Characterization of proinflammatory cytokine production and CD14 expression by murine alveolar macrophage cell lines

Summary Alveolar macrophages, which play a central role in lung defense, produce cytokines that help orchestrate local inflammatory responses. In sepsis and other pathological conditions, bacterial lipopolysaccharide endotoxin can induce alveolar macrophages (AM) to release proinflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1, and interleukin-6. Studying the mechanisms that control alveolar macrophage cytokine production may lead to better therapies for conditions involving inflammatory lung injury. We and others have noted significant differences between alveolar macrophages and peritoneal macrophages, but large numbers of human or murine alveolar macrophages are rarely available for detailed mechanistic studies. We have obtained three murine alveolar macrophage cell lines (AMJ2C8, AMJ2C11, and AMJ2C20) and have begun to characterize their cytokine responses to proinflammatory stimuli. We measured the effects of endotoxin, interferon gamma, and the combination of the two on production of tumor necrosis factor, interleukin-1 beta, and interleukin-6 in each line. We also studied the expression of the endotoxin receptor CD14 by these cells, and investigated the effect of serum on their endotoxin responsiveness. We show here that all three of the cell lines responded in a manner comparable to that of primary murine alveolar macrophages. Observed variations between these lines may reflect the documented heterogeneity seen in populations of primary alveolar macrophages. These cell lines should expand the repertoire of tools available to investigators studying regulation of murine alveolar macrophage responses.     

Surfactant protein A enhances alveolar macrophage phagocytosis of apoptotic neutrophils

Surfactant protein A (SP-A) is an innate immune molecule that binds foreign organisms that invade the lungs and targets them for phagocytic clearance by the resident pulmonary phagocyte, the alveolar macrophage (AM). We hypothesized that SP-A binds to and enhances macrophage uptake of other nonself particles, specifically apoptotic polymorphonuclear neutrophils (PMNs). PMNs are recruited into the lungs during inflammation, but as inflammation is resolved, PMNs undergo apoptosis and are phagocytosed by AMs. We determined that SP-A increases AM phagocytosis of apoptotic PMNs 280 +/- 62% above the no protein control value. The increase is dose dependent, and heat-treated SP-A still enhanced uptake, whereas deglycosylated SP-A had significantly diminished ability to enhance phagocytosis. Surfactant protein D also increased phagocytosis of apoptotic PMNs by approximately 125%. However, other proteins that are structurally homologous to SP-A, mannose-binding lectin and complement protein 1q, did not. SP-A enhances phagocytosis via an opsonization-dependent mechanism and binds apoptotic PMNs approximately 4-fold more than viable PMNs. Also, binding of SP-A to apoptotic PMNs does not appear to involve SP-A's lectin domain. These data suggest that the pulmonary collectins SP-A and SP-D facilitate the resolution of inflammation by accelerating apoptotic PMN clearance.     

Surfactant protein D reduces alveolar macrophage apoptosis in vivo

Surfactant protein D (SP-D) is a molecule of the innate immune system that recognizes the patterns of surface carbohydrate on pathogens and targets them for phagocytosis and killing. SP-D-deficient mice show an increased number of macrophages in the alveolar space, excess surfactant phospholipid, overproduction of reactive oxygen species, and the development of emphysema. We report here that SP-D-deficient mice have a 5- to 10-fold increase in the number of apoptotic and necrotic alveolar macrophages, as defined by annexin V and propidium iodine staining, respectively. Intrapulmonary administration of a truncated 60-kDa fragment of human recombinant SP-D reduces the number of apoptotic and necrotic alveolar macrophages and partially corrects the lipid accumulation in SP-D-deficient mice. The same SP-D fragment binds preferentially to apoptotic and necrotic alveolar macrophages in vitro, suggesting that SP-D contributes to immune homeostasis in the lung by recognizing and promoting removal of necrotic and apoptotic cells.     

Vitamin D inhibits monocyte/macrophage proinflammatory cytokine production by targeting MAPK phosphatase-1

It is estimated that 1 billion people around the world are vitamin D deficient. Vitamin D deficiency has been linked to various inflammatory diseases. However, the mechanism by which vitamin D reduces inflammation remains poorly understood. In this study, we investigated the inhibitory effects of physiologic levels of vitamin D on LPS-stimulated inflammatory response in human blood monocytes and explored potential mechanisms of vitamin D action. We observed that two forms of the vitamin D, 1,25(OH)(2)D(3), and 25(OH)D(3), dose dependently inhibited LPS-induced p38 phosphorylation at physiologic concentrations, IL-6 and TNF-α production by human monocytes. Upon vitamin D treatment, the expression of MAPK phosphatase-1 (MKP-1) was significantly upregulated in human monocytes and murine bone marrow-derived macrophages (BMM). Increased binding of the vitamin D receptor and increased histone H4 acetylation at the identified vitamin D response element of the murine and human MKP-1 promoters were demonstrated. Moreover, in BMM from MKP1(-/-) mice, the inhibition of LPS-induced p38 phosphorylation by vitamin D was completely abolished. Vitamin D inhibition of LPS-induced IL-6 and TNF-α production by BMM from MKP-1(-/-) mice was significantly reduced as compared with wild-type mice. In conclusion, this study identified the upregulation of MKP-1 by vitamin D as a novel pathway by which vitamin D inhibits LPS-induced p38 activation and cytokine production in monocytes/macrophages.     

Surfactant proteins A and D bind CD14 by different mechanisms

Surfactant proteins A (SP-A) and D (SP-D) are lung collectins that are constituents of the innate immune system of the lung. Recent evidence (Sano, H., Sohma, H., Muta, T., Nomura, S., Voelker, D. R., and Kuroki, Y. (1999) J. Immunol. 163, 387-395) demonstrates that SP-A modulates lipopolysaccharide (LPS)-induced cellular responses by direct interaction with CD14. In this report we examined the structural elements of the lung collectins involved in CD14 recognition and the consequences for CD14/LPS interaction. Rat SP-A and SP-D bound CD14 in a concentration-dependent manner. Mannose and EDTA inhibited SP-D binding to CD14 but did not decrease SP-A binding. The SP-A binding to CD14 was completely blocked by a monoclonal antibody that binds to the SP-A neck domain but only partially blocked by an antibody that binds to the SP-A lectin domain. SP-A but not SP-D bound to deglycosylated CD14. SP-D decreased CD14 binding to both smooth and rough LPS, whereas SP-A enhanced CD14 binding to rough LPS and inhibited binding to smooth LPS. SP-A also altered the migration profile of LPS on a sucrose density gradient in the presence of CD14. From these results, we conclude that 1) lung collectins bind CD14, 2) the SP-A neck domain and SP-D lectin domain participate in CD14 binding, 3) SP-A recognizes a peptide component and SP-D recognizes a carbohydrate moiety of CD14, and 4) lung collectins alter LPS/CD14 interactions.     

HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.     

Surfactant strengthens the inhibitory effect of C-reactive protein on human lung macrophage cytokine release

In this study we investigated the effect of acute-phase levels of C-reactive protein (CRP) on cytokine production by pulmonary macrophages in the presence or absence of pulmonary surfactant. Both human alveolar and interstitial macrophages as well as human surfactant were obtained from multiple organ donor lungs. Precultured macrophages were stimulated with LPS alone or together with IFN-gamma in the presence or absence of CRP, surfactant, and combinations. Releases of TNF-alpha and of IL-1beta to the medium were determined. We found that CRP could modulate lung inflammation in humans by decreasing the production of proinflammatory cytokines by both alveolar and interstitial macrophages stimulated with LPS alone or together with IFN-gamma. The potential interaction between CRP and surfactant phospholipids did not overcome the effect of either CRP or surfactant on TNF-alpha and IL-1beta release by lung macrophages. On the contrary, CRP and pulmonary surfactant together had a greater inhibitory effect than either alone on the release of proinflammatory cytokines by lung macrophages.     

Surfactant-associated protein A inhibits LPS-induced cytokine and nitric oxide production in vivo

The role of surfactant-associated protein (SP) A in the mediation of pulmonary responses to bacterial lipopolysaccharide (LPS) was assessed in vivo with SP-A gene-targeted [SP-deficient; SP-A(-/-)] and wild-type [SP-A(+/+)] mice. Concentrations of tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein-2, and nitric oxide were determined in recovered bronchoalveolar lavage fluid after intratracheal administration of LPS. SP-A(-/-) mice produced significantly more TNF-alpha and nitric oxide than SP-A(+/+) mice after LPS treatment. Intratracheal administration of human SP-A (1 mg/kg) to SP-A(-/-) mice restored regulation of TNF-alpha, macrophage inflammatory protein-2, and nitric oxide production to that of SP-A(+/+) mice. Other markers of lung injury including bronchoalveolar fluid protein, phospholipid content, and neutrophil numbers were not influenced by SP-A. Data from experiments designed to test possible mechanisms of SP-A-mediated suppression suggest that neither binding of LPS by SP-A nor enhanced LPS clearance are the primary means of inhibition. Our data and others suggest that SP-A acts directly on immune cells to suppress LPS-induced inflammation. These results demonstrate that endogenous or exogenous SP-A inhibits pulmonary LPS-induced cytokine and nitric oxide production in vivo.     

IL-10 inhibits cytokine production by activated macrophages

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.     

CD47 engagement inhibits cytokine production and maturation of human dendritic cells

Upon encounter with bacterial products, immature dendritic cells (iDCs) release proinflammatory cytokines and develop into highly stimulatory mature DCs. In the present study, we show that human monocyte-derived DCs functionally express the CD47 Ag, a thrombospondin receptor. Intact or F(ab')2 of CD47 mAb suppress bacteria-induced production of IL-12, TNF-alpha, GM-CSF, and IL-6 by iDCs. 4N1K, a peptide derived from the CD47-binding site of thrombospondin, also inhibits cytokine release. The inhibition of IL-12 and TNF-alpha is IL-10-independent inasmuch as IL-10 production is down-modulated by CD47 mAb and blocking IL-10 mAb fails to restore cytokine levels. CD47 ligation counteracts the phenotypic and functional maturation of iDCs in that it prevents the up-regulation of costimulatory molecules, the loss of endocytic activity, and the acquisition of an increased capacity to stimulate T cell proliferation and IFN-gamma production. Interestingly, regardless of CD47 mAb treatment during DC maturation, mature DC restimulated by soluble CD40 ligand and IFN-gamma, to mimic DC/T interaction, produce less IL-12 and more IL-18 than iDCs. Finally, CD47 ligation on iDCs does not impair their capacity to phagocytose apoptotic cells. We conclude that following exposure to microorganisms, CD47 ligation may limit the intensity and duration of the inflammatory response by preventing inflammatory cytokine production by iDCs and favoring their maintenance in an immature state.     

Surfactant protein D regulates the cell surface expression of alveolar macrophage beta(2)-integrins

The beta(2)-integrin receptors (CD11a/CD18, CD11b/CD18, and CD11c/CD18) are expressed on the surface of alveolar macrophages and are important for the phagocytic clearance of pathogens. In the present study, we demonstrate that surfactant protein D (SP-D) modulates surface expression of CD11b and CD11c, but not CD11a or CD18, on alveolar macrophages. While cell surface receptors were reduced, CD11b and CD11c mRNAs were increased by SP-D deficiency. CCSP-rtTA(+)/(tetO)(7)-rSPD(+)/SP-D(-/-) mice, which conditionally express SP-D in the lung, were used to study the kinetics and reversibility of beta(2)-integrin receptors in response to changes in alveolar SP-D. Surface CD11b and CD11c were reduced on the alveolar macrophages within 3 days of SP-D deficiency and were restored with 3 days for CD11b and 7 days for CD11c of repletion of SP-D. SP-D deficiency caused a loss of cellular CD11b and CD11c content, indicating that the decrease in total cell content of the receptors was related to degradation rather than to redistribution of the receptor within the macrophage. CD11b and CD11c staining colocalized with Lamp-1 during SP-D deficiency, supporting the concept that reduced macrophage receptor levels resulted from increased lysosomal trafficking. Hydroxychloroquine, a lysomotropic agent, prevented the reduction of cellular and surface CD11b and CD11c. SP-D regulates surface CD11b and CD11c levels on the alveolar macrophage by modulating receptor trafficking, providing a mechanism by which SP-D mediates phagocytic activity in the alveolar macrophage.     

Surfactant protein A prevents silica-mediated toxicity to rat alveolar macrophages

Silicosis is a serious occupational lung disease associated with irreversible pulmonary fibrosis. The interaction between inhaled crystalline silica and the alveolar macrophage (AM) is thought to be a key event in the development of silicosis and fibrosis. Silica can cause direct injury to AMs and can induce AMs to release various inflammatory mediators. Acute silicosis is also characterized by a marked elevation in surfactant apoprotein A (SP-A); however, the role of SP-A in silicosis is unknown. We investigated whether SP-A directly affects the response of AMs to silica. In this study, the degree of silica toxicity to cultured rat AMs as assessed by a (51)Cr cytotoxicity assay was shown to be dependent on the time of exposure and the concentration and size of the silica particles. Silica directly injured rat AMs as evidenced by a cytotoxic index of 32.9 +/- 2.5, whereas the addition of rat SP-A (5 microg/ml) significantly reduced the cytotoxic index to 16.6 +/- 1.2 (P < 0. 001). This effect was reversed when SP-A was incubated with either polyclonal rabbit anti-rat SP-A antibody or D-mannose. These data indicate that SP-A mitigates the effect of silica on AM viability, and this effect may involve the carbohydrate recognition domain of SP-A. The elevation of SP-A in acute silicosis may serve as a normal host response to prevent lung cell injury after exposure to silica.     

Tannin mediated alveolar macrophage protein phosphorylation

Summary Experiments were designed to determine if cotton bract tannin, a component of cotton mill dust, would promote the phosphorylation of alveolar macrophage proteins in doses potentially achievable in vivo. Rabbit alveolar macrophages were loaded with ³²PO₄ and challenged with various doses of tannin for time periods ranging from two seconds to 120 minutes. Changes in protein phosphorylation began after two seconds and were maximal at five to fifteen minutes. Dose response studies using an exposure time of one hour showed phosphorylation changes began at 1 µg/mL and were maximal at 10 to 30 µg/mL. Phosphorylation changes were similar to but not identical to those induced by the protein kinase C activator, phorbol myristate acetate (PMA). Calcium ionophore, A-23187 had no clear effect either alone or in conjunction with PMA. These results indicate that cotton bract tannin is able to rapidly promote protein phosphorylation of alveolar macrophages at doses potentially achievable in vivo. Other mechanisms in addition to those of protein kinase C appear to be involved in this protein phosphorylation process.     

Biotinylation of heat shock protein 70 induces RANTES production in HEK293 cells in a CD40-independent pathway

Biotinylated proteins and peptides have been used as popular ligands for characterization of cell surface receptors by a variety of methods including flow cytometry. The number and the location of biotin moieties incorporated could alter the structural and physicochemical properties of ligands, although biotin is thought to be such a small molecule (244Da) that it is capable of being conjugated to most proteins without affecting their activity. Here, we demonstrate that the biotinylated HSP70 molecule via primary amines bound to epithelium-like HEK 293 cells in a saturable manner whereas the unlabeled counterparts of HSP70 other than mouse Hsp72 do not. This binding was not competed by either HSP70 or the biotin entity itself. Interestingly, the biotinylated HSP70 also elicited the production of CC-chemokine RANTES independent of CD40 signaling. This response occurred regardless of sequence diversity of HSP70 derived from different species, and neither the biotinylated ovalbumin nor the unlabeled HSP70 cross-linked with a biotinylated protein stimulated a significant level of RANTES production which was induced by biotinylated HSP70 itself. Our findings suggest that modification of HSP70 such as biotinylation may function as a biological alarm signal in the innate immune system.     

Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages

Surfactant protein-A (SP-A) plays multiple roles in pulmonary host defense, including stimulating bacterial phagocytosis by innate immune cells. Previously, SP-A was shown to interact with complement protein C1q. Our goal was to further characterize this interaction and elucidate its functional consequences. Radiolabeled SP-A bound solid-phase C1q but not other complement proteins tested. The lectin activity of SP-A was not required for binding to C1q. Because C1q is involved in bacterial clearance but alone does not efficiently enhance the phagocytosis of most bacteria, we hypothesize that SP-A enhances phagocytosis of C1q-coated antigens. SP-A enhanced by sixfold the percentage of rat alveolar macrophages in suspension that phagocytosed C1q-coated fluorescent beads. Furthermore, uptake of C1q-coated beads was enhanced when either beads or alveolar macrophages were preincubated with SP-A. In contrast, SP-A had no significant effect on the uptake of C1q-coated beads by alveolar macrophages adhered to plastic slides. We conclude that SP-A may serve a protective role in the lung by interacting with C1q to enhance the clearance of foreign particles.