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A Monoclonal Antibody Directed Against the Murine Macrophage Surface Molecule F4/80 Modulates Natural Immune Response to Listeria monocytogenes

Department of Immunology, Robert Koch Institute, Berlin, Germany

	Abstract

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Whole spleen cell cultures from SCID mice release high levels of IFN- when exposed to heat-killed Listeria monocytogenes (HKL). This microbe-induced and T cell-independent response depends on both macrophages (M) and NK cells: HKL-stimulated M release TNF- and IL-12, which together activate NK cells for IFN- release. We show here that this cytokine-mediated activation cascade can be modulated by a mAb against the M surface glycoprotein F4/80. HKL-induced IL-12, TNF-, and IFN- in SCID whole spleen cell cultures was inhibited by coincubation with anti-F4/80 mAb whereas IL-1 and IL-10 were enhanced. Both effects were apparent at mRNA and protein release levels. Whereas inhibitory activities were F4/80 Ag specific, stimulatory effects were Fc dependent and nonspecific. Furthermore, cytokine inhibition by anti-F4/80 was only apparent when M and NK cells were present simultaneously and in close vicinity, indicating that direct cell-to-cell contact is a prerequisite. These data suggest a novel pathway for microbe-induced M/NK cell interaction involving direct cell-to-cell signaling and give the first evidence for a functional role of the M surface glycoprotein F4/80.

	Introduction

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Nonspecific or natural immune responses form a first line of defense against invading pathogens and have decisive regulatory functions for subsequent Ag-specific T cell-dependent immune reactions. IFN- plays a central role in regulating both stages of defense and is the crucial cytokine for initiating the effector phase of cell-mediated immunity by activating M for enhanced microbicidal activity (1, 2). Murine listeriosis is a widely used model for studying the mechanisms of natural cellular immune response to microorganisms. During infection with the Gram-positive facultative intracellular bacterium Listeria monocytogenes, IFN- production is necessary for control of bacterial growth both in immunocompetent and in SCID mice, which lack functional T and B cells (3, 4). In SCID mice or SCID whole spleen cell (WSC)² cultures, L. monocytogenes can induce IFN- release only in the presence of both M and NK cells (5). As activation pathway, a sequence of steps has been described in which Listeria first stimulate M to release IL-12 and TNF-, which in combination activate NK cells for IFN- release (6, 7, 8). In a positive feedback circle, IFN- may further activate M for enhanced cytokine-synthesis and release of antimicrobial and cytotoxic metabolites such as reactive oxygen and nitrogen intermediates (9, 10).

In vitro experiments performed in our laboratory as well as by others (6) revealed that the level of IFN- release not only depends on the relative numbers of microorganisms but also on the culture system itself. Direct coincubation of L. monocytogenes organisms or heat-killed L. monocytogenes (HKL) with M and NK cells results in 2-fold higher concentrations of IFN- compared with Listeria-stimulated M and NK cells cultured in the same well but separated by a semipermeable membrane. The latter system allows free passage of cytokines but inhibits cell-to-cell contact between the two cell types. Moreover, in vitro experiments with Pneumocystis carinii show that IFN- is not produced by NK cells at all when these are separated by a semipermeable membrane from M cultured with this extracellular fungal pathogen. However, when P. carinii organisms are given to nonseparated M and NK cells, IFN- release can be observed at levels comparable with those produced in response to stimulation with L. monocytogenes (11). These results led to the hypothesis that cell contact-dependent costimulatory signals might also play a role in initiating cellular cooperation in natural immunity.

We approached this question in the in vitro listeriosis model and SCID WSC with functional inhibition studies using a panel of mAbs against different M surface molecules. Incubation with an Ab directed against the murine M surface glycoprotein F4/80 (12) resulted in markedly reduced levels of IFN- released into the supernatant (SN). Because of its highly restricted expression, the F4/80 Ag is widely used as a specific marker for murine M. The Ag is well expressed by M of the spleen (red pulp), lung, liver, peritoneal cavity, and nervous system (12, 13, 14, 15). It is a glycoprotein of 160 kDa and has recently been cloned (16). The deduced amino acid sequence indicates a seven-transmembrane molecule with homologies to human EMR1 (17) and CD97 (18). As F4/80 is down-regulated in vivo after infection with bacillus Calmette-Guerin (19, 20), its expression seems to depend on the activation status of the M. To date, specific ligands and biologic functions of the F4/80 Ag have not been described.

Here, we report on experiments showing that a mAb directed against F4/80 was able to modulate Listeria-induced cytokine signaling between M and NK cells. For it to be effective, both cell populations had to be present simultaneously, moreover in close vicinity, possibly in direct cell-to-cell contact. The data suggest an important costimulatory mechanism involving cell-bound molecules in natural cellular immune reactions to L. monocytogenes.

	Materials and Methods

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Animals and microorganisms

Male and female C.B-17^scid/scid (SCID) mice 6–8 wk old (21), provided by the Deutsches Krebsforschungsinstitut (Heidelberg, Germany) and bred at the Zentrale Versuchstierzucht, Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin (Berlin, Germany), were kept at the Robert Koch Institute in a separate animal room in filter-top cages within laminar air flow cabinets. All material, including water, bedding, and food was sterilized before use, and the filter tops were removed only within class II safety cabinets. The mice were negatively screened for serum Ab and were age and sex matched within individual experiments. The mouse-pathogenic L. monocytogenes strain EGD, serotype 1/2b (22), was raised in tryptone soya broth (36°C, 24 h; Oxoid, Basingstoke, U.K.), washed three times in HBSS (2000 x g, 6°C, 12 min), counted as CFU on tryptone soya agar (Oxoid), and stored in aliquots at -70°C. For HKL, bacteria were exposed to 62°C in a water bath for 60 min. Before killing, viable bacteria were assessed as CFU and the concentration of HKL estimated accordingly. Aliquots of HKL were frozen in HBSS at -20°C until use.

Cells

For WSC preparations, spleens from SCID mice (six per experiment) were homogenized and red cells removed by hypotonic lysis. For purification of NK cells, adherent cells were removed by incubating WSC in nylon wool columns (Vygon, Lyon, France) as described (23). Purity of cells in the effluent was assessed according to morphologic, antigenic, and functional criteria. SCID mouse-derived, nonadherent cells of large granular lymphocyte morphology, negative for nonspecific esterase (Sigma, Deisenhofen, Germany) and F4/80, positive for asialo GM1 (Wako Chemicals, Neuss, Germany) (24, 25), and toxic for YAC-1 cells (26) were designated NK cells. Addition of low concentrations of IL-2 (10 U/ml; a concentration below activation threshold) was necessary to prevent cell death (apoptosis) over the assay period. For preparation of splenic M, WSC were resuspended in RPMI 1640 without antibiotics, supplemented with 10% FCS (herein called R10 medium; Boehringer Mannheim, Mannheim, Germany), and incubated in tissue culture-grade petri dishes for 90 min. Nonadherent cells were stringently removed by repeated rinsing with warm (37°C) R10, the dishes chilled for 1 h at 4°C, and the now loosely adherent cells harvested by flushing with cold R10 pressed through an 18 gauge needle. These cells consisted >98% of M as judged from their morphology in Diff-Quik-stained micrographs, plastic adherence, positivity for nonspecific esterase, and binding of the M-specific mAb F4/80. Before experiments, cell viability was assessed by Trypan blue exclusion. After harvest of SNs, viability was again controlled in an MTT assay according to Ref. 27.

Functional assays

For all functional assays, 5 x 10⁵ M or NK cells and 1 x 10⁶ nucleated WSC were seeded in 1 ml R10/well in 24-well plates (Falcon Multiwell, Becton Dickinson, Oxnard, CA). Cultures were stimulated with HKL for 48 h (18 h for TNF testing) before harvest of SN. Cell culture inserts of polyethylene terephthalate (Falcon) were used to separate M (lower chamber) from NK cells (upper chamber) when indicated. For stimulation of NK cells by M SN, M were incubated for 18 h (or longer; not shown). M culture SN were then centrifuged, filtered, and given to freshly isolated NK cells for another 48 h. Monoclonal Abs against M surface molecules were added simultaneously with HKL at varying concentrations as indicated: rat anti-mouse CD11a (IgG2a) clone M17/4, rat anti-mouse CD86 (IgG2a) clone GL1 (both PharMingen, Hamburg, Germany), rat anti-mouse-CD11b (IgG2b) clone M1/70, rat anti-mouse-MHC class II (IgG2b) clone M5/114 (both hybridomas were kindly provided by Paul Kaye, London School of Hygiene and Tropical Medicine, U.K.), rat anti-mouse CD54 (IgG2a) clone KAT-1 (Immunokontact, Frankfurt am Main, Germany), and rat anti-mouse F4/80 (IgG2b) clone CI:A3–1 (Serotec, Oxford, U.K.). For isotype controls rat mAb clones R35–95 (IgG2a) and R35–38 (IgG2b; both PharMingen) were used. When necessary, sodium azide was removed by centrifugation dialysis (Centricon, Amicon, Beverly, MA). Ag-nonspecific binding of Ab to FcIII or FcII receptors was inhibited by giving 10 µg/ml rat anti-mouse-CD16/CD32 (IgG2b) clone 2.4G2 (Fc-block; PharMingen) to cell cultures 10 min before adding test Ab. Fab fragments of F4/80 were obtained by cleavage with preactivated papain (Sigma). In short, papain was preactivated at 37°C for 30 min with 10 mM L-cysteine (Sigma) in 0.1 M acetic buffer (pH 6.0). After removal of free cysteine by gel filtration (Sephadex G-25, Pharmacia), Ig was mixed with papain at a ratio of 20:1 and incubated at 37°C for 2 h. Reaction was stopped with 0.03 M iodoacetamide (Sigma). Papain and iodoacetamide were removed by dialysis (Centricon), and digestion was controlled by SDS-PAGE.

Detection of inorganic NOs (iNOs) and cytokines

iNOs were quantitated with Griess reagent according to Reference 28 , as described elsewhere (29) (lower detection threshold, 5 µM). IL-1ß was measured by ELISA using different polyclonal rabbit anti-mouse-IL-1ß sera (Immunogenetics, Zwijndrecht, Belgium) for coating and biotinylated for detection (threshold, 160 pg/ml). Production of IL-10 was measured by ELISA using the rat anti-mouse-IL-10 clone JES.5-2A5.1.1 (IgG1; kindly provided by Greg Bancroft, London School of Hygiene and Tropical Medicine) for coating and biotinylated rat anti-mouse-IL-10 clone SXC-1 (IgM; PharMingen) for detection (threshold, 40 pg/ml). IFN- was quantitated by ELISA using the rat-anti-mouse clone R4–6A2 (IgG1; provided by Paul Kaye) for coating and biotinylated AN-18 (IgG1; kindly provided by Hans-Ulrich Weltzien, Max Planck-Institut für Immunbiologie, Freiburg, Germany) for detection (threshold, 40 pg/ml). Production of IL-12 (p40/p70) was measured by ELISA using the rat-anti-mouse clone C15.6 (IgG1) for coating and biotinylated C17.8 (IgG2a; both PharMingen) for detection (threshold, 80 pg/ml). TNF- was quantitated by ELISA using the rat anti-mouse TNF clone G281-2626 (IgG1) for coating and biotinylated MP6-XT3 (IgG1; both PharMingen) for detection (threshold, 80 pg/ml).

RT-PCR

Total RNA was extracted from 5 x 10⁶ WSC with Trizol (Life Technologies, Karlsruhe, Germany) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1–5 µg total RNA by 200 U Superscript II reverse transcriptase (Life Technologies) with 100 ng random primers (Life Technologies). First-strand cDNA was amplified by PCR using 2 µM sense/antisense primers with 2.5 U Taq polymerase (Goldstar, Eurogentec, Seraing, Belgium) in a Genius Thermocycler (Genius, Thermo-Dux, Wertheim, Germany) in a total volume of 50 µl. The reaction buffer consisted of 75 mM Tris-HCL, 2.5 mM MgCl₂, 20 mM (NH₄)₂SO₄, 0.01% Tween 20, and 20 µM dNTP. Thirty-six PCR cycles were performed (1 min at 94°C, 1 min at 61°C, and 3 min at 72°C). First cycle denaturing was conducted for 2 min at 94°C, and a final extension was performed for 4 min at 72°C. The sense and antisense primers were as follows: IL-1ß sense, 5'-GCAACTGTTCCTGAACTCA-3'; IL-1ß antisense, 5'-CTCGGAGCCTGTAGTGCAG-3'; IL-10 sense, 5'-TACCTGGTAGAAGTGATGCC-3'; IL-10 antisense, 5'-CATCATGTATGCTTCTATGC-3'; IL-12 sense, 5'-CGTGCTCATGGCTGGTGCAAAG-3'; IL-12 antisense, 5'-CTTCATCTGCAAGTTCTTGGGC-3'; IFN- sense, 5'-TACTGCCACGGCACAGTCATTGAA-3'; IFN- antisense, 5'-GCAGCGACTCCTTTTCCGCTTCCT-3'; TNF- sense, 5'-ATGAGCACAGAAAGCATGATC-3'; TNF- antisense, 5'-TACAGGCTTGTCACTCGAATT-3'; inducible NO synthase (iNOS) sense, 5'-CATGGCTTGCCCCTGGAAGTTTCTCTTCAAAG-3'; iNOS antisense, 5'-GCAGCATCCCCTCTGATGGTGCCATCG-3'; hypoxanthine phosphoribosyltransferase (HPRT) sense, 5'-GTTGGATACAGGCCAGACTTTGTTG-3'; and HPRT antisense, 5'-GAGGGTAGGCTGGCCTATAGGCT3'. RT-PCR products were electrophoresed through a 1.0% agarose gel containing 0.83 µg/ml ethidium bromide with a 100 bp DNA marker (Life Technologies) run in parallel. The gel was read in a UV transilluminator and documented by photography (Biometra Göttingen, Germany). All samples of an experiment were first analyzed for HPRT mRNA only. The HPRT bands were compared densitometrically (WinCam Software, Cybertech, Berlin, Germany), and the samples were diluted accordingly to achieve a common initial cDNA concentration before being analyzed for cytokine message.

Statistics

Significance of results was determined according to Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Release of HKL-induced NK-derived IFN- is mediated by soluble M-derived factors but is reduced when cell contact is inhibited

M and NK cells are both necessary and sufficient for HKL particles to induce IFN-. As shown in Table I, the concentrations of IFN- released by equal numbers of these cell types differ according to the experimental design. The highest levels of IFN- are reached when HKL, M, and NK cells are cocultivated in the same well. When NK cells are cultivated in the SN of HKL-treated M, IFN- release is appreciable, but reduced compared with the coculture system. To ensure that this reduced performance of NK cells was not due to decay of biologic activities in the M-SN, HKL-treated M and purified NK cells were incubated in the same well, only separated by a semipermeable membrane. Again, NK-derived IFN- was reduced to similar levels, or was slightly elevated, compared with those achieved when culturing purified NK cells in SN of HKL-treated M. In all three experimental setups, the amount of IFN- released was proportional to the relative numbers of HKL particles. In parallel experiments, viable L. monocytogenes organisms (5 x 10⁶/well) induced similar concentrations of IFN- in M/NK cell cocultures (5.44 ± 0.46 ng/ml). IFN- release was reduced when cell contact between M and NK cells was inhibited by a semipermeable membrane (3.86 ± 0.75) or when medium from M stimulated with viable L. monocytogenes organisms was transferred to NK cells (3.56 ± 0.60). HKL did not stimulate IFN- production in purified NK cells alone (see Table II). These results indicated that, apart from M induction of NK cell activation via soluble cytokines, a costimulatory pathway might exist that requires close vicinity between M and NK cells, possibly even direct cell-to-cell contact.

View this table: [in this window] [in a new window]   Table I. HKL-induced NK-derived IFN- is mediated by M-derived soluble factors but diminished when cell contact is inhibited1

View this table: [in this window] [in a new window]   Table II. Effect of anti-F4/80 mAb on HKL-induced M-mediated IFN--production by NK cells1

Ab against the M surface Ag F4/80 inhibits NK cell-derived IFN- release

Assuming that cell contact promotes an important costimulatory mechanism in microbe-induced M/NK cell interaction, HKL-stimulated SCID WSC were incubated with a panel of mAb directed against different M surface molecules. As shown in Fig. 1, coincubation with mAb against the F4/80 molecule had a significant inhibitory effect on HKL-induced IFN- release, whereas mAb directed against other M surface Ags or the IgG2b-isotype control had neither inhibitory nor enhancing effects.

View larger version (47K): [in this window] [in a new window]   FIGURE 1. Anti-F4/80 mAb specifically inhibits IFN- production in HKL-stimulated SCID WSC cultures. SCID WSC (1 x 106/ml/well) were incubated with HKL alone (1 x 107/well; solid) or together with mAb (12 µg/well; gray) against the listed M surface Ags for 48 h. SNs were tested for IFN- by ELISA. Spontaneous IFN- release was below threshold (40 pg/ml). A representative experiment of four is shown. Values express means of triplicates ± SD; *, p < 0.0001 vs control.

Semiquantitative RT-PCR was performed on anti-F4/80 mAb-treated and control cultures of HKL-stimulated SCID WSC to determine whether IFN- release was affected at the pre- or posttranscriptional level and whether other cytokines involved in natural cellular immunity were affected in a similar manner. Densitometric estimates of the cytokine PCR products shown in Fig. 2 suggest that presence of anti-F4/80 mAb already inhibited IFN- at pretranscriptional levels. Whereas the mRNA expression of TNF- and IL-12 was inhibited, that of IL-1ß and IL-10 was increased. The mRNA expression of iNOS was not obviously affected. Fab fragments of anti-F4/80 mAb showed inhibitory effects on IFN-, TNF-, and IL-12 release that were similar to those of the complete Ab, although IL-1ß and IL-10 release was now similar to that of controls without mAb.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Anti-F4/80 mAb differentially modulates cytokine mRNA expression in HKL-stimulated SCID WSC cultures. SCID WSC (1 x 106/ml/sample) were incubated for 24 h alone (lane 1), with HKL (1 x 107/ml; lane 2), with HKL and anti-F4/80 mAb (20 µg/ml; lane 3), or with HKL and anti-F4/80 Fab (lane 4). Total RNA was extracted and semiquantitative RT-PCR performed using HPRT mRNA expression to normalize sample first-strand cDNA. A, PCR products run in parallel with 100-bp marker (lane 5), visualized by ethidium bromide. B, Densitometric comparison of individual cytokine mRNA PCR products (bands) shown in A.

To further characterize the effect of anti-F4/80 mAb on HKL-induced natural immune response mechanisms, the release of further relevant cytokines was analyzed. Fig. 3 illustrates the levels of IFN-, TNF-, IL-1ß, IL-10, IL-12, and iNO induced by given HKL numbers. Anti-F4/80 mAb inhibited release of IFN-, IL-12, and TNF- in a dose-dependent manner. Control isotype-matchedmAb or anti-LFA mAb showed no significant effect. In contrast to its effect on IFN-, TNF-, and IL-12, anti-F4/80 mAb enhanced release of IL-1ß and IL-10. Synthesis of iNO, often used as an indicator of murine M activation, was not modulated significantly. Interestingly, anti-F4/80-matched isotype control mAb R35–38 had similar, if lower, enhancing effects on the release of IL-1ß and IL-10, whereas incubation with anti-LFA-1 mAb again had no effect. These data indicate that the modulatory effects of anti-F4/80 mAb on HKL-induced cytokine release by WSC differ according to the individual cytokine.

View larger version (76K): [in this window] [in a new window]   FIGURE 3. Anti-F4/80 mAb differentially modulates cytokine release in HKL-stimulated SCID WSC cultures. SCID WSC (1 x 106/ml/well) were incubated with HKL (1 x 107/well; dark gray) and anti-F4/80 mAb (filled), an IgG2b isotype control (light gray), or anti-LFA-1 mAb (open) at the concentrations indicated for 24 h (TNF) or 48 h (other factors), and the SNs were tested for cytokines by ELISA. Spontaneous release: IFN-, 0; IL-12, 0.12 ± 0.1; TNF-, 0; iNO, 0; IL-1ß, 0.24 ± 0.1; and IL-10, 0. A representative experiment of four is shown. Values in ng/ml (iNO in µM) express means of triplicates ± SD; *, p < 0.0001; **, p < 0.001 vs control.

For further elucidation of HKL-induced reaction of spleen cells in absence of functional T or B cells, the order of appearance of individual cytokines and the modulatory effects of anti-F4/80 were analyzed (Fig. 4). In the absence of anti-F4/80, IL-10, IL-12, and IFN- could be detected at 6 h, and iNO at 8 h, after addition of HKL. Concentrations of these three factors increased further or reached a plateau during the 72 h they were observed. TNF- was detectable already after 2 h, peaked at 24 h, and then rapidly diminished. Constitutive levels of IL-1ß were not modified by HKL; they remained low over the study period. For all factors, the effects of anti-F4/80 were detectable with their first appearance in the SN. The specific modulatory effects did not change in quality over time. Incubation with anti-F4/80 isotype control mAb had no significant effect on HKL-induced cytokine release, again with the exception of IL-10 and IL-1ß, which were induced both by anti-F4/80 and by its isotype control.

View larger version (57K): [in this window] [in a new window]   FIGURE 4. Kinetics of cytokine release in HKL-stimulated SCID WSCs and modulating effects of anti-F4/80 mAb. SCID WSC (1 x 106/ml/well) were incubated with HKL (1 x 107/well) alone (open), with HKL plus anti-F4/80 mAb (12 µg/ml; filled), or HKL plus an IgG2b isotype control (12 µg/ml; gray) for the times indicated, and the SNs were tested for cytokines by ELISA. A representative experiment of four is shown. Values in ng/ml (iNO in µM) express means of triplicates ± SD; no asterisk, p < 0.0001; *, p < 0.05; **, p < 0.001; ***, not significant vs control.

Inhibitory effects of F4/80 mAb are specific and mediated by Fab fragment

To further clarify the diverging effects of anti-F4/80 mAb on HKL-induced WSC cytokine release, Fab and Fc fragments of the Ab were tested individually. Fig. 5 illustrates that the inhibitory effect of F4/80 mAb was mediated by the Ag-specific Fab-fraction. Moreover, F4/80 Fab also inhibited induction of IL-10. On the other hand, the stimulatory effect of F4/80 mAb, specifically on IL-10 release, was mediated by its Fc fragment only. Release of IL-10 might be responsible for the marginal, nevertheless significant inhibition of cytokine release observed for Fc fragment alone.

View larger version (62K): [in this window] [in a new window]   FIGURE 5. Inhibitory effect of anti-F4/80 mAb is mediated by Fab fragment. SCID WSC (1 x 106/ml/well) were incubated with HKL (1 x 107/well) with either the Fab or the Fc fragment of anti-F4/80 mAb at the concentrations indicated for 24 h (TNF) or 48 h (other factors), and the SNs were tested for cytokines by ELISA. Spontaneous release: IFN-, 0; IL-12, 0.12 ± 0.1; TNF-, 0; iNO, 0; IL-1ß, 0.24 ± 0.1; IL-10, 0. A representative experiment of three is shown. Values express means of triplicates ± SD; *, p < 0.0001; **, p < 0.001 vs WSC + HKL alone.

Inhibition of IFN- release in the presence of anti-F4/80 is only apparent when direct cell contact between M and NK cells is possible

As a first investigation into the mechanism by which anti-F4/80 modulates HKL-induced NK-derived IFN- release, different culture systems were employed, allowing or preventing direct contact between M and NK cells. As shown in Table II, detectable amounts of IFN- were induced when HKL were given to cocultures of M and NK cells, when HKL-stimulated M and NK cells were separated by a semipermeable membrane, and when cell culture SN from HKL-stimulated M was given to NK cells. HKL-stimulated cocultures revealed at least twice the amount of IFN- found in the other culture systems. Addition of anti-F4/80 mAb reduced IFN- release only in the coculture system. Anti-F4/80 mAb had no effect when direct cell contact between HKL-stimulated M and NK cells was inhibited.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In a previous publication we suggest that auxiliary, cell contact-dependent, pathways might be involved in microbe-induced, M-mediated NK cell activation. For example, P. carinii organisms stimulate IFN- production in SCID WSC cultures only, when both M and NK cells are present and cell contact is not inhibited (5). We now provide evidence that binding of the murine M surface glycoprotein F4/80 with specific mAb modulates cytokine levels induced by HKL in SCID WSC or in cocultures of M and NK cells. This report shows that in SCID WSC: 1) inhibition of HKL-induced IFN-, TNF-, and IL-12 by anti-F4/80 mAb or Fab fragment is Ab specific and dose dependent; 2) this inhibition is apparent at mRNA and protein release levels; 3) modulation of cytokine release by anti-F4/80 is effective as soon as the cytokines are detectable in the SN; 4) anti-F4/80 only inhibits release of IFN-, TNF-, and IL-12 when both M and NK cells are present in the culture system; and 5) anti-F4/80 is only effective when cell contact between M and NK cells is not inhibited. These data further indicate that an auxiliary microbe-induced signaling pathway between M and NK cells may exist involving cell surface molecules.

The natural or innate immune system that includes potent effector cells such as M, granulocytes, and NK cells is responsible for the early partial resistance to the facultative intracellular bacterial pathogen L. monocytogenes in SCID mice (30). Cytokine signaling plays a major role in activation of this T cell-independent immune response. M respond directly to organisms of large taxonomic variety by releasing TNF- and IL-12 (31, 32, 33, 34, 35). In combination, TNF- and IL-12 activate NK cells for release of IFN-, which in turn augments M microbicidal activity. This cytokine-mediated activation cycle is under negative control of IL-10, which inhibits IL-12 release by M as well as IFN- production by NK cells in response to TNF- plus IL-12 (5, 7, 36).

In a variety of experiments, however, we observed that soluble cytokine signaling might not cover all aspects of microbe-induced M/NK cell interactions. First, stimulation of purified M with P. carinii organisms, opportunistic fungal agents of pneumonia in severely immunocompromised individuals, does not lead to TNF- or IL-12 release. Nevertheless, P. carinii induces maximum levels of IFN- in cocultures of M and NK cells or in SCID WSC cultures similar to those of L. monocytogenes (5, 11). These data seem irreconcilable with the above-outlined model of NK cell activation by microbe-induced, M-released cytokines. Second, the amount of IFN- induced by HKL depends on whether HKL-stimulated M and NK cells are incubated together or are separated by a semipermeable membrane, with the former giving at least 2-fold higher concentrations of IFN- than the latter (Table I). Initial experiments had shown that, with respect to the in vitro experiments presented here, viable L. monocytogenes organisms and HKL particles have a similar capacity for inducing cytokine release in SCID WSC or in purified M and NK cell cocultures. Furthermore, anti-F4/80 mAb exerted the same inhibitory effects whether these cell cultures were stimulated with viable L. monocytogenes or with HKL.

These qualitative and quantitative deviations from the current model of a microbe-induced cytokine cascade led to the hypothesis that costimulatory signaling pathways may exist that require direct contact between M and NK cells and act via cell surface molecules.

To examine this hypothesis, blocking experiments were performed with a panel of mAb for murine M surface molecules added to SCID WSC in the presence of HKL (Fig. 1). Abs directed against the mouse M surface glycoprotein F4/80 specifically inhibited HKL-induced, NK cell-derived IFN-. The remaining levels of IFN- were similar to those found when HKL-stimulated M and NK cells were kept separated by a semipermeable membrane or when cell-free SNs from HKL-stimulated M were given to purified NK cell populations (Table I). Anti-F4/80 mAb is widely used for immunohistochemical or cytofluorometric detection of mouse M. The mAb was tested for cytotoxic effects on M or NK cells in MTT assays (27). No differences in cell viability in the absence or presence of anti-F4/80 mAb were observed for either cell population over 48 h (data not shown). As certain mAb have been reported to induce apoptosis (37), a cell death detection assay using the TUNEL method was employed to address this possibility. The results gave no indication for anti-F4/80-mediated apoptosis (data not shown).

Semiquantitative RT-PCR performed on anti-F4/80 mAb-treated and control cultures of HKL-stimulated SCID WSC revealed that inhibition of IFN- occurred already at pretranscriptional levels. Furthermore, mRNA expression of TNF- and IL-12 was also inhibited, whereas IL-1ß and IL-10 were enhanced, and iNOS mRNA was not obviously affected (Fig. 2). Cocultures in the presence of anti-F4/80 Fab showed similar inhibition of IFN-, TNF-, and IL-12 mRNA expression, whereas IL-1ß, IL-10, and iNOS were not biased. The inhibitory effects of anti-F4/80 were clearly concentration dependent (Fig. 3) and evident as soon as the respective cytokine was detectable in the SN (Fig. 4).

Release of certain cytokines that are not strongly induced by HKL under the given experimental conditions, such as IL-1 and IL-10, were enhanced when anti-F4/80 mAb was added (Fig. 2). This differential modulation of cytokine release by anti-F4/80 could be mediated by at least three different mechanisms. First, M could be nonspecifically stimulated by anti-F4/80 mAb via their Fc receptors. Accordingly, nonAg-specific binding was blocked by preincubating cultures with specific, inhibitory anti-FcIII (CD16)/FcII (CD32) mAb (Fc block). Whereas absence or presence of Fc block had no significant effect on inhibition of IFN- release by anti-F4/80, it annulled the agonistic effects of anti-F4/80 on IL-10 release. We investigated the possibility of nonspecific effects of the anti-F4/80 mAb by separating its Fab from its Fc fragments. The Fab fragments inhibited TNF-, IL-12, and IFN- production in HKL-stimulated SCID WSC cultures as described for the whole Ab (Fig. 5), indicating that these effects were Ag specific. On the other hand, only Fc fragment of anti-F4/80 enhanced release of IL-10 or IL-1ß, as found for whole mAb, indicating stimulation of IL-10, and IL-1 could indeed have been mediated nonspecifically by FcR binding. These data also show that inhibition of IFN-, TNF-, and IL-12 by anti-F4/80 mAb did not result simply from enhanced IL-10 release. Direct effects of anti-F4/80 mAb on NK cells can probably be ruled out. When NK cells were stimulated with TNF- plus IL-12, addition of anti-F4/80 mAb had no effect on the levels of IFN- produced (data not shown).

Second, specific binding of mAb to the F4/80 Ag might act indirectly by initiating signal transduction leading to down-regulation of as-yet-unknown costimulatory ligands or inducing production of molecules inhibitory for NK cell functions. Experiments performed to date give no indication for either pathway. For instance, release of TNF- or IL-12 by HKL-stimulated M alone was not altered in the presence of anti-F4/80 mAb nor was HKL-induced release of IL-10 or IL-1 affected by anti-F4/80 Fab. Analysis at the mRNA level gave the same results (data not shown). Apparently, the inhibitory activity of anti-F4/80 related only to M/NK interaction and not to inducible functions of either cell population alone.

Third, specific binding of mAb to the M surface Ag F4/80 might directly inhibit contact with an as-yet-unknown ligand on NK cells. With strong M stimulants such as HKL that can directly induce TNF- and IL-12 release, binding of F4/80 to this putative ligand may act as an additional stimulus, enhancing NK cell activation and IFN- release. With weak M stimulants such as P. carinii organisms (5, 11), which do not directly induce TNF- and IL-12 release, binding of F4/80 to its NK cell ligand is an essential step. Following a two-signal hypothesis for M activation, the first signal (priming) would be delivered by adherence/phagocytosis of certain microorganisms. The second could either be delivered by other elements of the same microorganism (strong stimulants) or by accessory stimuli such as binding of F4/80 to its putative ligand. The subsequent activation cascade would then be controlled by mutual cytokine release as observed in both HKL or P. carinii-stimulated NK/M cocultures. Of the three, the latter mechanism seems best supported by the available data.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Albrecht F. Kiderlen, Robert Koch-Institut, Abteilung Immunologie, Nordufer 20, D-13353 Berlin, Germany. E-mail address:

² Abbreviations used in this paper: WSC, whole spleen cells; M, macrophages; HKL, heat-killed Listeria monocytogenes; iNO, inorganic NOs; iNOS, inducible NO synthase; SN, supernatant; HPRT, hypoxanthine phosphoribosyltransferase.

Received for publication December 22, 1998. Accepted for publication July 9, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Schreiber, R. D., L. J. Hicks, A. Celada, N. A. Buchmeier, P. W. Gray. 1985. Monoclonal antibodies to murine -interferon which differentially modulate macrophage activation and antiviral activity. J. Immunol. 134:1609.[Abstract]
2. Bancroft, G. J., R. D. Schreiber, G. C. Bosma, M. J. Bosma, E. R. Unanue. 1987. A T cell-independent mechanism of macrophage activation by interferon-. J. Immunol. 139:1104.[Abstract]
3. Bancroft, G. J., R. D. Schreiber, E. R. Unanue. 1991. Natural immunity: a T-cell-independent pathway of macrophage activation, defined in the scid mouse. Immunol. Rev. 124:5.[Medline]
4. Dunn, P. L., R. J. North. 1991. Early interferon production by natural killer cells is important in defense against murine listeriosis. Infect. Immun. 59:2892.[Abstract/Free Full Text]
5. Warschkau, H., H. Yu, A. F. Kiderlen. 1998. Activation and suppression of natural cellular immune functions by Pneumocystis carinii. Immunobiology 198:343.[Medline]
6. Wherry, J. C., R. D. Schreiber, E. R. Unanue. 1991. Regulation of interferon production by natural killer cells in scid mice: roles of tumor necrosis factor and bacterial stimuli. Infect. Immun. 59:1709.[Abstract/Free Full Text]
7. Tripp, C. S., S. F. Wolf, E. R. Unanue. 1993. Interleukin 12 and tumor necrosis factor are costimulators of interferon production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiological antagonist. Proc. Natl. Acad. Sci. USA 90:3725.[Abstract/Free Full Text]
8. Bancroft, G. J., J. P. Kelly, P. M. Kaye, V. McDonald, C. E. Cross. 1994. Pathways of macrophage activation and innate immunity. Immunol. Lett. 43:67.[Medline]
9. Ding, A. H., C. F. Nathan, D. J. Stuehr. 1988. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: comparison of activating cytokines and evidence for independent production. J. Immunol. 141:2407.[Abstract]
10. Beckerman, K. P., H. W. Rogers, J. A. Corbett, R. D. Schreiber, M. L. McDaniel, E. R. Unanue. 1993. Release of nitric oxide during the T cell-independent pathway of macrophage activation. J. Immunol. 150:888.[Abstract]
11. Warschkau, H., H. Yu, A. F. Kiderlen. 1997. Activation of natural immune functions by Pneumocystis carinii. APMIS (Suppl.) 77:14.
12. Austyn, J. M., S. Gordon. 1981. F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur. J. Immunol. 11:805.[Medline]
13. Hume, D. A., S. Gordon. 1983. Mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80: identification of resident macrophages in renal medullary and cortical interstitium and the juxtaglomerular complex. J. Exp. Med. 157:1704.[Abstract/Free Full Text]
14. Hume, D. A., A. P. Robinson, G. G. MacPherson, S. Gordon. 1983. The mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80: relationship between macrophages, Langerhans cells, reticular cells, and dendritic cells in lymphoid and hematopoietic organs. J. Exp. Med. 158:1522.[Abstract/Free Full Text]
15. Lawson, L. J., V. H. Perry, P. Dri, S. Gordon. 1990. Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. Neuroscience 39:151.[Medline]
16. McKnight, A. J., A. J. Macfarlane, P. Dri, L. Turley, A. C. Willis, S. Gordon. 1996. Molecular cloning of F4/80, a murine macrophage-restricted cell surface glycoprotein with homology to the G-protein-linked transmembrane 7 hormone receptor family. J. Biol. Chem. 271:486.[Abstract/Free Full Text]
17. Baud, V., S. L. Chissoe, E. Viegas-Pequignot, S. Diriong, V. C. N'Guyen, B. A. Roe, M. Lipinski. 1995. EMR1, an unusual member in the family of hormone receptors with seven transmembrane segments. Genomics 26:334.[Medline]
18. Hamann, J., W. Eichler, D. Hamann, H. M. J. Kerstens, P. J. Poddighe, J. M. N. Hoovers, E. Hartmann, M. Strauss, R. A. W. van Lier. 1995. Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretin receptor superfamily with an unusual extracellular domain. J. Immunol. 155:1942.[Abstract]
19. Ezekowitz, R. A. B., J. Austyn, P. D. Stahl, S. Gordon. 1981. Surface properties of bacillus Calmette Guérin-activated mouse macrophages. Reduced expression of mannose-specific endocytosis, Fc receptors, and antigen F4/80 accompanies induction of Ia. J. Exp. Med. 154:60.[Abstract/Free Full Text]
20. Ezekowitz, R. A. B., S. Gordon. 1982. Down-regulation of mannosyl receptor-mediated endocytosis and antigen F4/80 in bacillus Calmette-Guérin-activated mouse macrophages: role of T lymphocytes and lymphokines. J. Exp. Med. 155:1623.[Abstract/Free Full Text]
21. Bosma, G. C., R. P. Custer, M. J. Bosma. 1983. A severe combined immunodeficiency mutation in the mouse. Nature 301:527.[Medline]
22. Kiderlen, A. F., S. H. E. Kaufmann, M.-L. Lohmann-Matthes. 1984. Protection of mice against the intracellular bacterium Listeria monocytogenes by recombinant immune interferon. Eur. J. Immunol. 14:964.[Medline]
23. Julius, M. H., E. Simpson, L. A. Herzenberg. 1973. A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol. 3:645.[Medline]
24. Kasai, M., M. Iwamori, Y. Nagai, K. Okumura, T. Tada. 1980. A glycolipid on the surface of mouse natural killer cells. Eur. J. Immunol. 10:175.[Medline]
25. Habu, S., H. Fukui, K. Shimamura, M. Kasai, Y. Nagai, K. Okumura, N. Tamaoki. 1981. In vivo effects of anti-asialo GM1. I. Reduction of NK activity and enhancement of transplanted tumor growth in nude mice. J. Immunol. 127:34.[Abstract]
26. Kiessling, R., E. Klein, H. Wigzell. 1975. "Natural" killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells: specificity and distribution according to genotype. Eur. J. Immunol. 5:112.[Medline]
27. Mosman, T.. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55.[Medline]
28. Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, S. R. Tannenbaum. 1982. Analysis of nitrate, nitrite, and [¹⁵N]nitrate in biological fluids. Anal. Biochem. 126:13.
29. Roach, T. I. A., A. F. Kiderlen, J. M. Blackwell. 1991. Role of inorganic nitrogen oxides and tumor necrosis factor in killing Leishmania donovani amastigotes in interferon-lipopolysaccharide-activated macrophages from Lsh^s and Lsh^r congenic mouse strains. Infect. Immun. 59:3935.[Abstract/Free Full Text]
30. Bancroft, G. J., M. J. Bosma, G. C. Bosma, E. R. Unanue. 1986. Regulation of macrophage Ia expression in mice with severe combined immunodeficiency: induction of Ia expression by a T cell-independent mechanism. J. Immunol. 137:4.[Abstract]
31. Gazzinelli, R. T., S. Hieny, T. A. Wynn, S. Wolf, A. Sher. 1993. Interleukin-12 is required for the T-lymphocyte-independent induction of interferon by an intracellular parasite and induces resistance in T-cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115.[Abstract/Free Full Text]
32. Flesch, I. E. A., J. H. Hess, S. Huang, M. Aguet, J. Rothe, H. Bluethmann, S. H. E. Kaufmann. 1995. Early interleukin 12 production by macrophages in response to mycobacterial infection depends on interferon and tumor necrosis factor . J. Exp. Med. 181:1615.[Abstract/Free Full Text]
33. Orange, J. S., B. Wang, C. Terhorst, C. A. Biron. 1995. Requirement for natural killer cell-produced interferon in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration. J. Exp. Med. 182:1045.[Abstract/Free Full Text]
34. Anguita, J., D. H. Persing, M. Rincón, S. W. Barthold., E. Fikrig. 1996. Effect of anti-interleukin 12 treatment on murine lyme borreliosis. J. Clin. Invest. 97:1028.[Medline]
35. Derrico, C. A., K. J. Goodrum. 1996. Interleukin-12 and tumor necrosis factor mediate innate production of interferon by group B Streptococcus-treated splenocytes of severe combined immunodeficiency mice. Infect. Immun. 64:1314.[Abstract]
36. Tripp, C. S., K. P. Beckerman, E. R. Unanue. 1995. Immune complexes inhibit antimicrobial responses through interleukin-10 production: effects in severe combined immunodeficient mice during Listeria infection. J. Clin. Invest. 95:1628.
37. Henke, C., P. Bitterman, U. Roongta, D. Ingbar, V. Polunovsky. 1996. Induction of fibroblast apoptosis by anti-CD44 antibody: implications for the treatment of fibroproliferative lung disease. Am. J. Pathol. 149:1639.[Abstract]

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