced by in vitro endotoxin treatment to express a 1.1-kb MIP-1 alpha mRNA. Expression of the mRNA could be elicited by treatment with 0.1 to 10.0 micrograms/ml of endotoxin in vitro with peak steady-state levels detectable up to 9 h after adding endotoxin to the media. Alveolar macrophages recovered by whole lung lavage from endotoxin-treated rats expressed increased amounts of the mRNA homologous to MIP-1 alpha mRNA when treated in vitro with endotoxin. We also found that rat neutrophils could be induced by endotoxin in vitro to express the MIP-1 alpha mRNA. We were able to identify MIP-1 alpha in culture supernatant from endotoxin-stimulated rat alveolar and bone marrow-derived macrophages by immunoprecipitation with a specific goat anti-murine MIP-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)