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Originally published online as doi:10.1189/jlb.0104014 on June 24, 2004

Published online before print June 24, 2004
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(Journal of Leukocyte Biology. 2004;76:577-584.)
© 2004 by Society for Leukocyte Biology

Activation of murine macrophages by Neisseria meningitidis and IFN-{gamma} in vitro: distinct roles of class A scavenger and Toll-like pattern recognition receptors in selective modulation of surface phenotype

Subhankar Mukhopadhyay, Leanne Peiser and Siamon Gordon1

Sir William Dunn School of Pathology, South Parks Road, Oxford, United Kingdom

1 Correspondence: Sir William Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, UK. E-mail: Christine.holt{at}path.ox.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Innate and adaptive immune activation of macrophages (M{phi}) by microorganisms and antigen-activated lymphoid cells, respectively, plays an important role in host defense and immunopathology. Antigen-presenting cells express a range of pattern recognition receptors including the class A types I and II scavenger receptors (SR-A) and Toll-like receptors (TLR). Recognition of microbial products by SR-A and TLR controls uptake, killing, altered gene expression, and the adaptive immune response; however, the contribution of each receptor and interplay with cytokine stimuli such as interferon-{gamma} (IFN-{gamma}) are not defined. We used Neisseria meningitidis (NM), a potent activator of innate immunity, and IFN-{gamma}, a prototypic T helper cell type 1 proinflammatory cytokine, to compare surface antigens, secretion of mediators, and receptor functions in elicited peritoneal M{phi} from wild-type and genetically modified mouse strains. We show that these stimuli regulate major histocompatibility complex type II (MHC-II) and costimulatory molecules differentially, as well as expression of the mannose receptor and of M{phi} receptor with collagenous structure (MARCO), a distinct SR-A, which provides a selective marker for innate activation. In combination, NM inhibited up-regulation of MHC-II by IFN-{gamma} while priming enhanced release of tumor necrosis factor {alpha} and nitric oxide. The SR-A contributes to phagocytosis of the organisms but not to their ability to induce CD80, CD86, and MARCO or to inhibit MHC-II. Conversely, studies with lipopolysaccharide (LPS)-deficient organisms and/or TLR-4 mutant mice showed that LPS and TLR-4 are at least partially required to induce CD80, CD86, and MARCO, but LPS is not required to inhibit MHC-II. These studies provide an experimental model and identify surface markers for analysis of innate and acquired immune activation of M{phi}.

Key Words: macrophages • cellular activation • cell-surface molecules • phagocytosis • bacterial infection


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Innate and T cell-dependent pathways contribute to the activation of macrophages (M{phi}) during infection. Myeloid-derived antigen-presenting cells (APC) initiate adaptive cellular and humoral immune responses through germ line-encoded pattern recognition receptors (PRR) for microbial constituents [1 ] and promote inflammation and repair through cellular interactions and secretion of bioactive products. In turn, cytokines released by activated lymphoid and other immune cells enhance M{phi} effector mechanisms critical in host defense against infection. Exposure to interferon-{gamma} (IFN-{gamma}) induces a classical, activated phenotype in M{phi}, which includes enhanced major histocompatibility complex type II (MHC-II) expression [2 ], which can be counteracted by interleukin (IL)-10, whereas IL-4 and IL-13 treatment induce alternative activation in these cells [3 4 ]. Much has been learned regarding the direct interactions of various pathogens with M{phi} in studies using proinflammatory microbial products such as lipopolysaccharides (LPS) [5 ] and various infection models in vivo and in vitro. However, the initial role of pathogen and immune mediators has not always been distinguished, living microorganisms often circumvent M{phi} effector mechanisms [6 ], and isolated microbial constituents do not represent the full range of surface and other products found in the intact organisms.

Recently, it has been shown that in addition to opsonic receptors for antibody (Ab) [7 ] and complement [8 ], APC express a range of nonopsonic receptors for modified proteins, lipids, and carbohydrates [9 ], which together with Toll-like receptors (TLR), profoundly alter gene expression and effector functions [10 ]. PRR include scavenger receptors (SR), which have been implicated in lipoprotein homeostasis [11 ] and apoptotic cell clearance as well as host defense against infection [12 , 13 ]. The class A SRs are collagenous transmembrane glycoproteins, which include type I and type II isoforms (here referred to as SR-A), alternative splice variants of the same gene, and M{phi} receptor with collagenous structure (MARCO), the product of a distinct gene expressed by different subpopulations of M{phi} [14 ]. SR-A is an adhesion and endocytic receptor for a range of polyanionic ligands, implicated in nonopsonic uptake of gram-negative and -positive bacteria [15 , 16 ], and MARCO has also been shown previously to bind Escherichia coli and Staphylococcus aureusin vitro and in vivo [17 ].

Neisseria meningitidis (NM), a gram-negative diplococcus, is an important cause of bacterial meningitis and septic shock in infected humans. The organism and its cell-wall derivatives are potent, immune adjuvants [18 ], able to stimulate proinflammatory cytokine secretion and Ab production and to activate complement [19 ]. Earlier studies with bone marrow culture-derived M{phi} (BMDM) from wild-type (WT) and SR-A–/– mice showed that SR-A mediates most of the phagocytosis of intact, unopsonizsed NM; uptake was independent of lipid A [20 ], a known ligand for SR-A on gram-negative bacteria [21 ]. By contrast, studies of lipid A-deficient NM [22 ] and TLR-4 mutant mice showed a marked reduction in the release of the proinflammatory cytokines tumor necrosis factor {alpha} (TNF-{alpha}), IL-6, and IL-12 but not of SR-A-mediated phagocytosis, indicating that SR-A and TLR could be functionally uncoupled [20 ].

Although direct activation of M{phi} by microorganisms has been studied extensively, we know little about alterations in surface phenotype, which are relevant to many immune cell interactions and lack surface markers convenient for isolation and characterization of such activated M{phi}. We therefore set out to identify novel, selective surface markers and to clarify the role of selected PRR in innate activation of M{phi}. We have developed an in vitro cell-culture model using ethanol-killed NM, which retains SR-A ligand activity [20 ] in comparison with IFN-{gamma} treatment. Peritoneal M{phi} (PM) were obtained from various WT and genetically modified mouse strains and treated with agent alone or in combination. We examined the surface, endocytic, phagocytic, and secretory phenotypes of these M{phi} and established the role of SR-A and TLR-4 in M{phi} activation. Our studies show that innate activation by NM is distinct from that of cytokine activation by IFN-{gamma}, that these agents are able to potentiate each other, and that each pathway of activation induces a selective remodeling of the surface phenotype of the M{phi}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
1,1'-Dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL) was obtained from Intracell (Rockville, MD), and Rhodamine Green X (RdGnX), from Molecular Probes (Eugene, OR). Enzyme-linked immunosorbent assay (ELISA) kits for murine TNF-{alpha} were obtained from BD PharMingen (San Diego, CA). OPTIMEM-1 culture media were obtained from Gibco (Paisley, UK). Unless stated otherwise, all other reagents were from Sigma (Poole, UK). Bacteriologic plastic (BP) products were obtained from Greiner (Gloucester, UK), and all other plastic products were from Becton Dickinson Labware (Oxford, UK).

Animals
We used the following mouse strains: SR-A (SR-A–/–) [23 ], IFN-{gamma} receptor (IFN-{gamma}R–/–), and TLR-4 mutant (C3H/HeJ) [24 , 25 ] and their corresponding WT control strains, ICR/129, ICR/svev, and C3H/HeN, respectively, as well as BALB/c mice. C3H/HeJ and C3H/HeN strains were bought from Harlan-Olac (Bicester, UK). All animals were bred and housed under specific pathogen-free conditions and according to Home Office guidelines.

M{phi} isolation and culture
Bio-gel-elicited PM (Bg-PM) were prepared by injecting 1 ml polyacrylamide gel P-100 (Bio-Rad, Hercules, CA) beads (2% w/v in endotoxin-free water) intraperitoneally. After 4 days, peritoneal cells were harvested by lavage with phosphate-buffered saline (PBS) and plated on BP dishes in a defined serum-free medium, OPTIMEM, supplemented with 50 IU/ml penicillin-streptomycin and 2 mM L-glutamine. After 3–4 h of plating, adherent cells were washed three times to remove nonadherent cells and Bg beads. Yields were generally lower in knockout (KO) animals and also varied with mouse strain. After washing, purity of M{phi} in adherent monolayer was >95%, characterized by morphology (not shown) and expression of the M{phi}-specific markers SR-A and mannose receptor (MR; see Fig. 1 ).



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  		Figure 1. IFN-{gamma} differentially regulates SR-A, MHC-II, and MR expression in Bg-PM, M{phi} from BALB/c and ICR/129 mice were incubated overnight with or without IFN-{gamma} (20 ng/ml). Total cellular expression of SR-A (left panel), MHC-II (middle panel), and MR (right panel) was compared between untreated control (thin line) and treated groups (thick line) in fixed and permeabilized cells. The dotted histograms show staining by an appropriate isotype-matched mAb. (For control showing no staining by 2F8 mAb in SR-A–/– M{phi}, see Fig. 2B .)

 
Bacterial culture and fluorescent labeling
The capsulated serogroup B strain of NM MC58 [26 ], 44/76 [27 ], and lipid-A mutant strain 44/76lpxA [28 ] was treated as described before [26 ]. For fluorescent labeling, NM were harvested and resuspended in 70% ethanol overnight at 4°C and labeled with RdGnX (RdGnX-NM), according to the manufacturer’s protocol. Briefly, ethanol was removed by washing with PBS. Bacteria were resuspended in 0.2 M sodium bicarbonate, 100 µl RdGnX (dissolved in dimethyl sulfoxide) was added per 1010 bacteria, and the mixture incubated at room temperature for 1–1.5 h; finally, 400 µl hydroxylamine hydrochloride (per 1010 bacteria) was added, and the organisms incubated for a further 1 h to stop the reaction. Bacteria were washed with ice-cold saline, resuspended in PBS, and stored at –80°C.

Association of ethanol-fixed NM with M{phi}
M{phi} (106) were plated in six-well BP dishes 24 h before assay. Some M{phi} were pretreated overnight with 20 ng/ml recombinant mouse (rm)IFN-{gamma} (R&D Systems, Minneapolis, MN) or 1 µg/ml E. coli LPS (Sigma). The optimal concentration of IFN-{gamma} was established by titration of MHC-II induction (not shown). To examine the uptake of bacteria, M{phi} were washed twice with PBS and incubated with OPTIMEM containing ethanol-fixed RdGnX-NM (20 NM/M{phi} for 90 min at 37°C). Some wells were preincubated for 30 min with a general SR inhibitor, poly-Inosinic acid (poly-I), or a cognate, nonligand poly-Cytidylate (poly-C) at 50 µg/ml and retained throughout the assay. The endocytosis of 5 µg/ml DiI-AcLDL was examined in all assays as a positive control to measure SR function. After incubation, the M{phi} were washed three times with PBS and detached from BP surfaces by means of PBS containing 5 mM EDTA and 4 mg/ml Lidocaine before fixation with 4% (w/v) formaldehyde. The mean fluorescence was analyzed by flow cytometry using Cell Quest software.

Western blot analysis of cellular antigens
M{phi} were cultured on BP dishes and treated with IFN-{gamma} and/or NM, as required. M{phi} were then lysed using lysis buffer containing 1% (v/v) Igepal (Nonidet P-40), 10 mM Tris, 0.15 M NaCl, 10 mM NaN3, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 5 mM Iodoacetamide). The total protein concentration was measured in each assay condition using a bicinchoninic acid kit (Pierce, Rockford, IL), and equal amounts of protein were used for each sample. Western blots were performed according to standard protocols, and samples were probed with the following rat monoclonal Ab (mAb) for SR-A (2F8), MR (5D3), or MARCO (ED31). Binding was detected with horseradish peroxidase-conjugated secondary Ab and developed using an enhanced chemiluminescence kit (Amersham Biosciences, UK). ED31 was a gift from Professor Georg Kraal, Free University, Amsterdam.

Measurement of TNF-{alpha} and nitric oxide (NO) release
M{phi} were plated in 96-well BP culture dishes. The monolayers were washed three times with PBS to remove nonadherent cells and Bg beads and were then incubated with different doses of NM in the presence or absence of 20 ng/ml IFN-{gamma} for 24 h. E. coli LPS (1 µg/ml) was used as a positive control stimulus for TNF-{alpha} and NO. N-Monomethyl arginine was included as a control to block NO release. The tissue-culture supernatants were harvested and centrifuged at 1300 g to remove particulate matter.

TNF-{alpha} was measured by ELISA, according to the manufacturer’s protocol, with rmTNF-{alpha} as standard. NO release was detected using the colorimetric Greiss reaction, and absorbance was measured at 550 nm with sodium nitrite as standard.

Assay for cellular antigen expression
M{phi} were cultured on BP dishes and treated overnight with 20 ng/ml rIFN-{gamma} or 20 NM/M{phi} or culture medium, as appropriate. The M{phi} were detached as described above and blocked with PBS containing 5% (v/v) goat serum, 5% (v/v) rabbit serum, and 1% (w/v) bovine serum albumin (BSA). The M{phi} were stained with the primary rat mAb 2F8 (SR-A), 5D3 (MR), M.5114 (MHC-II), 5C6 (CR3), 16-10A1 (CD80), PO3 (CD86), or MCA1143F (CD40). Biotin-conjugated Ab and unconjugated Ab were detected with a Streptavidin-phycoerythrin conjugate or fluorescein isothiocyanate-labeled secondary Ab, respectively. Finally, the M{phi} were fixed with 4% paraformaldehyde (PFA) and analyzed by flow cytometry. In some cases, prior to Ab staining, the M{phi} were fixed in 4% PFA and permeabilized with 0.25% (w/v) saponin in the blocking solution. M.5114, 16-10A1, and PO3 were obtained from BD PharMingen and MCA1143F from Serotec (Oxford, UK). All assay variables were examined in triplicate, and data shown are representative of at least three independent assays.

Statistical analysis
The statistical significance of results was determined by Student’s paired t-test, and significance tested at the 95% confidence level (P≤0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of IFN-{gamma} on SR-A expression and function in Bg-PM
Previous studies have shown that BMDM depend on SR-A for most of their ability to ingest unopsonized NM [20 ]. To establish a model with Bg-PM, M{phi} from WT and SR-A–/– mice were plated in BP dishes and treated with IFN-{gamma} and/or NM. Fixed and permeabilized M{phi} were labeled with the SR-A-specific mAb 2F8. Figure 1 shows that although the two WT strains BALB/c and ICR/129 expressed different levels ofSR-A, IFN-{gamma} treatment had no effect on receptor expression by either strain. M{phi} activation by IFN-{gamma} was confirmed by the induction of MHC-II and down-regulation of MR (Fig. 1) [2 , 4 , 29 ]. Morphologic studies revealed that IFN-{gamma} and NM induced spreading of WT and SR-A–/– Bg-PM (not shown), indicating that M{phi} spreading on BP was independent of SR-A adhesion [30 ]. Further experiments with Bg-PM from IFN-{gamma}R–/– mice confirmed that the receptor was required for spreading induced by IFN-{gamma} but not by NM (not shown). IFN-{gamma} treatment had little or no effect on the uptake of NM or of DiI-AcLDL (Fig. 2A ). Although SR-A–/– Bg-PM showed the expected marked reduction in endocytosis of DiI-AcLDL (Fig. 2B) , uptake of NM by these M{phi} was less dependent on SR-A than by BMDM (~45% reduction vs. ~90%) [20 ], irrespective of IFN-{gamma} treatment (not shown). However, poly-I, a nonspecific, polyanionic SR inhibitor, but not poly-C (not shown) reduced uptake of NM by Bg-PM efficiently (>90%), suggesting that Bg-PM express additional SR activities, independent of SR-A.



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  		Figure 2. IFN-{gamma} does not alter Poly-I sensitive (SR-A/SR-mediated) phagocytosis of NM and endocytosis of DiI-AcLDL. (A) Bg-PM of BALB/c, 129/ICR, and SR-A–/– mice were incubated overnight with 20 ng/ml rmIFN-{gamma} or media alone. Cells were washed three times with PBS and incubated with 5 µg/ml DiI-AcLDL or ethanol-fixed RdGnX MC58 (20/M{phi}) for 90 min. The endocytosis of DiI-AcLDL (right panel) and phagocytosis of RdGnX-NM (left panel) were measured by flow cytometry. NM and DiI-AcLDL uptake by IFN-{gamma}-treated (thick line) and untreated cells (thin lines) was compared for each strain. Bacterial uptake was compared in the presence (thick line) or absence (thin line) of poly-I (middle panel). M{phi} from the corresponding strain of mouse, incubated with media alone, are shown as dotted histograms. (B) SR-A expression (mAb 2F8 labeling) and uptake of RdGnX-NM and DiI-AcLDL by ICR/129 (thin line) and SR-A–/–(thick line) M{phi}. Thin, unmarked histograms indicate corresponding negative controls.

 
NM induces MARCO, a distinct class A SR
MARCO is an independent class A SR [14 ], which might contribute to the SR-A-independent poly-I-inhibitable phagocytic ability of Bg-PM. It is constitutively expressed on marginal zone M{phi} and on freshly isolated resident PM and also known to be induced by LPS in vitro [31 ]. Figure 3 shows that MARCO was barely detectable in Western blots of Bg-PM but strongly up-regulated after overnight incubation with NM. The effect of IFN-{gamma} by itself on MARCO expression was minimal, although there was some potentiation of NM-induced expression. This pattern of up-regulation was quite different from that of SR-A, which was minimally altered by either stimulus; the MR showed striking down-regulation after combined NM and IFN-{gamma} treatment, greater than with IFN-{gamma} alone. The up-regulation of MARCO by NM was independent of SR-A, as the SR-A–/– M{phi} had comparable induction. However, induction was absent in NM-treated C3H/HeJ compared with C3H/HeN M{phi} (Fig. 3B) , consistent with a role for TLR-4 in its up-regulation (Fig. 3B) .



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  		Figure 3. NM and IFN-{gamma} regulate SR-A, MR, and MARCO expression differentially. (A) Bg-PM from the ICR/129 strain were incubated overnight with NM (20 MC58/M{phi}), IFN-{gamma} (20 ng/ml), or a combination of both. Western blotting was used to detect SR-A and MR antigens in cell lysates of ICR/129 M{phi} with 2F8 and 5D3, respectively. Similarly, MARCO expression was detected in WT and SR-A–/– strains with mAb ED-31. Lanes 1–4 represent SR-A, MR, or MARCO expression in untreated, NM-, IFN-{gamma}-, and NM + IFN-{gamma}-treated cells, respectively. Similar amounts of lysate protein were loaded in different lanes. (B) Western blotting was performed to detect MARCO expression in cell lysates of Bg-PM from C3H/HeN and C3H/HeJ strains after overnight incubation with NM (20 NM/M{phi}).

 
Effects of NM and IFN-{gamma} on TNF-{alpha} and NO release; role of SR-A and TLR-4
We wanted to examine further the role of TLR-4 versus SR-A in M{phi} activation, and so we studied TNF-{alpha} and NO release in response to NM or IFN-{gamma} treatment, as they are important secretory products of M{phi} [32 , 33 ]. Figure 4 shows that NM alone was able to induce the release of TNF-{alpha} and NO by Bg-PM, which was markedly enhanced by combined treatment. IFN-{gamma} by itself did not evoke secretion nor did untreated M{phi} release detectable products (not shown). Experiments with IFN-{gamma}R–/– M{phi} showed a marked reduction in secretion, which was almost complete for TNF-{alpha} with some residual IFN-{gamma}R-independent NO release. TNF-{alpha} release by IFN-{gamma}R–/– M{phi} was reduced when NM was used alone, suggesting that low levels of endogenous IFN-{gamma} contribute to the response in WT M{phi} even when no exogenous IFN-{gamma} was added. Control experiments confirmed that endocytosis of Ac-LDL and uptake of NM were unaffected in IFN-{gamma}R–/– M{phi} and therefore notresponsible for reduced secretion (not shown). The role of SR-A and TLR-4 in mediator secretion is also shown in Figure 4 . We incubated WT and C3H/HeJ M{phi}, which lack a functional TLR-4, or WT and SR-A–/– M{phi} with NM in the presence or absence of IFN-{gamma}. Apart from a modest reduction in NO release in the absence of IFN-{gamma}, SR-A–/– M{phi} displayed identical responses after combined treatment. In TLR-4-deficient M{phi}, NM failed to induce NO or TNF-{alpha}; this could be overcome by IFN-{gamma} for NO release but not for TNF-{alpha}. These studies indicated that NM stimulated secretion of mediators through TLR-4 but not SR-A, that IFN-{gamma} could overcome some of the TLR-4-dependent functions, and that TNF-{alpha} and NO were regulated differentially.



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  		Figure 4. IFN-{gamma} and NM act synergistically to induce maximal NO and TNF-{alpha} release in Bg-PM. M{phi} SR-A–/–, IFN-{gamma}–/–, and C3H/HeJ mice and their corresponding WT strains ICR/129, C57/Bl6, and C3H/HeN were incubated with different concentrations (1, 10, 100 NM/M{phi}) of NM, alone or in combination with IFN-{gamma} at 37°C for 24 h. The culture supernatant was harvested, and TNF-{alpha} and NO release was measured by cytokine ELISA and the Greiss reaction, respectively. Increased levels of TNF-{alpha} and NO were detected in culture supernatant in a dose-dependent manner (not shown). The bar diagram represents TNF-{alpha} and NO release for a single dose (10 NM/M{phi}). As expected, unstimulated cells did not release either mediator. The results are representative of at least three experiments, and the average of triplicate assays is shown. *, No detectable TNF-{alpha} reslease. IFN-{gamma} significantly (P≤0.05) increased NO and TNF-{alpha} release compared with its untreated counterpart in all WT and KO strains described except conditions marked with $.

 
Innate and IFN-{gamma}-activated M{phi} differ in cell-surface phenotype
To extend our characterization of microbial versus immune cytokine activation via NM and IFN-{gamma}, we examined expression of MHC-II costimulatory molecules (CD-80, CD-86, CD-40) [34 ] and of another PRR, CR3 [35 ] (Fig. 5 ). Unexpectedly, the presence of NM prevented MHC-II induction by IFN-{gamma} but had no effect on basal expression in its absence. NM alone was more potent at inducing CD-80/CD-86/CD-40 than IFN-{gamma}, accounting for most of the enhanced expression of these costimulatory antigens under the conditions used. CR3 expression was unaffected by any treatment.



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  		Figure 5. IFN-{gamma} and NM affect expression of Bg-PM cell-surface antigens differentially. Bg-PM of BALB/c mice were incubated overnight with serogroup B NM, MC58 (20 bacteria/M{phi}), IFN-{gamma} (20 ng/ml), or a combination of IFN-{gamma} and MC58. Cells incubated with media alone served as control. Surface expression of CD80, CD86, CD40, CR3, and MHC-II was studied. The expression of each antigen was compared between untreated control (thin line) and treated groups (thick line). The dotted histogram in each group shows staining by an appropriate isotype-matched mAb.

 
We studied the role of LPS by comparing the effect of a lipid A mutant strain of NM lpxA [28 ] with WT NM on the expression of MHC-II, CD80, and CD86. Figure 6 shows that both strains down-regulated IFN-{gamma}-induced MHC-II expression equally; apart from a modest effect on CD-80 expression, LPS was not significant in the up-regulation of costimulatory antigens by NM, alone or with IFN-{gamma}. Further control experiments with permeabilized and SR-A–/– M{phi} showed that IFN-{gamma} up-regulated surface expression as well as total expression of MHC-II and that SR-A did not play a role in MHC-II down-regulation or in costimulatory antigen enhancement by NM (not shown).



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  		Figure 6. Neisserial LPS is partially responsible for induction of CD80 and CD86 but not for NM-mediated inhibition of MHC-II. Bg-PM of BALB/c mice were incubated overnight with IFN–{gamma} (20 ng/ml), lpxA (20 bacteria/M{phi}), or its WT control strain 44/76 (20 bacteria/M{phi}), alone or in combination. Cells incubated with media alone served as control. Surface expression of CD80, CD86, and MHC-II was compared in untreated control (thin line) and treated groups (thick line and also pointed by arrows). The thin, unmarked histograms show staining by an appropriate isotype-matched mAb.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The concept of pattern recognition by APC receptors and discovery of evolutionarily conserved pathways of signal transduction and gene activation in M{phi} and dendritic cells in response to microorganisms have renewed interest in innate immunity and its relation to adaptive immune responses. Here, we have used prototypic stimuli, intact NM, and IFN-{gamma} to compare their actions, alone and together, in murine-elicited PM. We have placed particular emphasis on the PRR, SR-A, and TLR and exploited genetically modified mouse strains to examine the surface phenotype and selected endocytic/phagocytic and secretory activities of M{phi} induced by these agents. Our main results are summarized in Table 1 and show that NM and IFN-{gamma} have distinct but potentiating effects on M{phi} functions; different PRR contribute independent roles to phagocytosis and secretory responses, and these cellular responses can be uncoupled from each other; the related SR MARCO is differentially regulated from SR-A, depends on TLR-4 functions for induction, and provides a valuable marker for innate activation of M{phi}; and NM prevent IFN-{gamma}-induced up-regulation of MHC-II and may therefore be able to modulate APC functions independent of their LPS content.


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  		Table 1. Effects of NM and IFN-{gamma} on M{phi} Activation; Role of SR-A and TLR-4

 
The in vitro model we have characterized is efficient and reproducible. Use of Bg-PM allows access to defined murine strains, with high yields of elicited M{phi}, which are readily purified by adhesion. Bg-PM are more representative of M{phi} recruited by inflammatory stimuli in vivo than BMDM, which express high levels of SR-A as a result of CSF-1 present in the L cell-conditioned growth medium [36 ]. The use of BP vessels and serum-free media avoids ligation of SR-A, which promotes divalent cation-independent adhesion to tissue-culture plastic [30 ] and facilitates detachment for further analysis by fluorescein-activated cell sorter-based assays. Ethanol-treated NM, the model organisms we studied, provide a potent and safe innate stimulus, more complex than isolated bacterial components, and one that is relevant to pathogenesis. Our earlier studies confirmed that SR-A plays a comparable role in uptake of live and fixed NM [20 ]. Ethanol fixation preserves key ligand functions for SR-A and TLR-4 and limits surface variation in the organism. The two agents were added simultaneously and incubated with Bg-PM overnight, allowing for sequential responses and interactions. The effects of IFN-{gamma} were validated by the use of M{phi} from IFN-{gamma}R–/– animals, and these studies made it possible to distinguish similar cellular responses such as spreading induced by NM and IFN-{gamma} itself. Earlier studies by Paulnock and colleagues [37 , 38 ] have used general SR ligands such as LPS, lipoteichoicacid, or maleylated BSA to study interactions with IFN-{gamma} and phenotypic changes in the RAW M{phi}-like cell line. However, these studies did not differentiate among SR and did not relate isolated microbial components to intact organisms able to use multiple PRR.

The role of the type A SR was defined in our studies by use of WT versus SR-A–/– mice and the specific mAb 2F8. SR-A was responsible for most of the endocytosis of Ac-LDL but contributed less to phagocytosis of unopsonized NM in Bg-PM than in previously used BMDM (Fig. 2) [20 ]. NM or IFN-{gamma} did not significantly affect SR-A expression and function (Figs. 1 and 3) . By contrast, the structurally related but independently encoded molecule MARCO was strikingly and selectively up-regulated by NM, and this induction was shown to be dependent on TLR-4 but not on SR-A function (Fig. 3) . Previous studies have noted the role of LPS and other microbial stimuli in MARCO up-regulation [39 ] and M{phi} binding of gram-negative and -positive bacteria [14 , 17 ], and its enhanced expression in activated cells has been detected by microarray studies [40 ]. Here, we provide the first evidence for TLR dependence for inducible but not constitutive MARCO expression. As the TLR-4 does not play a direct role in pathogen binding or phagocytosis, it may serve as a pathogen sensor to induce additional phagocytic receptors such as MARCO. Pretreatment of Bg-PM with E. coli LPS (not shown) enhanced binding of RdGnX-NM by WT and SR-A–/– M{phi}, suggesting a possible role for induced MARCO in NM binding and uptake after coincubation. Further studies with KO and double-KO mice are required to elucidate its possible contribution to SR-A-independent, poly-I-inhibitable uptake of NM by Bg-PM.

We confirmed the role of TLR-4 in induced release of TNF-{alpha} and NO, observing strong potentiation of NM-induced responses and some differences between the two products, and extended earlier studies about the independence of TLR-4 and SR-A functions in promoting secretory and phagocytic responses to NM, respectively [20 ]. Pridmore et al. [22 ] showed that lpx-A mutant NM elicit proinflammatory cytokines through TLR-2, but the induction of proinflammatory cytokine by these mutants is much weaker than by the WT NM strain. In our model system using WT NM and C3He/J M{phi}, we found that LPS is the major inducer of cytokines. However, in WT, NM LPS probably masks these TLR2 ligands, and TLR4-LPS plays the dominant role in cytokine release, as shown in Figure 4 . The role of IFN-{gamma} in potentiating immune responses to microbial constituents is well known. Recent studies by Mantovani and colleagues [41 ] indicated that IFN-{gamma} up-regulated surface expression of TLR-4, MD-2, and MyD-88 and enhanced LPS-dependent phosphorylation of IL-1R-associated kinase and DNA-binding activity of nuclear factor-{kappa}B, all ultimately leading to enhanced TNF-{alpha} and other proinflammatory cytokine secretion by human mononuclear cells [42 ]. Similar mechanisms may underlie synergism between IFN-{gamma} and TLR-4-dependent stimulation by NM in the present studies.

We have also added to earlier studies on MR and CR3, showing that these PRR are independently regulated [29 ], presumably reflecting differential gene and protein expression. Down-regulation of MR provided a sensitive and selective marker of IFN-{gamma} activity on M{phi} and was shown to be potentiated by NM, which had little, if any, effect on MR expression by itself (Fig. 3) . The expression of CR3 and SR-A, unaltered by either stimulus, as well as of a range of other surface antigens (not shown) reinforces the selectivity of surface phenotype changes reported here. These results highlight the value of MARCO as a specific marker induced by innate but not IFN-{gamma} activation.

The induction of costimulatory molecules (CD-80, CD-86, CD-40) by microbial agents is well established [43 ]. Here, we show that IFN-{gamma} by itself has little effect on expression of these antigens, although it potentiated the direct action of NM. However, the potent, inhibitory effect of NM on IFN-{gamma}-induced MHC-II expression was a striking, new observation. This was true for total as well as surface expression (not shown) and was independent of LPS, as shown with a lipid-A-deficient mutant strain of NM, which is readily phagocytosed via SR-A, as reported in earlier studies [20 ]. The inhibition of MHC-II up-regulation is not unique to NM. The 19-kD lipoprotein of Mycobacterium tuberculosis was shown to inhibit IFN-{gamma}-regulated human leukocyte antigen-DR and Fc receptor for immunogloublin G1 on human [44 ] and MHC-II expression on mouse [45 ] M{phi} through TLR-2 and to inhibit class II-transactivator expression [46 ], a potential mechanism for immune evasion. In other studies, Mycobacterium avium reduced IFN-{gamma}R expression and phosphorylation of the Janus tyrosine kinase/signal transducer and activator of transcription signaling pathway and targeted IFN-{gamma}-inducible genes to avoid immune surveillance [47 ]. Further investigations are needed to establish whether the effect on MHC-II is relevant to NM pathogenesis and could promote evasion of specific host immune responses.

Our study defines a novel model system for global analysis of gene expression during innate versus immune cytokine activation of M{phi}. Although more limited in scope than microarray studies, the present report focuses on the expression, function, and regulation of surface proteins, which contribute to recognition, microbial and cellular interactions, and immune responses. Further studies are needed about the mechanism of altered cell-surface phenotype to account for differential up- or down-regulation of individual plasma membrane receptors. We have shown that altered surface expression of several receptor antigens (MARCO, MR, MHC-II) reflected changes in total cellular protein and not simply redistribution or shedding. In addition to the selective remodeling of surface antigen and PRR expression described here, we require further markers to characterize innate activation of M{phi} in situ and ex vivo and to define the relative contribution of the microbe and the major adaptive immune cytokine, IFN-{gamma}, in further modulating functions of activated M{phi}.


   ACKNOWLEDGEMENTS
 
S. M. is supported by an Usher Cunningham Studentship, Exeter College, and L. P., by an EPA Junior Research Fellowship, St. Cross College, University of Oxford. S. G. is supported by a grant from the Medical Research Council, UK. We thank Prof. Richard Moxon and Dr. Katherine Makepeace, Department of Pediatrics, John Radcliffe Hospital, Oxford, for the kind gift of the MC58+ and lpxA mutant strains and Professor G. Kraal, Free University, Amsterdam, for providing ED-31 Ab.

Received January 11, 2004; revised March 18, 2004; accepted May 7, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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