Published online before print December 23, 2003
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receptor-mediated signaling do not require expression of the SLP-76 and SLP-65 adaptors
,1
,¶
* Pediatric Oncology, Children’s Hospital of Philadelphia, Pennsylvania;
Abramson Family Cancer Research Institute and Departments of
Microbiology and
¶ Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia; and
Max Planck Institute for Immunobiology, Freiburg, Germany
1 Correspondence: Children’s Hospital of Philadelphia, Pediatric Oncology, ARC 907C, 3615 Civic Center Boulevard, Philadelphia, PA 19104. E-mail: nicholsk{at}email.chop.edu
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R)-mediated activation. We hypothesized that the related adaptor SLP-65, which is also expressed in macrophages, may compensate for the loss of SLP-76 during FcR-mediated signaling and functional events. To address this hypothesis, we examined bone marrow-derived macrophages (BMM) from wild-type (WT) mice or mice lacking both of these adaptors. Contrary to our expectations, SLP-76-/- SLP-65-/- BMM demonstrated normal FcR-mediated activation, including internalization of Ig-coated sheep red blood cells and production of reactive oxygen intermediates. FcR-induced biochemical events were normal in SLP-76-/- SLP-65-/- BMM, including phosphorylation of phospholipase C and the extracellular signaling-regulated kinases 1 and 2. To determine whether macrophages functioned normally in vivo, we infected WT and SLP-76-/- SLP-65-/- mice with sublethal doses of Listeria monocytogenes (LM), a bacterium against which the initial host defense is provided by activated macrophages. WT and SLP-76-/- SLP-65-/- mice survived acute, low-dose infection and showed no difference in the number of liver or spleen LM colony-forming units, a measure of the total body burden of this organism. Taken together, these data suggest that neither SLP-76 nor SLP-65 is required during FcR-dependent signaling and functional events in macrophages.
Key Words: innate immunity • phagocytosis • ITAM • respiratory burst
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adaptor molecules, modular proteins that mediate specific protein-protein and protein-lipid interactions, play important roles during immunoreceptor-mediated signaling and cellular activation. The Src-homology 2 (SH2) domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) [1
] is one member of a family of three related hematopoietic adaptors, which includes SLP-65 [2
], also known as B cell-linker protein [3
] B cell adaptor-containing SH2 domain [4
5
], and cytokine-dependent hematopoietic cell linker (CLNK) [6
]/mast cell immunoreceptor signal transducer [7
]. These adaptors are comprised of several functionally relevant domains, such as an N-terminal acidic domain that includes sites of tyrosine phosphorylation, a central proline-rich protein-interaction domain, and a C-terminal SH2 domain. The signaling pathways involving SLP-76 have been most extensively characterized in T cells where T cell receptor (TCR) engagement leads to the phosphorylation of N-terminal tyrosine residues, followed by formation of a multiprotein complex involving phospho-SLP-76 and the guanine nucleotide-exchange factor Vav [8
9
]; other adaptor molecules such as the linker for activation of T cells (LAT) [10
11
], Grb2-related adaptor downstream of Shc [12
], Fyn-binding protein [13
]/SLP-76-associated protein of 130 kD [14
]/adhesion- and degranulation-promoting adaptor protein [15
16
], and Nck [17
]; and enzymatic effectors including phospholipase C (PLC) [18
] and the Tec family kinase Itk [19
20 ]. Following TCR ligation, SLP-76 relocates to lipid rafts, membrane-associated structures where macromolecular signaling complexes are formed, and downstream signaling events occur [21
]. The dependence of T cell activation and development upon SLP-76 expression were first established in SLP-76-deficient Jurkat T cells, which exhibit severe defects in TCR signaling [22
], and in SLP-76-/- mice, which fail to develop peripheral T lymphocytes [23
24
].
In addition to T cells, SLP-76 is expressed in mast cells, where it has been shown to regulate immunoglobulin (Ig) Fc
receptor (FcR)-mediated signaling and effector functions, including degranulation and cytokine production [25
26
]. Consequently, SLP-76-/- mice are resistant to IgE-mediated, passive, systemic anaphylaxis [26
]. SLP-76 is also present in phagocytes, including neutrophils, monocytes, and macrophages [27
], where it becomes rapidly phosphorylated following Fc receptor for IgG (FcR) ligation [28
29
]. The FcRs, which regulate important, functional activities in myeloid cells, including engulfment of IgG-coated particles, production of reactive oxygen intermediates (ROI), and secretion of cytokines [30
], signal via pathways that are similar to those induced by the TCR and FcR. In macrophages, FcR engagement leads to activation of the Src family kinases hck, fgr, fyn, lyn, yes, and src and phosphorylation of ITAMs within the FcR complex [31
32
33
]. Subsequent recruitment and activation of the tyrosine kinase Syk [34
] result in tyrosine phosphorylation of SLP-76 and other downstream-signaling molecules, mobilization of intracellular Ca2+, and activation of the RAS-MAPK pathway.
Although there are similarities among FcR-, TCR-, and FcR-associated, signaling pathways, SLP-76-deficient macrophages, unlike SLP-76-deficient T lymphocytes and mast cells, demonstrate normal, FcR-induced activation, including phagocytosis and ROI production [28
29
]. Consistent with these findings, FcR-mediated, biochemical events are normal in SLP-76-/- bone marrow (BM)-derived macrophages (BMM), including phosphorylation of Syk and PLC2 [28
, 29
] and up-regulation of extracellular-regulated kinases 1 and 2 (ERK1/2) kinase activity [29
]. To explain these observations, we and others [28
] noted that in addition to SLP-76, macrophages express SLP-65, a homologous adaptor that plays a central role in B cell development [35
36
37
] and activation [3
, 5
, 35
, 37
] but whose function in macrophages has not been extensively explored. In fact, SLP-65 becomes rapidly tyrosine-phosphorylated in BMM following FcR ligation [28
], suggesting that it might play an important role during proximal FcR-mediated signaling events. In support of this possibility, phosphorylated SLP-65 coprecipitates with PLC2 in FcR-stimulated macrophages [28
], a property potentially linking SLP-65 with PLC2 activation. Taken together, these observations suggested that SLP-65 might function in parallel with SLP-76 during FcR-induced macrophage activation, which prompted us to undertake the current investigation.
Using wild-type (WT) mice or mice lacking expression of SLP-76 and SLP-65, we sought to determine whether SLP-65 could compensate for the loss of SLP-76 during FcR-induced macrophage signaling and cellular activation. Contrary to our hypothesis, FcR ligation led to normal biochemical signaling and function in SLP-76-/- SLP-65-/- BMM. SLP-76-/- SLP-65-/- BMM also responded normally following in vitro stimulation with other agents, including the bacterial cell-wall component lipopolysaccharide (LPS), interferon- (IFN-), the formylated bacterial peptide f-Met-Leu-Phe (fMLP), and complement components C3a and C5a. Furthermore, WT and SLP-76-/- SLP-65-/- mice survived an acute low dose of Listeria infection and showed no difference in their ability to eliminate pathogenic organisms. These results suggest that SLP-76 and SLP-65 are not required for signaling and activation events in BMM.
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In vitro culture of BMM
Cell culture and experimental conditions were performed using endotoxin-free reagents. BMM were established according to a previously published protocol [29
]. Briefly, BM cells were obtained by flushing femurs and tibias with RPMI-1640 medium. Following washing, cells were plated into Petri dishes containing RPMI-1640 medium supplemented with 20% L929 cell-conditioned medium (LCM), 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 1000 units/ml streptomycin, and 20 mM L-glutamine (R10-LCM). After 2 days of culture at 37°C in 5% CO2, nonadherent cells were collected and replated in fresh R10-LCM. After an additional 3–4 days, the adherent cellular monolayer was used for experiments. Morphological and fluorescence-activated cell sorter (FACS) analysis revealed that 98–100% of cells were macrophages.
For analysis of surface-marker expression, BMM were left unstimulated or were stimulated with LPS (1 µg/ml; Sigma-Aldrich Chemical Co., St. Louis, MO) for 18 h. Cells were harvested by scraping, washed in FACS buffer [1x phosphate-buffered saline (PBS), 2% FBS], and resuspended in FACS staining buffer (1x PBS, 2% FBS, 20% rat serum) for 20–30 min. Cells were then incubated for an additional 20 min with fluorochrome-conjugated antibodies at 4°C in the dark. After washing in FACS buffer, cells were analyzed by flow cytometry. Antibodies used for these studies included those recognizing mouse CD3, CD4, CD8, B220, Gr-1, Mac-1 (CD11b), the FcRs (CD16/32), CD80, CD86, and CD69b (PharMingen, San Diego, CA), Toll-like receptor 4 (TLR4; eBioscience, San Diego, CA), and F4/80 (Caltag Laboratories, Burlingame, CA).
Western blotting and immunoprecipitation
BMM (6x106) were plated onto 60-mm dishes and allowed to adhere overnight in R10-LCM. Cells were washed twice in 1x PBS and allowed to rest in Hanks’ buffered saline solution, 10% FBS for 20–30 min at 37°C. Cells were left untreated or were stimulated with sheep red blood cells (SRBC; Elmira Biologicals, Iowa City, IA), IgG-coated SRBC, complement-opsonized zymosan (COZ), or pervanadate (PV), as described previously [29
]. Briefly, 0.5 ml stimuli was added to each dish, and plates were spun at 1200 rpm at 4°C for 1 min and then transferred to 37°C for the indicated times. For PV stimulation, 0.5 ml of a solution containing 0.1 mM Na3VO4 and 3 mM H2O2 was added to the appropriate dishes, followed by incubation at room temperature (RT) for 3–5 min. After stimulation, cells were rinsed twice with cold 1x PBS and were lysed in 250 µl 1% Nonidet P-40 (NP-40) lysis buffer (1% NP-40, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA) containing phosphatase inhibitors (400 nM Na3VO4, 10 mM NaF, 10 nM Na pyrophosphate) and a protease-inhibitor cocktail (Sigma-Aldrich Chemical Co.). Lysates were centrifuged at 14,000 rpm for 10 min at 4°C, and supernatants were collected for immunoprecipitation and Western blotting.
For Western blotting, 25 µl protein lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to nitrocellulose. Immunoblotting was performed using the antiphosphotyrosine antibody 4G10 (Upstate Biotechnologies, Lake Placid, NY), antimurine SLP-76 (Elmira Biologicals), antimurine SLP-65, antimurine CLNK, antiphospho-ERK1/2 (Cell Signaling Technologies, Beverly, MA), anti-ERK1/2 (Zymed Laboratories, San Francisco, CA), or antitubulin (Santa Cruz Biotechnology, Santa Cruz, CA), followed by detection using horseradish peroxidase-conjugated secondary antibodies (BioRad Laboratories, Hercules, CA) and the electrochemiluminescence detection reagent (Amersham Biosciences, Arlington Heights, IL). For detection of PLC2 phosphorylation, cell lysates were subjected to immunoprecipitation using an anti-PLC2 antibody (Santa Cruz Biotechnology), followed by Western blotting using 4G10. The rabbit polyclonal anti-SLP-65 antibody used in these experiments was generated against the peptide sequence CSQNNTKDSTRLKYAVKVS (Biosource International, Camarillo, CA). Immunizing chickens with the peptide sequence CZYENTNSEKPDPTKPDEKD generated an anti-CLNK antibody (Aves Laboratories, Tigard, OR).
Evaluation of FcR-associated phagocytosis
SRBC were prepared by washing three times in 1x PBS. Next, 1 x 109 SRBC were incubated with 0.1 mCi Cr51, with or without subagglutinating concentrations of rabbit anti-SRBC antibodies (ICN Biomedicals, Aurora, CA) at 37°C for 60 min. Cells were washed three times in cold 1x PBS and were resuspended in 25 ml RPMI-1640 medium supplemented with 10% fetal calf serum (FCS).
Prior to experiments, BMM were plated in triplicate at 2.5 x 105/well in 96-well flat-bottom plates. Following overnight incubation, the medium was gently aspirated, and 100 µl Cr51-labeled SRBC (negative control) or Cr51-labeled, IgG-opsonized SRBC (Cr51–IgG–SRBC; test conditions) were added to appropriate wells and incubated for the indicated times at 37°C. Subsequently, BMM were gently washed with cold RPMI-1640 medium, and the remaining medium was aspirated. Uningested SRBC were lysed by adding 100 µl hypotonic RBC lysis buffer (Sigma-Aldrich Chemical Co.) and incubating for an additional 5 min at RT. BMM were washed with cold RPMI-1640 medium supplemented with 10% FCS, and the cellular monolayer was lysed using 100 µl 0.5% SDS solution for 10 min at RT. Lysates were collected, and ingested radioactivity was measured using a -counter.
Measurement of FcR-mediated ROI production
BMM were rinsed with ice-cold PBS without calcium or magnesium. Cells were scraped from plates and resuspended at a concentration of 20 x 106 cells/ml in ice-cold PBS–glucose (125 mM NaCl, 8 mM Na2HPO4, 2 mM NaH2PO4, 5 mM KCl, 5 mM glucose). BMM (2.5x105) were transferred to cold Krebs-Ringer phosphate (KRP) buffer (125 mM NaCl, 8 mM Na2HPO4, 2 mM NaH2PO4, 5 mM KCl, 5 mM glucose, 1 mM CaCl2, 1.5 mM MgCl2) or KRP prewarmed to 37°C and were then added to an enclosed chamber, where they were gently stirred for 1–2 min. Subsequently, 60 µg FcOxyBURST Green® reagent (Molecular Probes, Eugene, OR) was added, and the change in green fluorescence was determined using a FACSCaliber flow cytometer (Becton Dickenson, Franklin Lakes, NJ).
Measurement of cytokine production
BMM were plated at 5 x 104 cells/well in 96-well flat-bottom plates for 18 h in R10-LCM or R10-LCM containing IFN- (100 units/ml; R&D Systems, Minneapolis, MN), LPS (1 µg/ml), CpG DNA (1 µM; kindly provided by Christopher Hunter, Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA), or a combination of IFN- and LPS or CpG DNA. Culture supernatants were harvested after 48 h and diluted 1:10 with RPMI 1640. Secretion of interleukin (IL)-12p40, IL-6, and tumor necrosis factor 
(TNF-) was determined using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocols (R&D Systems).
Evaluation of nitric oxide (NO) production and induction of inducible NO synthase (iNOS) protein expression
BMM were incubated at 5 x 104 cells/well in 96-well flat-bottom plates for 18 h in R10-LCM or R10-LCM containing varying concentrations of LPS, IFN-, or LPS plus IFN-. Culture supernatants were harvested, and NO production was determined using the Greiss reagents. For Western blot analysis of iNOS expression, 0.5 x 106 cells were plated in 12-well dishes in R10-LCM supplemented with the indicated concentrations of LPS and IFN-. Cells were harvested by scraping in 1% NP-40 lysis buffer, and cell lysates were fractionated by SDS-PAGE, transferred to nitrocellulose, and blotted using an anti-NOS2 antibody (Santa Cruz Biotechnology).
Macrophage migration assays
BMM were rinsed with 1x PBS and were allowed to rest for 2 h in RPMI-1640 medium supplemented with 10% FBS at 37°C. After scraping, cells were resuspended in the same medium, and 5 x 104 cells were plated in triplicate in Neuroprobe chambers according to the manufacturer’s protocols (Neuroprobe, Gaithersburg, MD). Cells were allowed to migrate into lower chambers containing RPMI-1640 medium supplemented with 10% FBS and varying concentrations of fMLP (Sigma-Aldrich Chemical Co.). After specified periods of time, nonmigrating cells were gently scraped off the top surface of the Neuroprobe membranes, and the membrane undersurface was stained using the PROTOCOL® reagent (Biotechnical Sciences, Swedesboro, NJ). The number of macrophages migrating through the membrane was determined by light microscopy. Three high power fields (HPFs) were counted/well by a blinded reader.
Infection of mice with Listeria monocytogenes (LM)
The WT LM strain 10403S was maintained as a –80°C stock in brain-heart infusion (BHI) medium with 50% glycerol. For experiments, bacteria were inoculated onto BHI agar and were grown overnight at 37°C with aeration. Mice were immunized intravenously (i.v.) with a sublethal dose of 5 x 103 colony-forming units (CFU; 
0.1 of the 50% lethal dose). After 3 days, the number of bacteria per liver and spleen was determined by plate count.
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Figure 1. Characterization of protein expression and splenic macrophage numbers in SLP-76-/- SLP-65-/- mice. (A) Western blot analysis was performed using whole-cell lysates derived from SLP-76+/+ SLP-65+/+, SLP-76+/- SLP-65+/-, or SLP-76-/- SLP-65-/- splenocytes or in vitro-cultured BMM. (B) Live splenocytes obtained from 8- to 12-week-old SLP-76+/+ SLP-65+/+ and SLP-76-/- SLP-65-/- mice were stained and evaluated by flow cytometric analysis for expression of the macrophage-specific surface marker F4/80. (B) Data shown are representative of four mice of each genotype analyzed.
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Rs, the TLR4, the costimulatory receptors CD80 and CD86, and the activation marker CD69b was comparable among all genotypes at baseline and following overnight stimulation with LPS (Fig. 2
). These data indicate that SLP-76 and SLP-65 are not required for M-CSF-induced maturation of BM progenitors in vitro or for LPS-induced modulation of surface-marker expression.
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Figure 2. . WT and SLP-76-/- SLP-65-/- BMM demonstrate similar surface-marker expression. Following overnight stimulation with medium or medium and LPS (1 µg/ml), BMM were harvested and analyzed by flow cytometric analysis. The histograms represent surface-marker expression of unstimulated cells (black, filled histograms) or LPS-treated cells (gray, open histograms), as compared with staining of unstimulated cells with isotype-control antibodies (light gray, open histograms). Data shown are representative of three separate experiments.
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R-induced phagocytosis proceeds normally in SLP-76-/- SLP-65-/- BMMRs. As SLP-76 and SLP-65 are tyrosine-phosphorylated following FcR engagement, we sought to evaluate whether FcR-dependent phagocytosis might be perturbed in SLP-76-/- SLP-65-/- cells. WT or SLP-76-/- SLP-65-/- BMM were cultured with Cr51-labeled, IgG-coated SRBC (Cr51–IgG–SRBC) for various times or with unopsonized Cr51–SRBC as a negative control. After treatment with hypotonic buffer to remove bound, noninternalized Cr51–IgG–SRBC, phagocytic activity was determined by measuring total ingested radioactivity. As shown in Figure 3A
, WT and SLP-76-/- SLP-65-/- BMM similarly ingested Cr51–IgG–SRBC at each of the time points tested, suggesting that SLP-76 and SLP-65 are not required for FcR-mediated phagocytosis. In contrast, WT or SLP-76-/- SLP-65-/- BMM did not internalize unopsonized Cr51–SRBC (data not shown).
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Figure 3. FcR-induced function proceeds normally in SLP-76-/- SLP-65-/- BMM. (A) SLP-76+/+ SLP-65+/+ and SLP-76-/- SLP-65-/- BMM were incubated with Cr51-labeled, IgG-opsonized SRBC for the indicated times. Following removal of bound, noninternalized IgG–SRBC, BMM were lysed, and phagocytic activity was determined by counting ingested radioactivity using a -counter. (B) SLP-76+/+ SLP-65+/+ and SLP-76-/- SLP-65-/- BMM were incubated in an enclosed chamber at 4°C or 37°C. The FcOxyBURST Green® reagent was added as indicated (arrow), and production of ROI was determined over time based on changes in green fluorescence. The data shown are representative of four (phagocytosis) or three (ROI) experiments performed.
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R-induced ROI production, we stimulated WT or SLP-76-/- SLP-65-/- BMM using the FcOxyBURST Green® reagent, an engineered, immune complex consisting of bovine serum albumin (BSA) that has been covalently linked with the oxidation-sensitive fluorescent dye dichlorohydrofluorescein diacetate and opsonized with polyclonal anti-BSA IgG. Following FcR engagement via FcOxyBURST Green®, this nonfluorescent immune complex is rapidly internalized. Subsequent production of ROI leads to generation of a green fluorescent signal that is measured by flow cytometry. Approximately 5–10% of the FcOxyBURST Green® reagent is present in a spontaneously oxidized and therefore fluorescent state. At 4°C, WT and SLP-76-/- SLP-65-/- BMM similarly bound the FcOxyBURST Green® reagent, as indicated by a five- to tenfold increase in the baseline green fluorescence that occurs within 2–4 s of adding the reagent (Fig. 3B
; 4°C). Once warmed to 37°C, WT and SLP-76-/- SLP-65-/- BMM similarly internalized and oxidized the FcOxyBURST Green® reagent, reflecting a comparable degree and kinetics of ROI production (Fig. 3B
; 37°C). These results indicate that FcR-induced ROI production can occur in the absence of SLP-76 and SLP-65 expression.
FcR-mediated biochemical events do not require SLP-76 or SLP-65
To determine whether SLP-76 and SLP-65 were required to promote Syk-dependent FcR-induced signaling events in macrophages, WT and SLP-76-/- SLP-65-/- BMM were left unstimulated or were stimulated with IgG–SRBC or the phosphatase inhibitor PV. Cells were also stimulated via the non-Syk kinase-dependent CR [34
] using COZ. Following activation, cells were lysed and analyzed by Western blotting. As shown in Figure 4A
, the kinetics and degree of total cellular tyrosine phosphorylation were comparable between both genotypes, suggesting that FcR- and CR-induced phosphotyrosine-dependent signaling proceeded normally in these cells.
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Figure 4. FcR-mediated biochemical events do not require expression of SLP-76 and SLP-65. WT or SLP-76-/- SLP-65-/- BMM were stimulated with unopsonized SRBC (15 min), Ig-opsonized SRBC (IgG) for the indicated times, COZ (15 min), or PV (3 min). Cells were lysed, and tyrosine phosphorylation of total cellular proteins (pY; A) or ERK1/2 (C) was analyzed by Western blot analysis using the antiphosphotyrosine antibody 4G10 or antiphospho-ERK1/2 antibodies, respectively. (B) Phosphorylation of PLC2 was determined by immunoprecipitation using PLC2-specific antibodies, followed by Western blotting using 4G10. Equivalent amounts of protein loading were determined by cell count (A, C) or by stripping membranes and reprobing with anti-PLC2-specific antibodies (B). Data are representative of three separate experiments.
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R, or glycoprotein VI (GPVI) collagen receptor-induced phosphorylation of PLC, a lipid kinase that hydrolyzes phosphatidylinositol-4,5 bisphosphate to generate inositol 1,4,5-trisphosphate and diacylglycerol, important second messengers in immune cell activation. To determine whether SLP-76 and SLP-65 were required for FcR-induced activation of PLC2, the PLC isoform expressed in macrophages, we measured PLC2 phosphorylation, a surrogate marker of enzyme activation. As shown in Figure 4B
, PLC2 phosphorylation proceeded normally in SLP-76-/- SLP-65-/- BMM and was comparably sustained for 15 min, the end-point for our assays.
In addition to activating PLC, SLP-76 and SLP-65 are also required for antigen receptor-induced activation of the serine threonine kinases ERK1/2 [22
]. To determine whether ERK phosphorylation proceeded normally in SLP-76-/- SLP-65-/- BMM, cells were stimulated through the FcR or with the potent phorbol ester, phorbol 12-myristate 13-acetate (PMA), which bypasses proximal phosphotyrosine-dependent signaling events to directly activate downstream signaling intermediates. Following activation, cells were lysed and analyzed by Western blotting using a specific antibody recognizing the phosphorylated isoforms of ERK1/2. IgG–SRBC (Fig. 4C)
and PMA (data not shown) strongly induced ERK1/2 phosphorylation in WT and SLP-76-/- SLP-65-/- BMM. Taken together, the results of these biochemistry experiments indicate that in BMM, FcR-dependent signaling events do not require the expression of SLP-76 and SLP-65.
Macrophage migration proceeds normally in SLP-76-/- SLP-65-/- BMM
Proinflammatory mediators induce macrophages to undergo cytoskeletal rearrangements, which facilitate changes in cell shape and promote locomotion into infected tissues. In TCR-stimulated T cells, SLP-76 plays an important role during cytoskeletal remodeling by facilitating formation of a multimolecular complex that includes Vav, the adaptor molecule Nck, and Pak1, an upstream effector of the Rho family GTPases Rac1 and CDC42 [42
, 43
]. To determine whether SLP-76 or SLP-65 might contribute to BMM locomotion in vitro, WT or SLP-76-/- SLP-65-/- cells were placed into the upper wells of Transwell chambers, and migration, in response to the chemotactic bacterial peptide fMLP, was monitored over time. WT and SLP-76-/- SLP-65-/- BMM demonstrated a comparable dose (data not shown)- and time-dependent increase in migration following exposure to fMLP (Fig. 5
), indicating that the presence of the SLP-76 and SLP-65 adaptors is not required to facilitate cytoskeletal rearrangements or locomotion in vitro.
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Figure 5. fMLP-induced macrophage migration proceeds normally in SLP-76-/- SLP-65-/- BMM, which were plated into the upper wells of Neuroprobe chambers and allowed to migrate through a membrane in response to 100 µM fMLP. After the indicated times, membranes separating the upper and lower chambers were removed, and nonmigrating cells were gently scraped from the upper membrane surface. The membrane undersurface was stained using the PROTOCOL® reagent, and the number of macrophages migrating per three HPFs was determined by visual inspection using a light microscope. Data represent the average of three experiments performed.
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synergistically enhances iNOS expression and NO production by LPS-activated macrophages [45
]. Although SLP-76 and SLP-65 have not been directly implicated in cytokine or TLR signaling, we sought to determine whether SLP-76-/- SLP-65-/- macrophages might be defective in these activating pathways. Therefore, WT and SLP-76-/- SLP-65-/- BMM were stimulated with IFN- and LPS, and they were assessed for up-regulation of iNOS protein expression and induction of NO production. As shown in Figure 6A
, WT and SLP-76-/- SLP-65-/- BMM demonstrated a similar dose-dependent up-regulation of iNOS protein expression when stimulated overnight with increasing doses of LPS or LPS and IFN-. As shown in Figure 6B
, WT and SLP-76-/- SLP-65-/- BMM also produced comparable levels of NO following overnight stimulation with LPS, IFN-, CpG DNA, or a combination of these agents. These combined data indicate that SLP-76 and SLP-65 are not required for the production of NO by activated BMM.
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Figure 6. NO production is normal in WT and SLP-76-/- SLP-65-/- BMM. (A) BMM were plated in medium alone, medium supplemented with increasing concentrations of IFN- (1, 10 units/ml), LPS (10, 100, 1000 ng/ml), or with IFN- (10 units/ml) and increasing concentrations of LPS (10, 100, 1000 ng/ml). Following harvesting in lysis buffer, cell lysates were fractionated by SDS-PAGE, transferred to nitrocellulose, and blotted using an anti-iNOS2 antibody. (B) BMM were incubated for 18 h in medium, LPS (1 µg/ml), IFN- (100 units/ml), LPS (1 µg/ml) plus IFN- (100 units/ml), CpG DNA (1 µM), or CpG DNA (1 µM) plus IFN- (100 units/ml). Supernatants were harvested, and NO production was determined using the Greiss reagent. Data are representative of two (iNOS induction) or three (NO production) experiments performed.
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, macrophages secrete proinflammatory cytokines including TNF-, IL-6, and IL-12, which play important roles in sustaining the innate-immune response and inducing adaptive immunity. Therefore, we measured cytokine secretion by unstimulated BMM or by cells stimulated with various combinations of LPS, CpG DNA, and IFN-. As shown in Figure 7A
, 48 h stimulation with single agents, including medium, LPS, IFN-, or CpG DNA, led to comparable secretion of low levels of IL-12, TNF-, and IL-6 by WT and SLP-76-/- SLP-65-/- BMM. In response to stimulation using a combination of IFN- and CpG DNA, WT and SLP-76-/- SLP-65-/- BMM secreted similarly increased levels of IL-12, TNF-, and IL-6. In contrast, after overnight stimulation with IFN- and LPS, SLP-76-/- SLP-65-/- BMM secreted 25% less IL-6 and 50% less IL-12p40 and TNF- compared with WT cells (in two out of four experiments performed; Fig. 7A
and data not shown). In these same experiments, serial measurements of IFN- and LPS-induced cytokine secretion by SLP-76-/- SLP-65-/- BMM again revealed decreased IL-12, TNF-, and IL-6 production (Fig. 7B)
. These data suggest that SLP-76 and SLP-65 may not be required for cytokine production following the stimulation of BMM with LPS, IFN-, or CpG DNA or with a combination of CpG DNA and IFN-. However, expression of one or more of these adaptors may contribute to the production of certain cytokines, including IL-12, TNF-, and IL-6, following treatment of macrophages with a combination of IFN- and LPS.
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Figure 7. Cytokine production in WT or SLP-76-/- SLP-65-/- BMM. (A) WT or SLP-76-/- SLP-65-/- BMM were incubated overnight in medium, LPS (1 µg/ml), IFN- (100 units/ml), LPS (1 µg/ml) plus IFN- (100 units/ml), CpG DNA (1 µM), or CpG DNA (1 µM) plus IFN- (100 units/ml). (B) BMM were stimulated with LPS (1 µg/ml) plus IFN- (100 units/ml) for the indicated times. Culture supernatants were harvested after 48 h (A) or at the indicated times (B), and cytokine secretion was determined via ELISA. Results shown represent one out of four experiments performed.
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, IL-12p40, and IL-6 following stimulation with LPS and IFN-. As secretion of these cytokines is critical during the induction and amplification of the innate immune response, we chose to investigate whether primary SLP-76-/- SLP-65-/- macrophages were capable of responding normally to pathogenic organisms in vivo. For these experiments, we infected mice with LM, a bacterium against which the initial host defense is largely provided by activated macrophages [46
]. Following low-dose, i.v., LM injection, normal mice develop hepatic and splenic granulomas, where macrophages clear infectious organisms. In mice with compromised innate immunity, such as those lacking expression of the TNF- receptor [47
], bacterial growth remains unchecked and results in the death of infected animals within days. In contrast, mice lacking the recombination-activating genes (RAG) 1 and 2 demonstrate enhanced, acute anti-LM immune responses as a result of an increase in the number of functionally normal macrophages and natural killer (NK) cells within peripheral lymphoid organs [48
]. Therefore, RAG1/2-/- mice serve as a useful comparison with which to illustrate the enhanced effectiveness of innate immune cell function. WT, SLP-76-/- SLP-65-/-, and RAG1/2-/- mice were injected with sublethal doses of LM and were observed for overall survival. After 3 days, surviving mice were killed, spleens and livers were harvested, and the number of LM CFU was determined. WT, SLP-76-/- SLP-65-/-, and RAG1/2-/- mice comparably survived acute, low-dose LM infection (data not shown). As shown in Figure 8A , similar numbers of Listeria organisms were recovered from the livers and spleens of WT and SLP-76-/- SLP-65-/- mice, providing indirect evidence that the effectiveness of the innate immune response was similar between these genotypes of mice. As anticipated, the enhanced innate immunity of RAG1/2-/- mice led to the isolation of 100-fold fewer bacterial colonies from infected livers and spleens.
 View larger version (36K): [in a new window] |
Figure 8. SLP-76-/- SLP-65-/- mice control acute Listeria infection in a manner similar to WT mice. Mice of the indicated genotypes were injected via tail vein with 5 x 103 LM (strain 10403S). After 3 days, the number of bacterial colonies recovered per liver and spleen was determined by plate count (A). Live splenocytes were examined for surface-marker expression using flow cytometric analysis (B). The data shown are representative of two separate experiments. In total, eight SLP-76+/+ SLP-65+/+, eight SLP-76-/- SLP-65-/-, and four RAG1/2-/- mice of similar age, sex, and where possible, genetic background were used for these experiments. Similar results (bacterial viability and flow cytometric data) were obtained from all surviving mice of each genotype. The histograms shown in (B) reflect surface expression of activation markers on Gr-1- Mac-1+ splenic macrophages.
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Rs and the CD80 and CD86 costimulatory receptors, was comparable among SLP-76-/- SLP-65-/-, RAG1/2-/-, and WT mice (Fig. 8B)
. These combined data suggest that innate immunity against LM, including macrophage migration into the spleen and up-regulation of surface-activation markers, is not impaired in the absence of SLP-76 and SLP-65 expression. It remains possible, however, that subtle macrophage-functional defects might have been missed in these assays as a result of increases in absolute macrophage number or potential compensatory anti-LM activity provided by SLP-76-/- SLP-65-/- neutrophils and/or NK cells. |
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R ligation induces SLP-76 and SLP-65 phosphorylation and coprecipitation with downstream signaling intermediates. Based on these observations, we hypothesized that SLP-76, SLP-65, or SLP-76 and SLP-65 might play a role in FcR-induced signaling events and cellular activation in macrophages. Previous experiments have shown, however, that SLP-76-/- BMM function normally following FcR ligation, indicating that the simultaneous presence of both molecules is not essential. These data have also suggested that SLP-76 and SLP-65 might play redundant roles in macrophage development and/or FcR-induced activation.
To address whether SLP-65 could functionally compensate for loss of SLP-76 during macrophage differentiation and functional events, we generated mice lacking expression of both adaptors. Contrary to our hypothesis, SLP-76-/- SLP-65-/- BMM developed normally and demonstrated normal FcR-mediated activation, including phagocytosis of IgG–SRBC, ROI production, and biochemical signaling. Furthermore, WT and SLP-76-/- SLP-65-/- BMM responded similarly to stimulation via most other activating receptors, including TLR4 and -9 and CR. Although SLP-76-/- SLP-65-/- mice occasionally demonstrated defects in the production of proinflammatory cytokines, WT and SLP-76-/- SLP-65-/- littermates survived acute LM infection and showed no differences in the number of recovered bacterial organisms. Taken together, these data suggest that macrophage development and activation can occur in the absence of SLP-76 and SLP-65.
Given the absolute requirement for SLP-76 and SLP-65 in mediating ITAM-associated receptor signaling in other lineages, our observation was surprising and suggests several possible explanations. First, retention of intact FcR function may be a result of additional redundancy existing among the molecules involved in FcR-induced signaling pathways in macrophages. This redundancy is not unexpected as a result of the central role played by macrophages in maintaining host defense. Such redundancy ensures that fundamentally important processes, such as phagocytosis, remain intact, thereby ensuring host survival. CLNK, an adaptor that is expressed in cytokine-stimulated, hematopoietic cells [6
], is a recently identified member of the same family of adaptors that includes SLP-76 and SLP-65. Like SLP-76 and SLP-65, forced expression of CLNK augments immunoreceptor signaling, as demonstrated in TCR-stimulated Jurkat T cells [6
] and FcRI-stimulated RBL-2H3 basophilic leukemia cells [7
]. Based on the possibility that CLNK might function in parallel with SLP-76 and SLP-65 in FcR-associated signaling pathways in macrophages, we sought to determine whether CLNK was expressed in BMM from WT or SLP-76-/- SLP-65-/- mice. Using RT-PCR and Western blotting, we found no expression of CLNK mRNA or protein (data not shown), indicating that intact FcR signaling is not a result of simultaneous CLNK expression.
An alternative explanation for our findings includes the possibility that in contrast to antigen receptor signaling in lymphocytes, FcR signaling in mast cells, and GPVI collagen receptor signaling in platelets, FcR signaling in macrophages does not directly rely on the presence of adaptors such as SLP-76 and SLP-65, but rather it relies on related adaptor molecules such as LAT. Recently, experiments using the human monocytic cell line THP-1 have shown that the FcR chain constitutively associates with LAT [49
]. Moreover, following FcR cross-linking, LAT becomes phosphorylated and interacts with other signaling molecules, including PLC1, the adaptor molecule Grb2, and the p85 subunit of phosphoinositide 3-kinase. The potential, functional relevance of the direct interaction between the FcR and LAT is supported by additional data from this same laboratory that combined expression of these molecules in heterologous COS-7 cells, which do not express SLP-76 or SLP-65, augments FcR-mediated phagocytosis. Although LAT has not been reported to be present in mouse macrophages, LAT-/- BMM demonstrate a 50% reduction in FcR-induced phagocytic activity, suggesting that LAT might function in at least some FcR-associated pathways [49
]. Based on these observations, it remains possible that by directly interacting with the FcR, LAT predominates over SLP-76 and/or SLP-65 in FcR-induced signaling and functional events in macrophages. Experiments are currently ongoing to test this possibility.
As a third explanation for our findings, we note that the requirements for adaptor molecule use in macrophages may vary depending on several factors, including which FcRs are being engaged or the type, maturation, or activation status of the macrophages under investigation [33
]. Macrophages express three different classes of FcR, including the activating receptors FcRI and FcRIII and the inhibitory FcRIIb [30
]. Although the signaling pathways initiated by these receptors share common features, including the absolute requirement for intact Syk tyrosine kinase activity [34
], data exist suggesting that the downstream signaling pathways initiated by these different receptor classes may not be completely identical [50
]. In our experiments, we used IgG–SRBC to induce FcR-mediated signaling and functional activation. As IgG–SRBC likely engage all three categories of FcR concurrently, we would not have been able to detect signaling defects in our cells that were specific to one or more FcR classes.
In conclusion, the results of our experiments indicate that SLP-76 and SLP-65 are not required for FcR-, CR-, and possibly TLR/IFN--induced activation of in vitro-derived BMM. Nonetheless, SLP-76 and SLP-65 may still contribute to other receptor-induced signaling and activation pathways in macrophages. For example, several new receptor families have been identified recently in myeloid cells, including the Ig-like receptors (also known as leukocyte Ig-like and monocyte-macrophage inhibitory receptors) [51
], signal regulatory proteins [52
, 53
], the mouse-paired Ig receptors [54
], and the triggering receptors expressed on myeloid cells [55
56
57
58
]. Activating members of these receptor families function by forming an association with ITAM-bearing transmembrane adaptors, and although little is known about the signaling events initiated by these receptors, it is conceivable that SLP-76 and/or SLP-65 may play nonredundant roles downstream of one or more of them. Future studies addressing the molecular requirements of these new receptor families or of other Syk kinase-dependent receptors including integrins [59
] and a more careful dissection of the molecular mechanisms underlying FcR-induced signaling in BMM may clarify the role of SLP-76 and SLP-65 in macrophage signaling and functional activation.
Received July 3, 2003; revised October 13, 2003; accepted October 21, 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/CD3 induction of interleukin-2 Immunity 6,155-164[CrossRef][Medline]1 (PLC-1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC- 1 and NFAT Mol. Cell. Biol. 21,4208-42181 in an SLP-76-deficient T cell Science 281,413-416RI) in mast cells Mol. Cell. Biol. 23,2395-2406 receptors I and II/III Proc. Natl. Acad. Sci. USA 97,1725-1730 receptor-mediated phagocytosis and phagosome-lysosome fusion J. Leukoc. Biol. 70,801-811 receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn J. Exp. Med. 191,669-682 receptor signaling in macrophages and neutrophils Mol. Cell. Biol. 18,4209-4220IIb ß3 and collagen receptors Proc. Natl. Acad. Sci. USA 97,12056-12061 receptor-mediated signal transduction in myeloid cells J. Biol. Chem. 275,20480-20487 receptor-mediated phagocytosis Immunol. Lett. 44,133-138[CrossRef][Medline]This article has been cited by other articles:
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