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The Journal of Immunology, 2003, 170: 1887-1893.
Copyright © 2003 by The American Association of Immunologists

Human Activation-Induced Cytidine Deaminase Is Induced by IL-4 and Negatively Regulated by CD45: Implication of CD45 as a Janus Kinase Phosphatase in Antibody Diversification 1

Cheng Zhou, Andrew Saxon and Ke Zhang2

Hart and Louis Laboratory, Division of Clinical Immunology/Allergy, Department of Medicine, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activation-induced cytidine deaminase (AID) plays critical roles in Ig class switch recombination and VH gene somatic hypermutation. We investigated the role of IL-4 in AID mRNA induction, the signaling transduction involved in IL-4-mediated AID induction, and the effect of CD45 on IL-4-dependent AID expression in human B cells. IL-4 was able to induce AID expression in human primary B cells and B cell lines, and IL-4-induced AID expression was further enhanced by CD40 signaling. IL-4-dependent AID induction was inhibited by a dominant-negative STAT6, indicating that IL-4 induced AID expression via the Janus kinase (JAK)/STAT6 signaling pathway. Moreover, triggering of CD45 with anti-CD45 Abs can inhibit IL-4-induced AID expression, and this CD45-mediated AID inhibition correlated with the ability of anti-CD45 to suppress IL-4-activated JAK1, JAK3, and STAT6 phosphorylations. Thus, in humans, IL-4 alone is sufficient to drive AID expression, and CD40 signaling is required for optimal AID production; IL-4-induced AID expression is mediated via the JAK/STAT signaling pathway, and can be negatively regulated by the JAK phosphatase activity of CD45. This study indicates that the JAK phosphatase activity of CD45 can be induced by anti-CD45 Ab treatment, and this principle may find clinical application in modulation of JAK activation in immune-mediated diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Activation-induced cytidine deaminase (AID), 3 a putative RNA-editing enzyme, is specifically expressed in germinal center B lymphocytes in vivo (1). AID is required for Ab diversification processes, including Ig class switch recombination (CSR) (2, 3, 4, 5, 6), somatic hypermutation (SHM) (2, 3, 4, 6, 7, 8, 9, 10), and gene conversion (11). In humans, mutations in the AID gene cause type II hyper-IgM immunodeficiency and deficiency of Ig V gene somatic mutations, confirming the role of AID in CSR and SHM (3). The AID gene-encoded product appears to be the only lymphoid-specific factor required for CSR and SHM, as CSR and SHM can be reconstituted in fibroblasts (12, 13), hybridomas (7), and Escherichia coli (14) that ectopically express AID. AID acts on post-germline transcription in CSR, because Ig germline transcription is not affected by AID deficiency (2, 3). In SHM, it has been speculated that steps both upstream and downstream of the V DNA double-strand breaks (DSB) are the AID targeting stages (15, 16), as SHM is dependent, whereas DSB is independent, of AID (15, 16). The mechanisms by which AID mediates CSR and SHM are currently unknown; however, the facts that AID is able to mutate E. coli DNA (14) and that the Ig SHM pathways can be altered by inhibition of uracil-DNA glycosylase (17) strongly suggest that AID functions as DNA deaminase to initiate Ab diversification processes, although several other possibilities or mechanisms also have been proposed (4, 13, 15, 16, 18). The expression and regulation of AID is of considerable interest given the apparently critical role of AID in Ab diversity. Characterization of AID expression in mice has shown that AID is induced or regulated by several components that drive Ig CSR, including cytokines, CD40 ligand (CD40L), LPS, and a combination of these factors (1). However, little is known about the regulation and signaling pathways involved in human AID expression.

CD45, a type I transmembrane glycoprotein that is highly expressed on hemopoietic cells, functions as both positive and negative regulators in Ag receptor signaling for T and B cell activation and development via its protein tyrosine phosphatase (PTPase) activity for Src family kinases (19, 20, 21, 22, 23). In addition, CD45 also participates in regulation of macrophage integrin-mediated adhesion (24), MHC class II-mediated signaling (25), control of T cell apoptosis (26), and IgE-mediated degranulation in mast cells (27), as well as in CD40L-induced microglial activation in microglial cells (28). Therefore, CD45 is implicated in regulation of immune responses (19, 20); prevention of transplant rejection (29); development of autoimmune diseases (30), immune deficiency (31), and leukemia (32); and pathogenesis of Alzheimer disease (33).

Recently, CD45 was defined as a Janus kinase (JAK) phosphatase that negatively regulates cytokine receptor signaling via JAK/STAT signaling pathways to control cytokine-mediated activation, differentiation, and proliferation (34). We have previously shown that CD45, via its JAK phosphatase activity, is able to negatively regulate IgE CSR in human B cells through inhibition of IL-4 plus anti-CD40-induced Ig {epsilon} germline transcription (35). However, whether CD45 plays a role in AID expression is not known. In this study, we examined the role of IL-4 in AID induction, its modulation by CD40 signaling, the signal pathway involved in IL-4-dependent AID induction, and the role of CD45 in regulating IL-4-mediated AID expression in human B cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Human recombinant IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, and TGF-{beta} were purchased from R&D Systems (Minneapolis, MN). Anti-CD40 mAb G28.5 (anti-CD40) was produced from a hybridoma cell obtained from the American Type Culture Collection (Manassas, VA) (36). Purified anti-CD45 mAb (anti-CD45) HI30, anti-CD45RA HI100, anti-CD45RB MT4, anti-CD45RO UCHL1, anti-CD23, and anti-CD54 were purchased from BD PharMingen (San Diego, CA). Anti-CD45RC was purchased from BioSource International (Camarillo, CA). Anti-JAK1 Ab, anti-JAK3 Ab, anti-STAT6 Ab, anti-STAT6 (M-200), and anti-phosphorylated JAK3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphorylated STAT6 Ab and anti-phosphorylated JAK1 Ab were purchased from Cell Signaling (Beverly, MA).

Construction of dominant-negative STAT6 (STAT6-DN)

Human STAT6 cDNA cloned in vector pcDNA3 was kindly provided by Dr. A. E. Nel (University of California, Los Angeles, School of Medicine). The HindIII-SacI fragment from the vector, containing aa 1–661 of STAT6 (STAT-DN), was cloned into a modified pDsRed1-N1 (Clontech Laboratories, Palo Alto, CA), which encodes a red fluorescent protein (RFP) in the plasmid backbone as an indicator of transfection efficiency. This construct, termed pST6-DN, was used for transfection.

Cells, cell lines, and transfection

Human PBMCs from healthy donors were obtained from Core Virology Laboratory (University of California, Los Angeles). Human resting B cells were prepared by magnetic beads as described previously (36). This resulted in >95% of IgD-positive, as determined by FACS. Human B cell lines BL-2, DG75, and Ramos 2G6 were maintained and cultured with the stimulation indicated in each figure. For transient transfection, 1 x 106 DG75 cells in 0.2 ml of medium were electroporated (200V, 975 µF) with 20 µg pST6-DN or with control plasmids as described previously (37).

RT-PCR

Total RNA was extracted by TRIzol reagent (Invitrogen, San Diego, CA) as described (37). RNA was digested with DNase I (Sigma-Aldrich, St. Louis, MO) to remove contaminating DNA. Total RNA (2 µg) was reverse-transcribed to cDNA by Moloney murine leukemia virus transcriptase priming with oligo-dT15 (Invitrogen). PCR was performed at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min for 30 cycles. For detection of AID expression, the primer AID1 (5'-ACTTGCAGGGAGGCAAGAAGACACTCT-3'), which is located in exon 1 and corresponds to the nucleotide sequence of 39–66, or primer AID3 (5'-GACCCTGGCCGCTGCTACC-3'), which is located in exon 3 and corresponds to 287–305 (GenBank access no. AB040431) (38), and primer AID4 (5'-CAAAAGGATGCGCCGAAGCTGTCTGGAG-3'), which is located in exon 4 and corresponds to the nucleotide sequence of 619–591, were used. The sizes of the PCR products are expected to be 581 bp (AID1-AID4 primer pair) and 332 bp (AID3-AID4 primer pair) for the normally spliced AID. For detection of Ig {epsilon} germline transcripts ({epsilon}GTs), the primers GM3 (5'-AGCTGTCCAGGAACCCGACAGGGAG-3') and C{epsilon}2B (5'-GTTGATAGTCCCTGGGGTGTA-3') were used to amplify a 518-bp PCR product (35). The primers for GAPDH have been described previously and amplify a 981-bp product (37).

RNase protection assay (RPA)

For analysis of AID mRNA expression by RPA, the 581-bp RT-PCR fragment cloned in PCR 2.1/TOPO vector (Invitrogen) was used as a DNA template to synthesize the probe. The recombinant vector was linearized with BamHI and transcribed into a digoxigenin-labeled cRNA probe using T7 RNA polymerase according to the manufacturer’s instructions (Roche Diagnostics, Indianapolis, IN). A human {beta}-actin DNA template (Roche Diagnostics) was also digoxigenin labeled as an internal control for RPA. The synthesized human AID and {beta}-actin cRNA probes were 693 bp and 255 bp, respectively. RPAs were performed following the manufacturer’s protocol. Briefly, 30 µg of total RNA was hybridized with digoxigenin-labeled cRNA probes (50 pg/sample) at 50°C overnight, and then completely digested by RNase T1 (2.5 U/sample) at 32°C for 1 h. The protected RNAs were precipitated in ethanol, resolved on 6% acrylamide-urea gel, and transferred to nylon membranes (positively charged; Roche Diagnostics). The blots were immunodetected by alkaline phosphatase-labeled anti-digoxigenin Ab (Roche Diagnostics), washed twice, and developed using a chemiluminescent assay (CDP-Star, ready-to-use; Roche Diagnostics). Densitometric analyses were performed for quantifying AID expression using the Molecular Analyst software (Bio-Rad, Hercules, CA).

Western blot analysis

BL-2 cells (1 x 106) were preincubated with anti-CD45 (2 µg/ml) for 1 h, and then stimulated with 3 ng/ml IL-4 for the times indicated in the corresponding figures. After washing with PBS, the cells were collected by centrifugation and lysed in ice-cold cell lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 2% Triton X-100, 0.1 mM sodium orthovanadate, 10 µg/ml leupeptin, and 1 mM PMSF) for 15 min. After centrifugation, the supernatants were collected as cell lysates. The lysates were boiled in Laemmli sample buffers and fractionated on 8% SDS-PAGE. The separated proteins in gels were transferred electrophoretically to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The blots were immunoprobed with Abs specific to phospho-JAK1, -JAK3, and -STAT6, followed by HRP-labeled secondary Abs. The blots were stripped and reprobed with the corresponding Abs (JAK1, JAK3, and STAT6) as protein loading controls. An ECL (Amersham, Piscataway, NJ) was used to detect the signal.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human AID expression is IL-4 inducible

Studies in mice have shown that optimal AID induction requires costimulation with IL-4, TGF-{beta}, and CD40L in the murine B cell line CH12F3-2, and LPS, LPS plus IL-4, or LPS plus TGF-{beta} in murine splenic B cells (1). IL-4 and CD40L alone or in combination exhibited little, if any, AID induction in splenic B cells, although these factors individually were able to induce low levels of AID in CH12F3-2 cells (1). To assess AID induction and regulation in human B cells, we tested the roles of several factors that are involved in Ig CSR for their AID induction in human primary B cells and B cell lines. The results presented in Fig. 1 show that AID expression in purified primary B cells, as assayed by RT-PCR, was clearly inducible by IL-4 and IL-13, but not by IL-5, IL-6, IL-9, IL-10, or anti-CD40 (Figs. 1A and 2A). TGF-{beta} was not able to induce AID expression in human purified B cells (Fig. 1A, lane 9), although it was able to do so in PBMC (data not shown) and the B cell line, BL-2 (Fig. 1B, lane 12).



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  		FIGURE 1. IL-4-dependent AID induction in human B cells. A, Effects of selected cytokines on AID mRNA expression in primary B cells. Magnetic bead-purified human resting B cells (IgD+ B cells) (1 x 106) were cultured with IL-4 (10 ng/ml), IL-5 (10 ng/ml), IL-6 (10 ng/ml), IL-9 (10 ng/ml), IL-10 (10 ng/ml), IL-13 (10 ng/ml), TGF-{beta} (10 ng/ml), and anti-CD40 (1 µg/ml) for 48 h, followed by RT-PCR with primer pair AID3 plus AID4. A diagram of the human AID gene structure, PCR primers, and RPA probe are shown. The numbered boxes represent the defined exons (38 ), and the arrows represent the PCR primers. B, Effects of selected cytokines on AID expression in BL-2 cells. The cells were cultured under the same conditions as described above. AID expression was quantified by RPA using 30 µg of total RNA. {beta}-Actin was included in the RPA reaction as RNA loading control. C, LPS in human AID induction. BL-2 cells were stimulated with IL-4 or LPS (75 µg/ml) for 48 h, followed by RPA. D, Dose dependence of IL-4-induced AID expression. BL-2 cells were cultured for 48 h with the IL-4 concentrations indicated, and AID mRNA levels were determined by RPA. Results shown are representative of three independent experiments.

 


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  		FIGURE 2. Synergistic effects of CD40 signaling on IL-4-dependent AID induction. The cells were stimulated with the protocol shown in Fig. 1A. Effects of CD40 signaling on IL-4-dependent AID induction in human PBMC, primary B cells, and BL-2 cells measured by RT-PCR. B, Effects of CD40 signaling on IL-4-dependent AID induction in BL-2 cells quantified by RPA. Shown are the representatives of three independent experiments.

 
To better quantify AID mRNA expression, RPA was used to determine the AID expression level in human B cell lines because large amounts of RNA are available in cell lines. As shown in Fig. 2B, unstimulated BL-2 cells (and Ramos 2G6, DG75, data not shown) spontaneously express low levels of AID (Fig. 1B, lane 3). IL-4 significantly induced AID expression in BL-2 cells (Fig. 1B, lane 4) in a dose-dependent fashion (Fig. 1D), whereas IL-13 showed a positive but weaker effect (Fig. 1B, lane 11). IL-9 and IL-10 did not induce AID (Fig. 1B, lanes 5 and 6). Of note, LPS, which is a strong AID inducer in murine B cells (1), was not able to induce AID expression in human B cells (Fig. 1C), nor was CD40 stimulation (by anti-CD40 mAb) (Fig. 2). Thus, among all the factors mentioned above, only IL-4 and to a lesser extent IL-13 are able to induce AID expression in both human B cells and B cell lines. We focused primarily on IL-4-dependent AID induction because both IL-4 and IL-13 use the IL-4R{alpha}-chain as a receptor component and activate common signaling pathways for many of their biological functions (39).

At least six protected products could be resolved in RPA gel with the cRNA probe used (Fig. 1, A and B). Partial cloning and sequence analysis revealed that at least four of these products were alternatively spliced AID isoforms (data not shown). These results suggest that multiple AID isoforms exist in human B cells. Complete cloning analysis and characterization of these AID isoforms are currently undertaken in our laboratory.

IL-4-dependent AID expression is enhanced by CD40 stimulation

CD40 signaling is required for cytokine-directed CSR (40). To examine whether CD40 signaling plays a role in human AID induction by synergizing with IL-4, AID induction by IL-4 or IL-4 plus anti-CD40 was examined. As shown in Fig. 2, IL-4 was able to induce AID expression in PBMC, purified B cells, and BL-2 cell lines, and this IL-4-dependent AID expression was enhanced by anti-CD40 (Fig. 2B).

Dominant-negative STAT6 (STAT6-DN) expression suppresses IL-4-dependent AID induction

The human AID gene promoter has, as yet, not been characterized. However, analysis of the available 947-bp nucleotides upstream of the transcriptional initiation site of the AID mRNA (human genome sequence database, NM_000264) indicates that multiple predicted transcriptional factor-binding sites, including one typical and at least four potential STAT6 binding sites, exist in this region. To test whether IL-4-induced AID expression is mediated via STAT6 signaling, we examined the effect of STAT6-DN on IL-4-induced AID expression. A C-terminal truncated form of STAT6 (pST6-DN), in which the transactivation domain of the STAT6 was deleted, was transiently transfected into DG75 cells. This truncated STAT6 has been shown to inhibit STAT6 phosphorylation and function as a dominant-negative STAT6 (41). Fig. 3A shows that the expected truncated form of STAT6 was expressed in DG75 cells transfected with pST6-DN, but not expressed in those cells transfected with the control vector. Inhibition of IL-4-induced endogenous STAT-6 phosphorylation by STAT6-DN demonstrated that the short form of STAT6 indeed functioned as a STAT6-DN (Fig. 3B). As shown in Fig. 3C, IL-4-induced AID expression was suppressed by pST6-DN, but was not suppressed by the control vector (compare the normalized data extracted from lanes 2 and 3, lower panel of Fig. 3C). As only a portion of cells were transfected (~50%, as determined by the expression of the built-in red fluorescence gene in the plasmid, data not shown), greater degrees of inhibition of STAT6 phosphorylation and AID suppression were not expected from these unfractionated cells. IL-4-induced AID expression was significantly inhibited in the sorted RFP-positive cells containing the pST6-DN (Fig. 3D, lane 4), but not in cells transfected with the control vector (Fig. 3D, lane 2). Such inhibition was not observed from the sorted RFP-negative cell populations from either pST6-DN or control vector transfections (Fig. 3D, lanes 6 and 8). These data showed that IL-4-induced AID expression in human B cells is mediated through the STAT6 signaling pathway.



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  		FIGURE 3. Effects of STAT6-DN on IL-4-induced AID expression. A, The expression of STAT6-DN in DG75 cells after transient transfection. Two days after transient transfection, Western blot analysis was performed with whole cell lysates from cells transfected with pST6-DN or control vectors. The blot was probed with an Ab against the DNA-binding domain of STAT6 (M-200). B, Effects of transiently transfected pST6-DN on phosphorylation of endogenous STAT6. The transiently transfected DG75 cells were stimulated with IL-4 (3 ng/ml) for 10 min, followed by Western blot analysis. C, Effects of STAT6-DN on IL-4-induced AID expression. Two days after transient transfection, DG75 cells were stimulated with IL-4 (3 ng/ml) for 48 h, followed by RPA. D, Effects of STAT6-DN on IL-4-induced AID expression in RFP-sorted cells. pST6-DN- and control vector-transfected cells were stimulated with or without IL-4 for 48 h, followed by FACS sorting based on the RFP expression. The RFP-positive and -negative sorted cells (6 x 105) were subjected to RNA preparation and RPA analysis. Shown are the representative data from two independent experiments.

 
IL-4-induced AID expression is negatively regulated by CD45 signaling

We have previously shown that CD45 functions as a JAK phosphatase to negatively regulate IL-4-dependent IgE CSR via inhibition of Ig {epsilon} germline transcription (35). To investigate whether IL-4-dependent AID induction is also regulated by CD45 signaling, we examined the effect of anti-CD45 on IL-4-dependent AID induction, as well as on Ig {epsilon} germline transcripts, in BL-2 cells. As shown in Fig. 4, IL-4-induced AID expression was inhibited by anti-CD45, but not by anti-CD23 or anti-CD54 (Fig. 4A), and the inhibitory effect was anti-CD45 dose dependent, with a maximum inhibitory effect at 2 µg/ml or higher (Fig. 4B). As a positive control for CD45’s effects on BL-2 cells, we also confirmed that anti-CD45 inhibited IL-4-induced Ig {epsilon} germline transcripts from BL-2 cells in a dose-dependent manner (Fig. 4C). Similarly, IL-4-induced AID expression in human primary B cells was inhibited by anti-CD45 in a dose-dependent fashion (Fig. 4D). These data revealed that IL-4-induced AID production could be inhibited by CD45 signaling.



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  		FIGURE 4. Regulation of IL-4-induced AID expression by anti-CD45. A, Effects of anti-CD45 on IL-4-induced AID expression in BL-2 cells. Cells were treated with anti-CD45 (2 µg/ml) and control Abs anti-CD23 (2 µg/ml) and anti-CD54 (2 µg/ml) at 37°C for 1 h, followed by stimulation with IL-4 (3 ng/ml) for 48 h. AID mRNA was quantified by RPA. B, Dose-dependence of anti-CD45 suppression of IL-4-induced AID expression. Cells were treated at 37°C for 1 h with various concentrations of anti-CD45 and control Ab as indicated (lane 1 was loaded with RPA probes and lane 10 was loaded with bacterial tRNA), followed by stimulation with IL-4 (3 ng/ml) for 48 h and subsequent RPA analysis. C, Effects of anti-CD45 on IL-4-induced {epsilon}GT expression in BL-2 cells. The cells were treated following the above protocol with the anti-CD45 concentration indicated. RT-PCR was performed to measure {epsilon}GTs as described (35 ). D, Effects of anti-CD45 on IL-4-induced AID expression in primary B cells. Human purified B cells (1 x 106) were also treated following the above protocol with various concentrations of anti-CD45 and control Ab as indicated, followed by RT-PCR for AID expression.

 
IL-4-induced JAK1, JAK3, and STAT6 phosphorylation is inhibited by anti-CD45

To determine whether CD45 functions as a JAK phosphatase in CD45-mediated AID suppression, we examined the role of anti-CD45 stimulation on IL-4-activated JAK1, JAK3, and STAT6 phosphorylation in human B cell lines. The results showed that IL-4-induced JAK1 phosphorylation was inhibited by anti-CD45 over a 2–10 min time course (Fig. 5A, top panel, lanes 1–4 vs 5–8). JAK3, which was partially phosphorylated in unstimulated BL-2 cells (Fig. 5A, third panel, lane 1), was further phosphorylated by IL-4 stimulation (Fig. 5A, third panel, lanes 2–4). Phosphorylation of JAK3, both spontaneous and IL-4-induced, was markedly inhibited by anti-CD45 treatment (Fig. 5A, lower panel, lanes 5–8). Thus, the dose of anti-CD45 that significantly suppressed IL-4-induced AID expression also inhibited IL-4-activated JAK1 and JAK3 phosphorylation in BL-2 cells. This IL-4-induced JAK1 and JAK3 phosphorylation was not affected by an anti-CD54 Ab (data not shown). These results confirmed that CD45 functioned as a JAK phosphatase to dephosphorylate IL-4-induced JAK1 and JAK3 phosphorylation in BL-2 cells, similar to what we have observed in Ramos 2G6 cells (35).



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  		FIGURE 5. Effects of anti-CD45 on IL-4-induced JAK1, JAK3, and STAT6 phosphorylation. A, Inhibitory effects of anti-CD45 on IL-4-induced JAK1 and JAK3 phosphorylation. BL-2 cells were treated with anti-CD45 (2 µg/ml) for 30 min, followed by IL-4 (3 ng/ml) for the time courses indicated. The cell lysates were applied to each lane and probed with anti-phosphorylated JAK1 and JAK3. The blots were stripped and reprobed with anti-JAK1 and anti-JAK3 as a protein loading control (lower panel). B, Inhibitory effects of anti-CD45 on IL-4-induced STAT6 phosphorylation. BL-2 cells were treated with the same protocol as indicated in A for the times indicated. The blots were probed with anti-phosphorylated STAT6, and reprobed with anti-STAT6 for a protein loading control (lower panel). Results shown are representative of two independent experiments.

 
Because STAT6 is the substrate for JAK1 and JAK3 in the JAK/STAT signaling pathway, and IL-4-induced STAT6 phosphorylation is mediated via activation of JAK1 and JAK3 (42), it was predictable that IL-4-induced STAT6 phosphorylation would be inhibited by the JAK phosphatase activity of CD45 (35, 39). Indeed, IL-4-induced STAT6 phosphorylation (Fig. 5B, lanes 2–4) was strongly inhibited by anti-CD45 treatment (Fig. 5B, lanes 6–8), indicating that anti-CD45 could trigger CD45’s JAK phosphatase activity to negatively regulate IL-4-mediated biological effects, including IL-4-dependent AID induction, via blocking JAK/STAT signaling in BL-2 cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrated that IL-4 was able to induce AID expression in human B cells, and IL-4-dependent AID expression was synergistic with CD40 signaling. Using a STAT6-DN construct to block endogenous STAT6, we showed that STAT6-DN suppressed IL-4-dependent AID induction, thereby indicating that IL-4-induced AID expression occurs via the JAK/STAT signaling pathway. We subsequently showed that IL-4-induced AID expression was inhibited by CD45 triggering, and that this CD45-mediated inhibition of IL-4-induced AID expression correlated with the ability of CD45 to suppress IL-4-activated JAK1, JAK3, and STAT6 phosphorylation. These results revealed that IL-4 alone was sufficient to drive AID expression via JAK/STAT pathways in human B cells, and that IL-4-dependent AID induction was subject to negative regulation by CD45 via CD45’s JAK phosphatase activities. However, the possible contribution of CD45’s PTPase activities or JAK phosphatase-dependent activities on other substrates to anti-CD45-mediated AID regulation also cannot be ruled out.

Our findings reveal that the requirements for AID induction in mice and humans differ. In mice, IL-4 alone had no detectable effects on AID induction in splenic B cells, whereas CD40L exhibited a weak effect (1). Furthermore, IL-4 plus CD40L did not significantly synergize for murine AID induction (1). In humans, IL-4 alone clearly was able to induce AID expression in primary B cells and all three B cell lines tested. Although anti-CD40 itself was not able to induce human AID, IL-4-induced AID expression was significantly augmented by anti-CD40. In mice, LPS was a powerful AID inducer (1), suggesting that LPS-activated signaling, presumably through Toll-like receptors (43), plays an important control role for murine AID production. As LPS is not able to induce AID expression in humans, an LPS-dependent signaling pathway is not important for human AID induction. Overall, these results show that the pathways regulating AID expression diverge significantly between mice and humans. LPS-activated pathways appear to play an important role in mediating AID expression in mice, whereas IL-4 and/or CD40L activation possibly is less efficient. Compared with that in murine cells, LPS-mediated signaling in human B cells is quite distinct and limited while it appears that IL-4 plus anti-CD40 signaling has become a major route for human AID expression. The fact that LPS is a strong CSR inducer in mice (44), whereas IL-4 plus CD40 signaling, but not LPS, controls CSR in humans (44), is consistent with this hypothesis. Given that IL-4 plus CD40 signaling, but not LPS, was important in human AID expression, our finding that IL-4-activated JAK/STAT signal pathway was involved in the production of AID in human B cells was not unexpected, as IL-4-dependent biological functions are mainly mediated through STAT6 signaling pathways. Failure of response to LPS in human AID induction is likely to account for LPS’s inability to induce CSR in human B cells (45), because AID appears to be the only B cell-specific factor required for CSR (2, 12).

CD45 can function as a PTPase to regulate Ag receptor signaling through Src family protein tyrosine kinases (46), and also as a JAK phosphatase to regulate cytokine receptor signaling via JAK/STAT pathways (34, 35). Recently we have shown that CD45, via its JAK phosphatase activity, could inhibit Ig {epsilon} germline transcription, and subsequent CSR to IgE in human B cells (35). Combining with our current results that CD45 via its JAK phosphatase activity was able to suppress IL-4-dependent AID production, CD45 as a JAK phosphatase is capable of functioning on at least two independent levels, inhibition of cytokine-dependent germline transcription and expression of AID to control CSR (Fig. 6).



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  		FIGURE 6. Model for the action stages of cytokines and CD45 on CSR in human B cells. Ig germline transcription (GT) is directed by cytokine(s), such as IL-4 or TGF-{beta}. Some cytokines are also able to induce AID expression for AID-dependent DSB in switch region DNA. The generated DSB will be repaired by the nonhomologous end joining (NHEJ) pathway to accomplish the CSR processes that eventually lead to IgG, IgA, or IgE production. CD45, via its JAK phosphatase activity, is able to negatively regulate CSR through inhibition of cytokine-dependent GT and AID induction.

 
Previous studies have shown that IL-4 could drive Ig CSR to {gamma}3, {gamma}1, {gamma}4, and {epsilon} via induction of germline transcription for these isotypes (36, 44, 47, 48), indicating that IL-4 induces chromatin remodeling so as to selectively determine IgH locus accessibility control for CSR (47, 48). Our current data that IL-4 also induces AID production reveals that IL-4 functions in CSR by more than simply affecting accessibility control (Fig. 6), because AID is not required for germline transcription (2). Accumulating data suggest that AID is likely to be one component of the putative switch recombinase (13, 18); therefore, AID as a component of switch recombinase is inducible, at least by IL-4 in humans. In this sense, the switch recombinase activity is, at least in part, not fully preformed as suggested by the original "accessibility model" (36, 44, 47). This conclusion agrees with the results obtained from a murine cell line where optimal induction of switch recombinase activity required IL-4 plus TGF-{beta} (49), as well as de novo protein synthesis (1). Based on the fact that AID is also critical for SHM (2), IL-4 might also be involved in the up-regulation of the SHM process in vivo in humans via AID induction, and the phosphatase activity of CD45 might also be involved in the down-regulation of SHM via suppression of AID production (Fig. 6).

How CD45 molecules execute their biological function under the physiological conditions remains elusive. It has long been speculated that CD45 might execute its functions through binding of its extracellular domain with its potential ligand(s). However, a specific ligand(s) for CD45 has not been identified despite intensive search for many years. Therefore, whether such ligands indeed exist remains speculative. Recent advances in understanding the mode of action of CD45 suggest that CD45-mediated function may be regulated via differential homodimerization of the alternative spliced CD45 isoforms (50). Dimerization of CD45 inhibits phosphatase activity through symmetrical interaction between the putative inhibitory structural wedge in the juxtamembrane region of one monomer and the catalytic site in the cytoplasmic domain of its partner (51). Thus, when a mutation that disrupts the inhibitory wedge of CD45 is introduced into mice, CD45’s PTPase activity is inactivated, resulting in lymphoproliferative syndrome and severe autoimmune nephritis with autoantibody production (52). Modulation of the homodimerization by extracellular sialyation and O-glycosylation of CD45 is able to change CD45’s enzymatic activity (50). As a PTPase and/or JAK phosphatase regulating signal transduction, CD45 must be coupled with the targeting molecule in such a way as to precisely regulate the targeted signaling molecule while not disturbing other signal pathways. Dynamic inclusion or exclusion of different CD45 isoforms in stimulation-induced lipid rafts during immune synapse formation has been proposed to be a mechanism for CD45 to mediate its biological functions (53). For example, CD26-mediated signaling for T cell activation has been shown to associate with CD45RO in lipid rafts (54). If such a hypothesis is true, one can envision that interacting surface molecules, such as CD30-CD153 (55), negatively regulate Ig CSR through inclusion of CD45 (or its isoforms) in the interaction-induced lipid rafts.

Even though the mechanism by which CD45 executes its regulatory functions under the physiological conditions is unclear, CD45 triggering with anti-CD45 has been shown to alter Ab responses and immune responses in both humans and mice (56, 57, 58, 59). Our results showing that anti-CD45 is able to negatively regulate Ig CSR by inhibition of Ig {epsilon} germline transcription (35) and AID via CD45’s JAK phosphatase activity (in this study) indicate that CD45 triggering is able to activate CD45’s JAK phosphatase activity, and this CD45-dependent JAK phosphatase activity may play an important negative regulatory role in controlling JAK/STAT-dependent Ab responses. These results suggest that conformational changes in CD45 (induced by anti-CD45 Abs) may be required for activation or optimal activation of JAK phosphatase activity. If homodimerization of CD45 is a mechanism preventing CD45’s JAK phosphatase activity, CD45 triggering by anti-CD45 probably functions to dissociate such homodimers through induced conformational changes to activate JAK phosphatase activity. Thus, uncovering the role of CD45’s JAK phosphatase activity in immune responses provides not only a reasonable explanation for alteration of immune and Ab responses induced by anti-CD45 treatment (56, 57, 58, 59), but also the rationale for direct clinical application of anti-CD45 treatment in transplantation (29), autoimmune diseases (53, 56, 59), IgE-mediated allergic diseases (35, 59), or microglial activation-associated Alzheimer disease (28, 33).


   Acknowledgments
 
We thank Dr. A. E. Nel for pcDNA3-STAT6, Dr. Q. Pan for BL-2 and DG75 cells, Dr. M. Zhu and M. Jyrala for excellent technical assistance, and M. Rainof for her assistance in preparing this manuscript.


   Footnotes
 
1 This work was supported by Department of Health and Human Services Grants AI 15251, AI 40945, and AI 40551. Back

2 Address correspondence and reprint requests to Dr. Ke Zhang, Division of Clinical Immunology/Allergy, Department of Medicine, University of California, Los Angeles, School of Medicine, 52-175 Center for Health Science, 10833 Le Conte Avenue, Los Angeles, CA 90095-1680. E-mail address: kzhang{at}mednet.ucla.edu Back

3 Abbreviations used in this paper: AID, activation-induced cytidine deaminase; CSR, class switch recombination; SHM, somatic hypermutation; JAK, Janus kinase; DSB, double-strand break; CD40L, CD40 ligand; PTPase, protein tyrosine phosphatase; {epsilon}GT, {epsilon} germline transcript; RPA, RNase protection assay; RFP, red fluorescent protein. Back

Received for publication August 8, 2002. Accepted for publication December 16, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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