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The Journal of Immunology, 2008, 180, 8020 -8029
Copyright © 2008 by The American Association of Immunologists, Inc.
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CD45 Regulates TLR-Induced Proinflammatory Cytokine and IFN-β Secretion in Dendritic Cells1

Jennifer L. Cross, Katharine Kott, Tatjana Miletic and Pauline Johnson2

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD45 is a leukocyte-specific protein tyrosine phosphatase and an important regulator of AgR signaling in lymphocytes. However, its function in other leukocytes is not well-understood. In this study, we examine the function of CD45 in dendritic cells (DCs). Analysis of DCs from CD45-positive and CD45-null mice revealed that CD45 is not required for the development of DCs but does influence DC maturation induced by TLR agonists. CD45 affected the phosphorylation state of Lyn, Hck, and Fyn in bone marrow-derived DCs and dysregulated LPS-induced Lyn activation. CD45 affected TLR4-induced proinflammatory cytokine and IFN-β secretion and TLR4-activated CD45-null DCs had a reduced ability to activate NK and Th1 cells to produce IFN-{gamma}. Interestingly, the effect of CD45 on TLR-induced cytokine secretion depended on the TLR activated. Analysis of CD45-negative DCs indicated a negative effect of CD45 on TLR2 and 9, MyD88-dependent cytokine production, and a positive effect on TLR3 and 4, MyD88-independent IFN-β secretion. This indicates a new role for CD45 in regulating TLR-induced responses in DCs and implicates CD45 in a wider regulatory role in innate and adaptive immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A leukocyte-specific protein tyrosine phosphatase, CD45 is an abundant cell surface protein on lymphocytes where it functions to regulate the threshold of Ag receptor (AgR) signaling (1). In T cells, CD45 dephosphorylates the negative regulatory site of Lck, the Src family kinase primarily responsible for CD3 phosphorylation and initiation of the TCR-signaling cascade. The loss of CD45 in mice results in defective T cell development attributed to inefficient pre-TCR and TCR signaling (2, 3, 4). B cell development can occur in the absence of CD45, but there is an increased threshold for BCR signaling that impairs BCR-mediated proliferation and the deletion of autoreactive B cells (2, 3, 5). Analysis of a juxtamembrane wedge mutant of CD45 indicates that CD45 can both positively and negatively regulate signal transduction thresholds at multiple checkpoints in B cell development (6). In contrast, much less is known about the function of CD45 in other leukocytes. The lack of CD45 in macrophages affects their adhesion and the phosphorylation state of the Src family kinases Hck and Lyn (7); the loss of CD45 affects FcR signaling in mast cells (8) and chemotaxis in neutrophils (9). Although CD45 is best known for its ability to regulate specific Src family kinases in leukocytes, it has also been implicated in the regulation of Jaks that mediate cytokine signaling (10). In this study, we investigate the function of CD45 in dendritic cells (DCs).3

DCs play a crucial role in linking the innate and adaptive immune systems. Microbial Ags are recognized by pattern recognition receptors expressed by DCs and this triggers a maturation program in the DC that ultimately determines how a naive T cell will respond to Ag (11). Signaling from TLRs leads to the up-regulation of costimulatory molecules required for naive T cell activation and the generation of proinflammatory cytokines, such as IL-12, IL-6, and TNF-{alpha}, that influence the type of T cells generated (12). There are several subsets of DCs that can influence the type of T cells activated, as well as the type of response generated (13, 14). Splenic DCs can be broadly divided into CD11chigh DCs and CD11clow B220+ plasmacytoid DCs. CD11chigh DCs can be further subdivided based on their expression of CD8{alpha} or CD11b, which distinguish functional differences (13, 15). Plasmacytoid DCs represent a different functional subset responsible for the rapid production of type I IFNs ({alpha}/β/{omega}) upon viral stimuli. Given that DCs express CD45 and specific CD45 isoforms (B220 and CD45RB) mark specific DC subsets, it was of interest to determine how DCs would develop and function in the absence of CD45.

TLR signaling can trigger both MyD88-dependent and -independent signaling pathways but the extent to which each pathway is used varies between TLRs. Recruitment of the adaptor protein MyD88 to TLR4 is necessary for LPS-induced proinflammatory cytokine release, whereas recruitment of MyD88-independent adaptors Toll/IL-1R-containing adaptor inducing IFN-β (TRIF) and TRIF-related adaptor molecule leads to the up-regulation of costimulatory molecules and IFN-β release (16, 17). This contrasts with TLR2 and 9 signaling that use only the MyD88-dependent pathway for both cytokine production and up-regulation of costimulatory molecules, and with TLR3 signaling, which is MyD88 independent. Although the core elements of these signaling pathways have been elucidated, the contribution of other signaling molecules such as PI3K and Src family kinases remains unclear. For example, bone marrow-derived DCs (BMDC) from Lyn-null mice produce less IL-12 after exposure to LPS (18, 19), but bone marrow-derived macrophages from Lyn-, Hck-, and Fgr-null mice produce normal levels of IL-1, IL-6, and TNF-{alpha} in response to LPS and IFN-{gamma} (20).

The tyrosine phosphatase CD45 is a major phosphatase that regulates Src family kinases such as Lck, Lyn, Fyn, and Hck in leukocytes. Here, the function of CD45 in DC development and maturation was assessed by analysis of splenic and BMDC from CD45-deficient mice. CD45 was found to either positively or negatively regulate TLR-mediated proinflammatory cytokine and IFN-β secretion, the effect depending upon which TLR was activated. Unstimulated CD45–/– BMDC showed dysregulation of Lyn and SHIP phosphorylation and in LPS-stimulated DCs, Lyn activation was compromised. LPS-activated CD45–/– DCs were less efficient in activating NK and Th1 cells to produce IFN-{gamma}. This indicates a new role for CD45 in regulating innate and adaptive immune responses by regulating TLR-induced responses in DCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

CD45+/+, CD45 exon 9 knockout mice (CD45–/– mice), and TCR-transgenic mice specific for OVA323–339 peptide (OTII) were purchased from The Jackson Laboratory and bred at the University of British Columbia (UBC) Animal Unit. CD45–/– mice were backcrossed for a total of nine generations onto the CD45+/+ background before homozygous matings were established. All mice were used between 8 and 16 wk of age. Animal experimentation was conducted in accordance with protocols approved by the University Animal Care Committee and Canadian Council of Animal Care guidelines.

Abs and flow cytometry

Abs specific for mouse CD11c (N418), CD40 (HM40-3), CD80 (16-10A1), CD86 (GL1), MHC class II (MHCII) I-A/I-E (M5/114.15.2) CD4 (RM40-3), CD11b (M1/70), DX5, CD8{alpha} (53-6.7), IL-12p40 (C17.8), TNF-{alpha} (MP6-XT22), IFN-{gamma} (XMG1.2), and TLR4 (MTS510) were all purchased from eBioscience conjugated to either FITC, PE, PE-Cy5, or allophycocyanin. Plasmacytoid DCs were detected with the mPDCA-1 Ab from Miltenyi Biotec. DC FcRs were blocked using anti-CD16/32 (2.4G2; American Type Culture Collection) tissue culture supernatant before Ab labeling. Labeled cells were analyzed either on a FACScan or a FACSCalibur (BD Biosciences) and analyzed using FlowJo (Tree Star). For signaling studies, Abs specific for Jak1, Jak2, Lyn (sc-15), Hck (sc-72), Fyn (sc-16), Fgr (sc-17), and NF-{kappa}B p65 were all purchased from Santa Cruz Biotechnology. Anti-Src (no. 2123S) as well as the phosphospecific Abs to Erk, p38, Akt (Ser473), Jnk, phospho-Src416 (no. 2101S), and phospho-Lyn507 (no. 2731S) were obtained from Cell Signaling Technology, as was the anti-phosphotyrosine mAb (4G10). Anti-SHIP sera was a gift from G. Krystal (Terry Fox Laboratories, Vancouver, British Columbia, Canada).

BMDC preparation

BMDC were prepared according to Lutz et al. (21). Briefly, cells were isolated from the femurs and tibias of the mice and plated in petri dishes (BD Biosciences) at 2 x 105 cells/ml in medium (RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, nonessential amino acids, 20 mM HEPES, sodium pyruvate, penicillin/streptomycin, and 50 µM 2-ME (all obtained from Invitrogen)) supplemented with 4% (v/v) of GM-CSF-containing J558L supernatant (22). On day 3, an equal volume of medium with fresh GM-CSF was added to the plates. On day 6, half of the culture was removed, spun down, and replaced with fresh medium and GM-CSF. Cells were harvested for use on day 7 or 8. To assess DC differentiation, nonadherent cells were harvested from the plates at different culture days, counted by hemocytometry, and analyzed for CD11c expression by flow cytometry. The total number of BMDC was calculated as (percent CD11c+ cells/100) x total number of nonadherent cells obtained. For analysis of costimulatory molecule expression, day 7 cells were replated on tissue culture plates with or without 1 µg/ml LPS (Escherichia coli 055:B5, L2880; Sigma-Aldrich), or 250 ng/ml Pam3Csk4 (InvivoGen), or 1 µM CpG-oligodeoxynucleotide 1668 (synthesized by UBC Nucleic Acid Protein Service Unit facility), or 20 µg/ml poly(I:C) (InvivoGen) and the nonadherent cells were analyzed by flow cytometry after 24 h using CD11c as a marker for BMDC. For cytokine analysis, BMDC were purified from the day 8 cultures by positive selection for CD11c using MACS magnetic beads (Miltenyi Biotec) and the purity of CD11c+ cells was always >95%. Supernatants for cytokine analysis were obtained by incubating 2 x 105 BMDCs with LPS (E. coli 055:B5; Sigma-Aldrich), Ultrapure LPS (E. coli 0111:B4), Pam3Csk4, poly(I:C), or CpG-oligodeoxynucleotide 1668 for 20–24 h and supernatant stored at –80°C for subsequent ELISA.

Analysis of splenic dendritic cell populations

Harvested spleens were digested with 1 mg/ml collagenase D (Roche Applied Science) for 30 min at room temperature in HBSS and 5% FCS followed by the addition of EDTA to 20 mM for 5 min. The digested spleens were then passed through a metal sieve to generate a single-cell suspension and RBCs were lysed using 0.84% ammonium chloride. The cell suspension was washed twice in wash buffer (HBSS, 5% FCS, and 5 mM EDTA) before resuspension in 14.5% Histodenz (Sigma-Aldrich) for density gradient separation. The partially purified DCs were collected from the top of the gradient and counted for flow cytometric analysis. To assess total DC percentages and numbers, the collagenase-digested single-cell suspension was stained for CD11c and MHCII expression. The total numbers of DCs were calculated as (percentage of CD11c+MHCII+ in the spleen/100) x the total number of splenocytes obtained.

In vivo DC maturation

Mice were injected with 25 µg of LPS (Sigma-Aldrich) i.v. through the tail vein. After 18 h, the mice were sacrificed and the spleens were harvested for collagenase digestion and Histodenz enrichment. Costimulatory molecule expression on CD11chigh cells was analyzed by flow cytometry.

Cytokine assays

IL-12p70, IL-6, and TNF-{alpha} cytokine secretion by BMDC was analyzed by ELISA (eBioscience), used according to the manufacturer’s instructions. The ELISA for IFN-β (23) used a mouse anti-IFN-β monoclonal (7F-D3; Abcam) for capture and a polyclonal rabbit anti-mouse IFN-β for detection (PBL Biomedical Laboratories). For intracellular cytokine staining of splenic DCs, collagenase-digested single-cell spleen suspensions were plated at 1 x 107 cells/ml in 96-well tissue culture plates with or without 10 µg/ml LPS (Sigma-Aldrich) for 5 h with the addition of GolgiPlug (BD Biosciences) for the last 4 h. FcRs were blocked and cells subsequently labeled with Abs specific for CD11c, MHCII, and DX5 before fixation and permeabilization (4% paraformaldehyde, 0.2% saponin, and 2% FCS) and the cell suspension was then incubated with cytokine-specific Abs. The percentage of cytokine-secreting cells was analyzed by flow cytometry where DCs were identified as CD11c+MHCII+DX5–.

NK cell cocultures

NK cells were isolated from spleens of CD45+/+ mice using MACS DX5-positive selection (Miltenyi Biotec) and were >90% pure. Cocultures were initiated with 2 x 105 purified NK cells and 1 x 105 CD45+/+ or CD45–/– purified BMDC and incubated overnight in the presence of 1 µg/ml LPS (InvivoGen or Sigma-Aldrich). Supernatants were harvested and stored at –80°C before analysis of IFN-{gamma} by ELISA (eBioscience).

In vitro assays for IFN-{gamma}

OTII CD4 T cells (105) were cocultured with 5 x 104 CD45+/+ or CD45–/– BMDC with the indicated amounts of OVA323–339 peptide and 100 ng/ml LPS (InvivoGen), or 250 ng/ml Pam3Csk4 (InvivoGen), or 1 µM CpG in round-bottom 96-well plates for 3 days. Supernatants were harvested for IFN-{gamma} ELISA (eBioscience). For LPS-treated cells, cocultures were expanded into fresh medium with 10 U/ml recombinant mouse IL-2 (eBioscience) for another 3 days. For IFN-{gamma} secretion, cells were washed and replated for activation with 20 ng/ml PMA and 1 µg/ml ionomycin for 4 h with the addition of GolgiPlug during the final 3 h. Intracellular IFN-{gamma} in CD4+ cells was analyzed by flow cytometry.

Immunoprecipitation and Western blotting

For phosphotyrosine blotting of cell lysates, 1 x 106 cells were lysed in buffer containing 1% Triton X-100, 20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 2 mM EDTA, 0.2 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml aprotinin, 1 µg/ml pepstatin, 0.2 mM sodium molybdate, and 0.5 mM sodium orthovanadate (1% Tx-TNE). For immunoprecipitation of Lyn, Jak1, and Jak2, 1 x 107 stimulated cells were lysed in 1% Nonidet P-40 TNE buffer (20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 2 mM EDTA) containing 0.2 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 0.2 mM sodium molybdate, and 0.5 mM sodium orthovanadate and immunoprecipitated with 1 µg of Ab. For immunoprecipitation of SHIP, 1 x 107 cells were lysed in 0.5% Nonidet P-40, 100 mM sodium fluoride, 2 mM tetrasodium pyrophosphate, 2 mM sodium orthovanadate, 2 mM EDTA, 2 mM sodium molybdate, and 50 mM HEPES (pH 7.3) and SHIP antisera was added. Immunoprecipitations were run on SDS-PAGE and transferred to polyvinylidene difluoride membranes. For Western blotting, all primary Abs were added at a 1/1000 dilution in 0.1% BSA and incubated for 1 h before the addition of HRP-labeled secondary Ab for 30 min. Membranes were then washed in TBS solution (20 mM Tris (pH 7.5), 150 mM sodium chloride) containing 1% Tween 20 and developed using ECL (GE Health Care).

NF-{kappa}B activation assays

Purified BMDC were activated with 100 ng/ml Ultrapure LPS. Nuclear extracts were prepared by lysing stimulated cells in 10 mM HEPES (pH 7.9), 50 mM sodium chloride, 0.5 M sucrose, 0.1 mM EDTA, 0.5% Triton X-100, 1 mM DTT, 0.2 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml aprotinin, 1 µg/ml pepstatin. Nuclei were pelleted at 2500 x g before lysis in 10 mM HEPES (pH 7.9), 500 mM sodium chloride, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% Nonidet P-40, 0.2 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin. Equal amounts of nuclear extract were subjected to SDS-PAGE after protein concentration was determined by the BCA assay. Proteins were transferred to a polyvinylidene difluoride membrane and NF-{kappa}B p65 was immunoblotted with a 1/1000 dilution of goat polyclonal anti-NF-{kappa}B p65 (C-20; Santa Cruz Biotechnology). The NF-{kappa}B ELISA was performed as described (24). Briefly, a biotinylated NF-{kappa}B consensus sequence (5'-AGTTGAGGGGACTTTCCCAGGC) was immobilized on streptavidin plates and incubated with cell lysates. NF-{kappa}B p65 was detected using goat polyclonal anti-p65 (C-20) and rabbit anti-goat HRP from The Jackson Laboratory.

Statistical analysis

Results are expressed as the mean ± SEM and analyzed for statistical significance using a two-tailed Student t test or with a two-tailed Student t test with Welch’s correction where variances were significantly different. Values of p < 0.05 were considered statistically significant (*, p < 0.05; **, p ≤ 0.01; and ***, p < 0.001).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD45 is not required for splenic or in vitro BMDC development

Splenic DCs from C57BL/6 and CD45-deficient mice with a targeted disruption of exon 9 (3), backcrossed nine times onto the C57BL/6 background (hereafter referred to as CD45+/+ and CD45–/–, respectively), were compared. CD45–/– mice had a slightly reduced frequency of DCs (identified as CD11chighMHCIIhigh), but this was not significant when averaged over several experiments (Fig. 1A). There was a slight increase in the absolute number of splenic DCs isolated from CD45–/– mice compared with CD45+/+ (3.8 ± 2.3 x 106 vs 2.5 ± 1.6 x 106, n = 5), which was likely due to the 2-fold increase in total cellularity of the CD45–/– spleen (Ref. 3 and data not shown). Analysis of Histodenz-enriched CD11chigh DCs from the spleen revealed a reproducible decrease in the percentage of CD11chighCD11b+ DCs and a concomitant increase in the CD11chighCD8{alpha}+ DCs isolated from the CD45–/– mice (Fig. 1, B and C). This altered pattern of DC subsets was not due to an altered splenic environment arising from the lack of functional T and B cells, as it was still observed in CD45–/–CD11chigh cells when irradiated mice were reconstituted with 1:1 CD45–/–:CD45+/+ bone marrow to generate functional T and B cells (data not shown). Plasmacytoid DCs are usually identified by B220, a specific glycosylated form of CD45RABC. In the absence of CD45, they were identified using the mPDCA-1 Ab and were CD11clow. The lack of B220 expression on CD45–/– DCs did not affect the percentage of plasmacytoid DCs present in the spleen of CD45–/– mice (Fig. 1D). Thus, CD45 is not required for the generation of splenic DCs or plasmacytoid DCs, but it can influence the distribution of the CD8{alpha}+/CD11b+ DC subsets.


Figure 1
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  		FIGURE 1. Flow cytometric analysis of splenic DC populations. A, Flow cytometric analysis of CD11c+MHCIIhigh splenic DCs present in a single-cell suspension after collagenase D digestion. One representative experiment of five is shown. Graphical analyses showing the average percentage (±SEM) over multiple experiments for each data set are presented on the right for A–D. B, Flow cytometric analysis of CD11b+ distribution within the CD11c+ population after Histodenz enrichment. One representative experiment of seven is shown; ***, p ≤ 0.001. C, Flow cytometric analysis of CD8{alpha}+ distribution within the CD11c+ population after Histodenz enrichment. One representative experiment of five is shown; **, p < 0.01. D, Flow cytometric analysis of the mPDCA-1+ plasmacytoid DC population following Histodenz enrichment. One representative experiment of three is shown.

 
To compare the in vitro development of CD45+/+ and CD45–/– DCs, bone marrow was isolated from the respective mice and cultured in GM-CSF-containing medium, essentially as described (21), and the production and maturation of BMDC was monitored over time. Fig. 2A shows a decrease in the percentage of nonadherent bone marrow cells expressing the granulocyte marker, Ly6G, together with the concomitant increase in cells expressing CD11c for both CD45+/+ and CD45–/– mice. By day 8, there were approximately equal percentages of adherent (data not shown) and nonadherent, CD11chigh cells from the bone marrow of wild-type (Wt) and CD45–/– mice (Fig. 2B), but the yield was slightly higher from CD45–/– bone marrow (Fig. 2C). As the CD11chigh cells mature in vitro, they also become increasingly MHCIIhigh (Fig. 2D) and by day 8 there was also a slight increase in the percentage of CD45–/–MHCIIhigh cells. Thus, in terms of numbers and percentages, the lack of CD45 did not grossly affect the in vitro generation of DCs, but did subtly increase the number of CD11chighMHCIIhigh cells. There were no obvious differences in the morphology of these DCs (data not shown).


Figure 2
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  		FIGURE 2. Graphical representation of BMDC development from CD45+/+ (Wt) and CD45–/– mice. Bone marrow was cultured in medium with GM-CSF for indicated times and the nonadherent cells were harvested for analysis. A, Analysis of granulocyte contamination (Ly-6G) as compared with BMDC generation (CD11c) in the nonadherent population over time. The percentage of cells highly expressing each marker is represented at each time point. B, The percentage of CD11chigh cells over time was obtained by flow cytometric gating of CD11chigh cells present in the culture. C, Total numbers of CD11chigh cells were obtained by multiplying the percentage of CD11chigh cells by the total number of nonadherent cells. D, Percentage of CD11chigh cells that are also MHCIIhigh over time. Experiments are the mean ± SEM from three independent experiments using one mouse each.

 
CD45 affects costimulatory molecule expression in splenic and BMDC

Flow cytometric analysis of the expression levels of the costimulatory molecules (CD40, CD80, and CD86) and MHCII on unstimulated day 8 BMDC (nonadherent, CD11chigh) revealed a higher level of expression of all these molecules on the CD45–/– DCs (Fig. 3A, upper panels and graph), although the levels of MHCII were more variable. Analysis of Histodenz-enriched splenic CD11chigh DCs also showed a slight increase in expression of costimulatory molecules (Fig. 3B, upper panels and graph). CD80 showed a 2-fold increase in expression, whereas levels of CD40 and CD86 were only slightly elevated in the CD45–/– DCs and MHCII expression was slightly decreased.


Figure 3
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  		FIGURE 3. Flow cytometric analysis of costimulatory molecule expression on splenic and BMDC. A, CD45+/+ (Wt, thin line) and CD45–/– (thick line) bone marrow cells from day 7 GM-CSF cultures were incubated overnight with or without (bottom and top panels, respectively) 1 µg/ml LPS (Sigma-Aldrich) before analysis of CD80 and CD86 on CD11chigh cells by flow cytometry. Expression levels are shown on a log scale. Flow cytometry experiments are representative of three independent experiments using one mouse each. Graphs show the average fold difference in mean fluorescence intensity ± SEM for CD40, CD80, CD86, and MHCII from three experiments. B, Costimulatory molecule expression on unstimulated and in vivo-activated splenic DCs. CD45+/+ (thin line) and CD45–/– (thick line) mice were injected either with PBS (top panel) or 25 µg of LPS (Sigma-Aldrich; bottom panel). Spleens were harvested after 18 h and DCs were enriched by separation on Histodenz. The expression of CD80 and CD86 on CD11chigh cells was analyzed by flow cytometry. Expression levels are shown on a log scale. One representative experiment of three is shown. Each experiment used two mice per condition. The graph depicts the mean fold difference (±SEM) in costimulatory molecule expression on CD45–/– compared with CD45+/+ DCs over three experiments.

 
DC maturation results in the up-regulation of costimulatory molecules and the induction of proinflammatory cytokine secretion, and is triggered by pathogen recognition. LPS is a potent stimulator of DCs and both in vitro exposure of BMDC (with either 100 ng/ml Ultrapure LPS (InvivoGen) or 1 µg/ml LPS (Sigma-Aldrich)) and in vivo exposure of splenic DCs to LPS (Sigma-Aldrich) resulted in a substantial up-regulation of CD40, CD80, CD86, and MHCII in both the CD45+/+ and CD45–/– DCs. Despite this, expression levels of the costimulatory molecules still remained slightly higher in the CD45–/– splenic and BMDC (Fig. 3, A and B, lower panels). This implies that CD45 may have a minor role in regulating the expression levels of costimulatory molecules before LPS stimulation.

LPS-induced cytokine secretion is altered in CD45–/– DCs

In addition to up-regulating costimulatory molecules on DCs, LPS also plays an important role in shaping the type of adaptive immune response generated by stimulating cytokine secretion in DCs. LPS stimulates the production of proinflammatory cytokines such as IL-6, TNF-{alpha}, and IL-12 in CD8{alpha}+ splenic and GM-CSF-derived BMDC. CD45+/+ and CD45–/– splenocytes were stimulated in vitro with LPS (Sigma-Aldrich) for 5 h and intracellular cytokine production was examined in splenic DCs (CD11chighMHCIIhighDX5–) by flow cytometry (Fig. 4A). The percentage of cells positive for IL-12p40 and TNF-{alpha} was noticeably lower in the CD45–/– DCs (Fig. 4A). These changes could not be accounted for by the altered subset distribution as CD45–/– DCs have more, not less, CD8{alpha}+ DCs that contribute to proinflammatory cytokine secretion. A similar reduction in IL-12p70, TNF-{alpha}, and IL-6 cytokine production was also observed when 1 µg/ml LPS (Sigma-Aldrich) was used to stimulate BMDC. The concentration of cytokine in the supernatant was measured by ELISA after 16–24 h of stimulation and Fig. 4B shows the average cytokine secreted from three independent experiments with one mouse each. This difference could not be attributed to differences in TLR4 expression, as CD45+/+ and CD45–/– DCs expressed similar levels when assessed by FACS analysis (data not shown). To further determine a specific effect on TLR4 signaling, a titration was performed on BMDC with Ultrapure LPS (InvivoGen). This revealed a more subtle difference between CD45+/+ and CD45–/– DCs that was dependent on the LPS concentration (Fig. 4C). At low levels (10 ng/ml) of LPS, CD45–/– DCs showed a slight increase in IL-12p70 and IL-6 secretion but no difference in TNF-{alpha} secretion. However, at higher LPS concentrations the trend was reversed. A slight, but significant, reduction in all three proinflammatory cytokines was reproducibly seen at 100 ng/ml LPS, but no significant difference was seen at 1 µg/ml Ultrapure LPS. The data shown in Fig. 4C are from a representative experiment showing the average cytokine production from three mice per condition. This indicates that the absence of CD45 in DCs can affect the amount of proinflammatory cytokine produced by both splenic and BMDC in response to the bacterial stimulus, LPS.


Figure 4
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  		FIGURE 4. Analysis of cytokine secretion by LPS-activated CD45+/+ (Wt) and CD45–/– splenic and BMDC. A, Flow cytometric analysis after in vitro stimulation of splenocytes with LPS (Sigma-Aldrich). Splenocytes were stimulated in vitro with 10 µg/ml LPS for 5 h. IL-12p40 and TNF-{alpha} production were assessed by flow cytometry of CD11chighMHCIIhighDX5– cells. Experiment is representative of three or four independent replicates and the mean ± SEM from all the experiments is shown graphically below. B, In vitro stimulation of BMDC with 1 µg/ml LPS (Sigma-Aldrich). After 22–24 h of stimulation, the amounts of IL-12p70, TNF-{alpha}, and IL-6 in the culture supernatant were determined by ELISA. Data are the mean ± SEM of three to five independent experiments with one mouse each. C, In vitro stimulation of BMDC with titrated amounts of Ultrapure LPS (InvivoGen). After 22–24 h of stimulation, the amounts of IL-12p70, TNF-{alpha}, and IL-6 in the culture supernatant were determined by ELISA. One representative experiment of the average from three mice ± SEM is shown and was repeated three times. *, p < 0.05; **, p ≤ 0.01; ***, p < 0.001.

 
LPS-stimulated CD45–/– BMDC are less efficient at priming Th1 cells and stimulating NK cells to produce IFN-{gamma}

Because DC-derived IL-12 is instrumental in directing the adaptive immune response toward a Th1 response, T cells were activated in vitro with CD45+/+ and CD45–/– BMDC to determine whether CD45 affected the generation of a Th1 response and subsequent IFN-{gamma} production. CD4 T cells isolated from TCR-transgenic mice specific for OVA323–339 peptide (OTII) were incubated with peptide loaded, Ultrapure LPS (100 ng/ml) activated BMDC and cocultured for 6 days. The cells were then restimulated for 3 h with PMA/ionomycin and the production of IFN-{gamma} was measured by intracellular staining in CD4+ cells. Stimulation with LPS-activated CD45–/– DCs generated a lower percentage of CD4+ T cells producing IFN-{gamma} over a range of peptide concentrations (Fig. 5A). The average fold increase in CD4+ T cells making IFN-{gamma} was consistently less when LPS-activated CD45+/+ and CD45–/– DCs were compared (Fig. 5B). This decrease in IFN-{gamma} production was not accompanied by an increase in IL-4 production, as no intracellular IL-4 was detected by flow cytometry (data not shown). This reduced activation of Th1 cells by LPS-stimulated CD45–/– DCs, as measured by IFN-{gamma} production, indicates a functional defect in LPS-activated CD45–/– DCs and is consistent with the LPS-stimulated CD45–/– DC producing less IL-12p70.


Figure 5
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  		FIGURE 5. Ability of CD45+/+ (Wt) and CD45–/– DCs to stimulate IFN-{gamma} production from T and NK cells. A, Flow cytometric analysis of IFN-{gamma} secretion from T cells cocultured with BMDC. OTII CD4 T cells were cocultured with purified unstimulated or 100 ng/ml Ultrapure LPS-stimulated BMDC for 6 days before restimulation with PMA/ionomycin to measure intracellular IFN-{gamma}. Experiment is one representative of three. B, Graphical representation of the mean fold increase (±SEM) in IFN-{gamma} secretion by OTII T cells normalized to the amount of IFN-{gamma} produced in OTII T cells incubated with unstimulated CD45+/+ DCs pulsed with 0.03 µM peptide. C, IFN-{gamma} release from NK cells cultured in vitro with BMDC. BMDC and NK cells were cocultured in the presence or absence of 1 µg/ml Ultrapure LPS for 18 h and IFN-{gamma} in the culture supernatant was measured by ELISA. The mean ± SEM from a representative experiment using three mice per condition is shown. **, p < 0.01. Four independent experiments using a total 14 mice were performed.

 
DC-derived IL-12p70 has also been shown to influence NK cell activation and subsequent IFN-{gamma} secretion (25), therefore the ability of the CD45–/– DCs to induce IFN-{gamma} from NK cells was investigated. Fig. 5C shows that NK cells cocultured with 1 µg/ml Ultrapure LPS-activated CD45–/– BMDC produced less IFN-{gamma} than those cultured with LPS-activated CD45+/+ DCs. The same effect was observed with 1 µg/ml LPS (Sigma-Aldrich; data not shown), but not reproducibly with 100 ng/ml Ultrapure LPS, suggesting that factors in addition to IL-12 are contributing to this effect. No IFN-{gamma} was detected with LPS-activated BMDC alone, or with NK cells alone plus or minus LPS or when NK cells were incubated with unstimulated BMDC (data not shown). These data indicate that LPS-stimulated CD45–/– DCs have a reduced ability to activate NK and Th1 cells to secrete IFN-{gamma}.

Phosphotyrosine levels are dysregulated in CD45–/– BMDC

To investigate how CD45 is exerting its effect in DCs, phosphotyrosine levels from CD45+/+ and CD45–/– BMDC were compared. Fig. 6A shows alterations in cellular phosphotyrosine levels from lysates of CD45–/– BMDC, in particular, at bands around 130 and 66 kDa. The 130-kDa band was hyperphosphorylated in the CD45–/– DC lysate, indicative of a potential CD45 substrate, whereas the 66-kDa band was hypophosphorylated, suggesting it is a substrate of a tyrosine kinase regulated by CD45.


Figure 6
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  		FIGURE 6. Tyrosine phosphorylation state of proteins present in unstimulated CD45+/+ (Wt) and CD45–/– BMDC. A, Basal tyrosine phosphorylation in whole cell lysate. BMDC were lysed in 1% Tx-TNE and phosphotyrosine assessed by immunoblotting with 4G10. Blot is one representative of three experiments. B, Jak phosphorylation in day 8 BMDC. BMDC were lysed and Jak 1or 2 was immunoprecipitated. Phosphotyrosine and Jak loading were determined by immunoblotting. The blot is representative of three experiments. C, Phosphorylation of SHIP in day 8 BMDC. SHIP1 was immunoprecipitated from day 8 BMDC and loading and phosphotyrosine levels were determined by immunoblotting. Blot is one representative of at least three experiments. D, Src family kinase phosphorylation and expression in BMDC. Various Src family kinases were immunoprecipitated from day 8 BMDC and loading and phosphotyrosine levels were determined by immunoblotting. One representative experiment of three is shown. E, Densitometric analysis of the average fold increase (±SEM) in Src family kinase phosphorylation in CD45–/– BMDC over three experiments. Integrated density values of the phosphotyrosine level were divided by those obtained for the loading control, the value for CD45+/+ was then set to one.

 
Because CD45 has been implicated in Jak regulation and Jaks are around 130 kDa, Jak1 and 2 were immunoprecipitated from unstimulated day 8 BMDC, but were not phosphorylated (Fig. 6B). Another potential candidate was the inositol polyphosphate 5'-phosphatase (SHIP). This was found to be hyperphosphorylated in CD45–/– BMDC (Fig. 6C), but was not the p130 protein.

Because the major substrates for CD45 in lymphocytes and macrophages are members of the Src family of tyrosine kinases, which migrate between 50 and 60 kDa, we next evaluated the phosphorylation status of Src family kinases in BMDC. Src family kinases were immunoprecipitated from DC lysates and their tyrosine phosphorylation was determined by blotting with the anti-phosphotyrosine mAb, 4G10. Fig. 6D shows the presence of Hck, Lyn, Src, Fgr, and low levels of Fyn in BMDC but only Lyn, Hck, and Fyn were hyperphosphorylated in the absence of CD45. Fig. 6E indicates the extent of hyperphosphorylation for Fyn, Hck, and Lyn over three independent experiments. The increased tyrosine phosphorylation of Lyn, Hck and Fyn suggests that their functional activities may be dysregulated in CD45–/– DCs. Although not well-defined, Lyn has been implicated in TLR4 signaling in DCs leading to the up-regulation of costimulatory molecules and proinflammatory cytokine production (18, 19).

Lyn kinase activation is dysregulated in CD45–/– DCs

To determine whether the lack of CD45 affects the ability of Lyn to participate in TLR4 signaling, we examined the activation and phosphorylation status of Lyn upon stimulation with 100 ng/ml Ultrapure LPS. In unstimulated CD45–/– BMDC, Lyn is hyperphosphorylated at the negative regulatory site, tyrosine 507, but not at the autophosphorylation site (see upper and middle panels of Fig. 7A). Upon LPS stimulation, Lyn was inducibly phosphorylated at the autophosphorylation site in CD45+/+ DCs, whereas no comparable induction was seen in CD45–/– DCs (Fig. 7A). Phosphorylation detected by the anti-phosphotyrosine mAb, 4G10, or with anti-phospho-Lyn 507 was always stronger in the CD45–/– BMDC and did not change in response to LPS. This indicates that Lyn is hyperphosphorylated at the negative regulatory site in the absence of CD45 and is not inducibly phosphorylated or activated upon LPS stimulation.


Figure 7
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  		FIGURE 7. LPS induced signaling events in CD45+/+ (Wt) and CD45–/– BMDC. A, Lyn activation was assessed by Lyn immunoprecipitation from BMDC stimulated with 100 ng/ml LPS for indicated times before SDS-PAGE. Phosphorylation at the activation site (AutoP) was determined using antisera to phospho-Src416 that cross-reacts with the corresponding activation site on Lyn (Y396), phosphorylation at the inhibitory tyrosine was determined by using an Ab specific to phospho-Lyn507. Detection was done first for phosphotyrosine (Y416 or Y507 or 4G10) and then stripped and reprobed for Lyn. One representative from three experiments is shown. B, Phosphorylation of various signaling molecules in response to 100 ng/ml Ultrapure LPS was assessed by separation of cell lysates by SDS-PAGE and immunoblotting with phosphospecific Abs. Loading was determined by blotting for actin. C and D, Activation of NF-{kappa}B p65. Pooled BMDC from three mice were activated with 100 ng/ml Ultrapure LPS for indicated times and then lysed and left unfractionated for ELISA (C) or the nuclear fraction isolated and separated on SDS-PAGE and immunoblotted for NF-{kappa}B p65 (D). Experiment is one representative of three.

 
To examine possible downstream effects of altered Lyn activation in TLR4 signaling, the induction of MAPK phosphorylation upon LPS stimulation was investigated. Fig. 7B shows no obvious differences in the phosphorylation status of Erk, Jnk, or p38, which were all phosphorylated with similar kinetics and intensities in both CD45+/+ and CD45–/– BMDC. Because SHIP phosphorylation was altered in unstimulated BMDC, we examined the possibility that the PI3K pathway may be dysregulated in CD45–/– BMDC. Like the Src family kinases, the involvement of PI3K pathway in LPS signaling is not well-understood with both positive and negative effects being reported (reviewed in Ref. 26). However, analysis of phospho-Ser473 of Akt did not reveal any major differences between CD45+/+ and CD45–/– BMDC (Fig. 7B).

Because LPS-induced cytokine production requires the MyD88-dependent signaling pathway leading to NF-{kappa}B activation, we assessed whether NF-{kappa}B activation was affected. SDS-PAGE analysis of the translocation of NF-{kappa}B p65 into the nucleus as well as its ability to bind DNA, assessed by ELISA, revealed no reproducible differences in the kinetics or levels of translocated NF-{kappa}B or its DNA-binding activity (Fig. 7, C and D). Thus, no obvious defect was found in the MyD88-dependent TLR4-signaling pathway in CD45–/– DCs.

CD45–/– BMDC show enhanced proinflammatory cytokine production in response to TLR2 and TLR9 ligands

To gain further insight into which molecules or pathways are affected by CD45 in TLR signaling leading to cytokine production, we examined the effect of the loss of CD45 on three other TLR-signaling pathways, TLR2, TLR3, and TLR9. TLR4 signaling uses both MyD88-dependent and -independent pathways whereas TLR3 is MyD88 independent and TLR2 and TLR9 are both strictly dependent on MyD88. Upon stimulation with the TLR9 ligand, CpG 1668, the TLR2 ligand, Pam3Csk4, and the TLR3 ligand poly(I:C), CD45–/– BMDC were able to up-regulate their costimulatory molecules to levels similar to those observed in Wt BMDC (Fig. 8A). Again, it was noted that the levels of CD80 and CD86 were slightly higher in the CD45–/– BMDC before stimulation, similar to what we had observed with LPS. However, when we examined the production of proinflammatory cytokines induced by these specific TLR ligands, we found that in contrast to LPS, CD45–/– BMDC produced significantly more proinflammatory cytokines (IL-12, TNF-{alpha}, and IL-6) in response to the synthetic TLR2 ligand (Fig. 8B) and in response to the TLR9 ligand, CpG 1668 (Fig. 8C). The data are representative of three experiments and show the average cytokine produced from BMDC from three mice per condition. This increase in IL-12p70, IL-6, and TNF-{alpha} occurred over a range of concentrations for Pam3Csk4 and for CpG up to 1 µM. Stimulation of either Wt or CD45–/– BMDC with up to 20 µg/ml poly(I:C) did not produce measurable levels of the proinflammatory cytokines (data not shown). To further examine the negative effect of CD45 on TLR2 stimulation, we compared downstream signaling events in CD45+/+ and CD45–/– DCs after TLR2 stimulation. Investigation into increases in MAPK and Akt phosphorylation after Pam3Csk4 stimulation failed to reveal any noticeable differences between CD45+/+ and CD45–/– DCs (data not shown). This demonstrates a clear negative regulatory effect of CD45 on TLR2 and 9 proinflammatory cytokine production.


Figure 8
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  		FIGURE 8. BMDC maturation and cytokine secretion in response to various TLR ligands. A, CD45+/+ (Wt, thin line) and CD45–/– (thick line) bone marrow cells from day 7 GM-CSF cultures were incubated overnight with PBS, 1 µM CpG, 250 ng/ml Pam3Csk4, or 20 µg/ml poly(I:C) before analysis of CD80 and CD86 on nonadherent CD11chigh cells by flow cytometry. Expression levels are shown on a log scale. Flow cytometry experiments are representative of three independent experiments using BMDC generated from a pool of three mice. B and C, Purified day 8 CD45+/+ (Wt) and CD45–/– BMDC were stimulated with the indicated concentrations of (B) Pam3Csk4 or (C) CpG for 20–24 h. The amount of IL-12p70, TNF-{alpha}, and IL-6 in the culture supernatant was determined by ELISA. Graphs are the mean ± SEM of three mice in an individual experiment. Experiments were repeated at least three times. D, Purified day 8 CD45+/+ (Wt) and CD45–/– BMDC were activated with the indicated concentrations of Ultrapure LPS, CpG, or poly(I:C) for 20–24 h. The amount of IFN-β in the supernatant was quantified by ELISA. Graphs are the mean ± SEM from a representative experiment from three mice. Experiments were repeated two to four times. For all panels: **, p < 0.01; ***, p < 0.001.

 
CD45 regulates TLR-induced IFN-β production in DCs

To investigate why CD45 had a differential effect on proinflammatory cytokine secretion when different TLRs were activated, we chose to examine IFN-β production. Although TLR4-induced proinflammatory cytokine production is MyD88 dependent (27), production of IFN-β can enhance IL-12 production as well as promote more IFN-β (28). Thus, we hypothesized that the observed differences in proinflammatory cytokine production may be linked to differences in IFN-β production. IFN-β is produced upon TLR3 and 4 activation via a MyD88-independent pathway (29, 30), and via a MyD88-dependent pathway upon TLR9 stimulation. TLR2 stimulation does not generate IFN-β. Fig. 8D shows that lack of CD45 reduces IFN-β production in response to LPS, suggesting that CD45 positively regulates this MyD88-independent arm of the pathway. Interestingly, no IFN-β was made when low concentrations of LPS were used (10 ng/ml). IFN-β production by the TLR3 ligand, poly(I:C), was also reduced in CD45–/– BMDC (Fig. 8D), consistent with a positive effect of CD45 on MyD88-independent signaling leading to IFN-β production. Conversely, TLR9-induced production of IFN-β, which like IL-12 production, occurs through a MyD88-dependent pathway (27), was enhanced in CD45–/– BMDC (Fig. 8D). These data are consistent with the hypothesis that CD45 negatively regulates MyD88-dependent TLR signaling and positively regulates MyD88-independent TLR signaling.

Pam3Csk4-stimulated CD45–/– BMDC are more efficient at stimulating T cells to secrete IFN-{gamma}

IL-12 production by DCs is known to provide a favorable environment for the development of Th1 cells that produce IFN-{gamma} upon activation. In Fig. 5, we showed that LPS-stimulated CD45–/– BMDC were less efficient at inducing IFN-{gamma} from cocultured OTII T cells upon restimulation. This was consistent with the reduced amount of IL-12 they produced. To determine whether Pam3Csk4 and CpG-stimulated CD45–/– BMDC (which produce significantly more IL-12 than Wt BMDC) were more effective at generating IFN-{gamma}-producing Th1 cells, stimulated DCs were cocultured with OTII T cells for 3 days and IFN-{gamma} production was measured by ELISA. Fig. 9 demonstrates that even after 3 days in coculture, OTII cells cultured with Pam3Csk4-activated CD45–/– BMDC induced higher levels of IFN-{gamma}, compared with LPS (100 ng/ml; Ultrapure) activated CD45–/– BMDC, which produced less IFN-{gamma} than Wt BMDC. Surprisingly, OTII T cells cultured with CpG-activated CD45–/– BMDC produced similar amounts of IFN-{gamma} to Wt BMDC. However, significantly less IL-2 was present in these cultures (data not shown), indicative of less T cell activation. This may be related to the increased amount of IFN-β produced by CpG-stimulated CD45–/– BMDC (~2- to 3-fold more than Wt CpG-stimulated BMDC, see Fig. 8D), as type 1 IFNs have been shown to inhibit peptide-stimulated T cell proliferation in vitro (31). Overall, these data demonstrate the ability of CD45–/– BMDC to affect the amount of cytokine secreted by activated T cells and suggests a role for CD45 in modulating Th1 responses, possibly via its ability to modulate TLR-induced secretion of proinflammatory cytokines in DCs.


Figure 9
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  		FIGURE 9. IFN-{gamma} levels from cocultures of CD4+ OTII T cells and LPS, CpG, or Pam3Csk4 activated BMDC. Purified day 8 BMDC were activated with either 100 ng/ml Ultrapure LPS, 1 µM CpG, or 250 ng/ml Pam3Csk4 in the presence of titrated doses of OVA peptide before the addition of CD4+ OTII T cells. After 72 h in coculture, supernatants were harvested for analysis by IFN-{gamma} ELISA. Experiments are representative of three repeats using BMDC pooled from three mice. **, p ≤ 0.001; ***, p ≤ 0.0001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have shown that splenic and BMDC can develop in the absence of CD45, but there is a skewing toward the CD8{alpha}+CD11b– population and a decrease in the CD8{alpha}–CD11b+ population in splenic CD11chigh DCs. Given that the CD8{alpha}+ population is most efficient at cross-presentation and CD8 T cell activation, one would predict that if CD45–/– mice had functional T cells, they would be more efficient at these processes and less efficient at CD4 Th cell responses, which is mediated by the CD8{alpha}– DC subset (15).

The loss of CD45 led to a slight increase in CD40, CD80, and CD86 costimulatory molecule expression in splenic and BMDC. This slight increase persisted after up-regulation of the costimulatory molecules by LPS but was not observed consistently after CpG, poly(I:C), or Pam3Csk4 stimulation of BMDC. Higher levels of expression of CD40, CD80, and MHCII were also observed by Piercy and colleagues (32) in unstimulated and CpG- and poly(I:C)-stimulated CD45–/– BMDC. It is not clear why their observations were different from ours, but it may relate to the 5-fold higher concentration of poly(I:C) and to the different CpG sequence (1828 vs 1668) used. The consistent higher level of expression of costimulatory molecules on unstimulated CD45–/– DC suggests that CD45 has a slight negative regulatory effect on costimulatory molecule expression, possibly resulting from a slight enhancement of spontaneous DC maturation in the absence of pathogenic stimuli. This effect is noticeably different from that observed in the Lyn–/– mice where costimulatory molecule expression is decreased before stimulation and upon LPS stimulation (18, 19). Although we have shown that Lyn is not activated upon LPS stimulation in CD45–/– BMDC, Lyn inactivation alone cannot account for all the effects observed in the CD45–/– BMDC. This is also supported by our finding that CD45 regulates the phosphorylation state of not just Lyn, but also Hck and Fyn in BMDC. Although previous studies identified Src in BMDC (33) and Lyn, Hck, and Fgr in the lysates of BMDC (19), this is the first to report low levels of Fyn after immunoprecipitation from BMDC.

A major finding from this work is that TLR-induced proinflammatory cytokine secretion and IFN-β production is regulated by CD45 and that both positive and negative effects of CD45 were observed, depending on which TLR was stimulated. The absence of CD45 in BMDC led to an increase in TLR2- and TLR9-driven proinflammatory cytokine secretion, which both signal via a MyD88-dependent pathway. Thus, CD45 has a negative regulatory effect on TLR2- and TLR9-induced MyD88-dependent cytokine secretion. CD45 also had a negative regulatory effect on TLR9-induced IFN-β secretion, which also proceeds via a MyD88-dependent signaling pathway. In contrast, the absence of CD45 in BMDC led to a decrease in TLR3- and TLR4-induced IFN-β production, which proceeds via a MyD88-independent pathway. Thus, our data are consistent with the hypothesis that CD45 has a negative regulatory effect on MyD88-dependent TLR-signaling pathways leading to cytokine production and a positive effect on TLR-induced, MyD88-independent signaling leading to IFN-β production. The differential use of MyD88-dependent and -independent pathways by different TLRs thus provides one plausible explanation for the observed positive and negative effects of CD45 on specific TLR activated cytokine production. However, it should be noted that CD45 has also been reported to both positively and negatively affect IFN-{alpha} signaling from the type I IFNR in other cell types (10, 34).

LPS-induced proinflammatory cytokine secretion is known to be MyD88 dependent (35), yet reports have shown that IFN-β (made by stimulation via a MyD88-independent, TRIF-dependent pathway (36)), activates a feedback loop by binding to the type I IFNR on the DC, which not only stimulates more IFN-β secretion, but also enhances IL-12p70 secretion (28). Hence, the levels of IFN-β made upon LPS signaling can affect proinflammatory cytokine secretion. The effect of CD45 on TLR4-induced cytokine production was less obvious and more complex than its effect on TLR2-, TLR3-, or TLR9-induced cytokine secretion. The absence of CD45 on BMDC led to an increase in IL-6 and IL-12 production upon stimulation with low Ultrapure LPS concentrations, whereas a decrease was observed with 100 ng/ml LPS and no significant difference at 1 µg/ml LPS. A negative regulatory effect of CD45 observed with 10 ng/ml LPS occurred in the absence of any detectable IFN-β production and is consistent with the negative effect of CD45 on MyD88-dependent, TLR2 and 9 activation. At the higher LPS concentrations, one has to consider the positive effect of CD45 on IFN-β production as well as its negative effect on MyD88-dependent proinflammatory cytokine secretion. We hypothesize that the outcome of these two opposing effects is an overall positive effect with 100 ng/ml LPS and a balanced effect with 1 µg/ml LPS.

While this work was in progress, another group reported the effects of the absence of CD45 on proinflammatory cytokine production in DCs and on plasmacytoid DCs (32, 37). In agreement with this study, they observed increased TNF-{alpha} and IL-6 production from CD45–/– BMDC in response to CpG. They also saw an increase in proinflammatory cytokines with 100 µg/ml poly(I:C) stimulation of CD45–/– BMDC whereas we did not detect any proinflammatory cytokines in either CD45+/+ or CD45–/– BMDC with 20 µg/ml poly(I:C) stimulation. In contrast to our data showing no observable difference in the percentage of mPDCA-1 plasmacytoid DCs in the spleen, Montoya et al. (32) found an increase in plasmacytoid DCs in CD45–/– mice, although it was not clear how they identified them in the absence of the CD45-dependent B220 marker. In addition, they found an impaired capacity of CD45–/– mice to produce type I IFNs (IFN-{alpha}) in response to lymphocytic choriomeningitis virus infection in vivo. We also found an effect of CD45 on type I IFN (IFN-β) production in BMDC that was dependent upon the TLR stimulated.

Another major finding of this study was the inability of LPS-activated (Ultrapure, 100 ng/ml) CD45–/– BMDC to effectively activate NK cells and Ag-specific CD4+ T cells to produce IFN-{gamma}. Conversely, Pam3Csk4-stimulated CD45–/– BMDC caused higher levels of IFN-{gamma} secretion by Ag-specific T cells. Thus, it is clear that DC CD45 has the potential to modify the outcome of both innate and adaptive, Th1-mediated, immune responses. Although IL-12 is known to promote NK and Th1 IFN-{gamma} secretion (38), type I IFNs can also promote NK activation and affect T cell proliferation (31, 39). Thus, the effect of DC CD45 on these responses will depend on the overall effect of CD45 on cytokine production by DCs. It will be of interest to determine the response of CD45–/– DCs to an infection, where multiple TLRs are engaged and to determine the outcome of an in vivo immune response primed by CD45–/– DCs.

Investigation of the molecular defects occurring in BMDC in the absence of CD45 revealed the hyperphosphorylation of Hck, Lyn, Fyn, and SHIP. LPS stimulation induced transient Lyn activation in the CD45+/+ bone marrow cells but not in the CD45–/– cells, showing dysregulation of Lyn in the absence of CD45. However, further analysis of the phosphorylation states of MAPKs and Akt, as well as NF-{kappa}B p65 translocation and DNA binding, did not reveal any major differences between LPS (100 ng/ml Ultrapure) activated CD45+/+ and CD45–/– BMDC. Both Lyn and PI3K are activated upon LPS stimulation of human monocytes (40) and treatment of human monocyte-derived DCs with a Src family kinase inhibitor, PP1, decreased TNF-{alpha}, IL-6, and IL-12p40 production in response to LPS, indicating a role for Src family kinase activity in proinflammatory cytokine secretion (41). Analysis of BMDC from Lyn–/– mice also showed a positive role for Lyn, as reduced levels of IL-12 were produced in response to 1–4 ng/ml LPS and 5–80 ng/ml CpG (19). In contrast to this, macrophages from Lyn, Hck, and Fgr triple knockout mice show normal or slightly enhanced cytokine responses to LPS and IFN-{gamma} (20), suggesting dispensable or opposing roles for Src family kinases in LPS signaling. Recent data indicate that Hck and Lyn are activated in response to CpG in a TLR9-independent manner, yet are required for the subsequent TLR9-MyD88-dependent cytokine production in a human monocytic cell line, THP-1 (42). In addition, Src is activated and associates with TLR3 upon stimulation with dsRNA in human monocyte-derived DCs (43). Thus, the precise roles of Src family kinases in the various TLR signaling pathways leading to costimulatory molecule and cytokine production are complex and not yet fully understood. It is possible that the effect of CD45 on the MyD88-dependent pathway could be mediated via its dysregulation of Lyn, although the role of Lyn in TLR signaling remains poorly defined. In the CD45–/– BMDC, there was dysregulated phosphorylation of Lyn, Hck, and Fyn implying that the effect of CD45 is not just restricted to Lyn. Thus, the precise mechanism of CD45 in MyD88-dependent and -independent TLR signaling and its regulation of Src family kinases in DCs awaits further analysis.

Overall, this work identifies CD45 as a regulator of TLR-induced proinflammatory cytokine secretion in DCs. DC CD45 can impact subsequent NK and Th1 activation and thus has the potential to influence the outcome of both innate and adaptive immune responses. Thus, in addition to its well-established role in lymphocytes, CD45 may influence the outcome of an immune response by modulating TLR signaling in DCs.


   Acknowledgments
 
We thank the University of British Columbia FACS Facility for assistance with flow cytometric analysis and Gerry Krystal at the Terry Fox Laboratories for the anti-SHIP antisera.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Canadian Institutes of Health Research (to P.J.). Back

2 Address correspondence and reprint requests to Dr. Pauline Johnson, Department of Microbiology and Immunology, University of British Columbia, 2350 Health Sciences Mall, Life Sciences Institute, Vancouver, British Columbia V6T 1Z3, Canada. E-mail address: pauline{at}interchange.ubc.ca Back

3 Abbreviations used in this paper: DC, dendritic cell; TRIF, Toll/IL-1R-containing adaptor inducing IFN-β; BMDC, bone marrow-derived DC; MHCII, MHC class II; Wt, wild type. Back

Received for publication August 3, 2007. Accepted for publication April 9, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Hermiston, M. L., Z. Xu, A. Weiss. 2003. CD45: a critical regulator of signaling thresholds in immune cells. Annu. Rev. Immunol. 21: 107-137. [Medline]
  2. Kishihara, K., J. Penninger, V. A. Wallace, T. M. Kundig, K. Kawai, A. Wakeham, E. Timms, K. Pfeffer, P. S. Ohashi, M. L. Thomas, et al 1993. Normal B-lymphocyte development but impaired T-cell maturation in CD45-exon6 protein tyrosine phosphatase-deficient mice. Cell 74: 143-156. [Medline]
  3. Byth, K. F., L. A. Conroy, S. Howlett, A. J. H. Smith, J. May, D. R. Alexander, N. Holmes. 1996. CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and in B cell maturation. J. Exp. Med. 183: 1707-1718. [Abstract/Free Full Text]
  4. Mee, P. J., M. Turner, M. A. Basson, P. S. Costello, R. Zamoyska, V. L. Tybulewicz. 1999. Greatly reduced efficiency of both positive and negative selection of thymocytes in CD45 tyrosine phosphatase-deficient mice. Eur. J. Immunol. 29: 2923-2933. [Medline]
  5. Cyster, J. G., J. I. Healy, K. Kishihara, T. W. Mak, M. L. Thomas, C. C. Goodnow. 1996. Regulation of B-lymphocyte negative and positive selection by tyrosine phosphatase CD45. Nature 381: 325-328. [Medline]
  6. Hermiston, M. L., A. L. Tan, V. A. Gupta, R. Majeti, A. Weiss. 2005. The juxtamembrane wedge negatively regulates CD45 function in B cells. Immunity 23: 635-647. [Medline]
  7. Roach, T., S. Slater, M. Koval, L. White, E. D. Cahir McFarland, M. Okumura, M. Thomas, E. Brown. 1997. CD45 regulates Src family member kinase activity associated with macrophage integrin-mediated adhesion. Curr. Biol. 7: 408-417. [Medline]
  8. Berger, S. A., T. W. Mak, C. J. Paige. 1994. Leukocyte common antigen (CD45) is required for immunoglobulin E-mediated degranulation of mast cells. J. Exp. Med. 180: 471-476. [Abstract/Free Full Text]
  9. Harvath, L., J. A. Balke, N. P. Christiansen, A. A. Russell, K. M. Skubitz. 1991. Selected antibodies to leukocyte common antigen (CD45) inhibit human neutrophil chemotaxis. J. Immunol. 146: 949-957. [Abstract]
  10. Irie-Sasaki, J., T. Sasaki, W. Matsumoto, A. Opavsky, M. Cheng, G. Welstead, E. Griffiths, C. Krawczyk, C. D. Richardson, K. Aitken, et al 2001. CD45 is a JAK phosphatase and negatively regulates cytokine receptor signalling. Nature 409: 349-354. [Medline]
  11. Janeway, C. A., R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20: 197-216. [Medline]
  12. Medzhitov, R.. 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1: 135-145. [Medline]
  13. Shortman, K., Y. J. Liu. 2002. Mouse and human dendritic cell subtypes. Nat. Rev. Immunol. 2: 151-161. [Medline]
  14. Shortman, K., S. H. Naik. 2007. Steady-state and inflammatory dendritic-cell development. Nat. Rev. Immunol. 7: 19-30. [Medline]
  15. Dudziak, D., A. O. Kamphorst, G. F. Heidkamp, V. R. Buchholz, C. Trumpfheller, S. Yamazaki, C. Cheong, K. Liu, H. W. Lee, C. G. Park, et al 2007. Differential antigen processing by dendritic cell subsets in vivo. Science 315: 107-111. [Abstract/Free Full Text]
  16. Kawai, T., S. Akira. 2007. TLR signaling. Semin. Immunol. 19: 24-32. [Medline]
  17. Trinchieri, G., A. Sher. 2007. Cooperation of Toll-like receptor signals in innate immune defence. Nat. Rev. Immunol. 7: 179-190. [Medline]
  18. Beavitt, S. J., K. W. Harder, J. M. Kemp, J. Jones, C. Quilici, F. Casagranda, E. Lam, D. Turner, S. Brennan, P. D. Sly, et al 2005. Lyn-deficient mice develop severe, persistent asthma: Lyn is a critical negative regulator of Th2 immunity. J. Immunol. 175: 1867-1875. [Abstract/Free Full Text]
  19. Chu, C. L., C. A. Lowell. 2005. The Lyn tyrosine kinase differentially regulates dendritic cell generation and maturation. J. Immunol. 175: 2880-2889. [Abstract/Free Full Text]
  20. Meng, F., C. A. Lowell. 1997. Lipopolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn. J. Exp. Med. 185: 1661-1670. [Abstract/Free Full Text]
  21. Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rossner, F. Koch, N. Romani, G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223: 77-92. [Medline]
  22. Stockinger, B., T. Zal, A. Zal, D. Gray. 1996. B cells solicit their own help from T cells. J. Exp. Med. 183: 891-899. [Abstract/Free Full Text]
  23. Weinstein, S. L., A. J. Finn, S. H. Dave, F. Meng, C. A. Lowell, J. S. Sanghera, A. L. DeFranco. 2000. Phosphatidylinositol 3-kinase and mTOR mediate lipopolysaccharide-stimulated nitric oxide production in macrophages via interferon-β. J. Leukocyte Biol. 67: 405-414. [Abstract]
  24. Renard, P., I. Ernest, A. Houbion, M. Art, H. Le Calvez, M. Raes, J. Remacle. 2001. Development of a sensitive multi-well colorimetric assay for active NF{kappa}B. Nucleic Acids Res. 29: E21[Medline]
  25. Alli, R. S., A. Khar. 2004. Interleukin-12 secreted by mature dendritic cells mediates activation of NK cell function. FEBS Lett. 559: 71-76. [Medline]
  26. Fukao, T., S. Koyasu. 2003. PI3K and negative regulation of TLR signaling. Trends Immunol. 24: 358-363. [Medline]
  27. Kaisho, T., O. Takeuchi, T. Kawai, K. Hoshino, S. Akira. 2001. Endotoxin-induced maturation of MyD88-deficient dendritic cells. J. Immunol. 166: 5688-5694. [Abstract/Free Full Text]
  28. Gautier, G., M. Humbert, F. Deauvieau, M. Scuiller, J. Hiscott, E. E. Bates, G. Trinchieri, C. Caux, P. Garrone. 2005. A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells. J. Exp. Med. 201: 1435-1446. [Abstract/Free Full Text]
  29. Yamamoto, M., S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O. Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, S. Akira. 2003. Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science 301: 640-643. [Abstract/Free Full Text]
  30. Kawai, T., O. Takeuchi, T. Fujita, J. Inoue, P. F. Muhlradt, S. Sato, K. Hoshino, S. Akira. 2001. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J. Immunol. 167: 5887-5894. [Abstract/Free Full Text]
  31. Havenar-Daughton, C., G. A. Kolumam, K. Murali-Krishna. 2006. Cutting edge: the direct action of type I IFN on CD4 T cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection. J. Immunol. 176: 3315-3319. [Abstract/Free Full Text]
  32. Montoya, M., R. Dawes, D. Reid, L. N. Lee, J. Piercy, P. Borrow, E. Z. Tchilian, P. C. Beverley. 2006. CD45 is required for type I IFN production by dendritic cells. Eur. J. Immunol. 36: 2150-2158. [Medline]
  33. Wong, B. R., D. Besser, N. Kim, J. R. Arron, M. Vologodskaia, H. Hanafusa, Y. Choi. 1999. TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src. Mol. Cell 4: 1041-1049. [Medline]
  34. Petricoin, E. F., S. Ito, B. L. Williams, S. Audet, L. F. Stancato, A. Gamero, K. Clouse, P. Grimley, A. Weiss, J. Beeler, et al 1997. Antiproliferative action of interferon-{alpha} requires components of T-cell-receptor signalling. Nature 390: 629-632. [Medline]
  35. Kaisho, T., S. Akira. 2001. Dendritic-cell function in Toll-like receptor- and MyD88-knockout mice. Trends Immunol. 22: 78-83. [Medline]
  36. Pietras, E. M., S. K. Saha, G. Cheng. 2006. The interferon response to bacterial and viral infections. J Endotoxin Res. 12: 246-250. [Medline]
  37. Piercy, J., S. Petrova, E. Z. Tchilian, P. C. Beverley. 2006. CD45 negatively regulates tumour necrosis factor and interleukin-6 production in dendritic cells. Immunology 118: 250-256. [Medline]
  38. Watford, W. T., M. Moriguchi, A. Morinobu, J. J. O'Shea. 2003. The biology of IL-12: coordinating innate and adaptive immune responses. Cytokine Growth Factor Rev. 14: 361-368. [Medline]
  39. Fitzgerald-Bocarsly, P., D. Feng. 2007. The role of type I interferon production by dendritic cells in host defense. Biochimie 89: 843-855. [Medline]
  40. Herrera-Velit, P., N. E. Reiner. 1996. Bacterial lipopolysaccharide induces the association and coordinate activation of p53/56lyn and phosphatidylinositol 3-kinase in human monocytes. J. Immunol. 156: 1157-1165. [Abstract]
  41. Napolitani, G., N. Bortoletto, L. Racioppi, A. Lanzavecchia, U. D'Oro. 2003. Activation of src-family tyrosine kinases by LPS regulates cytokine production in dendritic cells by controlling AP-1 formation. Eur. J. Immunol. 33: 2832-2841. [Medline]
  42. Sanjuan, M. A., N. Rao, K. T. Lai, Y. Gu, S. Sun, A. Fuchs, W. P. Fung-Leung, M. Colonna, L. Karlsson. 2006. CpG-induced tyrosine phosphorylation occurs via a TLR9-independent mechanism and is required for cytokine secretion. J. Cell Biol. 172: 1057-1068. [Abstract/Free Full Text]
  43. Johnsen, I. B., T. T. Nguyen, M. Ringdal, A. M. Tryggestad, O. Bakke, E. Lien, T. Espevik, M. W. Anthonsen. 2006. Toll-like receptor 3 associates with c-Src tyrosine kinase on endosomes to initiate antiviral signaling. EMBO J. 25: 3335-3346. [Medline]




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