c-Met and Its Ligand Hepatocyte Growth Factor/Scatter Factor Regulate Mature B Cell Survival in a Pathway Induced by CD74

This information is current as Maya Gordin, Melania Tesio, Sivan Cohen, Yael Gore, Frida of September 28, 2021. Lantner, Lin Leng, Richard Bucala and Idit Shachar J Immunol published online 16 July 2010 http://www.jimmunol.org/content/early/2010/07/16/jimmun ol.0902566 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 16, 2010, doi:10.4049/jimmunol.0902566 The Journal of Immunology

c-Met and Its Ligand Hepatocyte Growth Factor/Scatter Factor Regulate Mature B Cell Survival in a Pathway Induced by CD74

Maya Gordin,* Melania Tesio,* Sivan Cohen,* Yael Gore,* Frida Lantner,* Lin Leng,† Richard Bucala,† and Idit Shachar*

The signals regulating the survival of mature splenic B cells have become a major focus in recent studies of B cell immunology. Durable B cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism involved in mature B cell homeostasis, the hepatocyte growth factor/scatter factor (HGF)/c-Met pathway. We demonstrate that c-Met activation by HGF leads to a survival cascade, whereas its blockade results

in induction of mature B cell death. Our results emphasize a unique and critical function for c-Met signaling in the previously Downloaded from described macrophage migration inhibitory factor/CD74-induced survival pathway. Macrophage migration inhibitory factor recruits c-Met to the CD74/CD44 complex and thereby enables the induction of a signaling cascade within the cell. This signal results in HGF secretion, which stimulates the survival of the mature B cell population in an autocrine manner. Thus, the CD74–HGF/c-Met axis defines a novel physiologic survival pathway in mature B cells, resulting in the control of the humoral immune response. The Journal of Immunology, 2010, 185: 000–000. http://www.jimmunol.org/ uring their development, B cells encounter various check- brane region and an ∼150-aa lumenal domain. The CD74 chain points that control cell survival. Under steady-state con- was originally thought to function mainly as an MHC class II chap- D ditions, the number and distribution of B cells are under erone, promoting the exit of MHC class II molecules from the en- homeostatic control maintained by a balance between survival and doplasmic reticulum, directing them to endocytic compartments, apoptosis. preventing binding within the , and Regulation of mature B cell survival involves multiple mecha- contributing to peptide editing in the MHC class II compartment (8). nisms. The BCR provides survival signals essential for maintaining In addition to its chaperone function, CD74 was shown to have the mature B cell pool. Deletion of the Igh (1) or conditional a role as an accessory signaling molecule. A small proportion of deletion of either Igh or the signal-transducing Cd79a (Iga) CD74 is modified by the addition of chondroitin sulfate, and this by guest on September 28, 2021 (2) leads to a loss of B cells. In addition, survival of mature naive form of CD74 is expressed on the surface of APCs, including B cells depends on signals delivered by the ligand–receptor pair monocytes and B cells. Ab blocking studies have shown that CD74 B cell-activating factor (BAFF) and BAFFR (3, 4). Mice that lack modified by the addition of chondroitin sulfate interacts with CD44 BAFF expression or that are subjected to treatments designed to (9, 10). In addition, it was shown that macrophage migration in- block the action of BAFF fail to produce or to maintain a mature hibitory factor (MIF) binds to the CD74 extracellular domain on B cell pool (5–7). macrophages, a process that results in initiation of a signaling Recently, we described an additional mechanism that regulates pathway in these cells (11). B cell survival, which depends on CD74 (invariant chain, Ii). CD74 is Our studies have shown that CD74 expressed on B cells is di- a type II integral membrane , containing a short N-terminal rectly involved in shaping the peripheral B cell populations by cytoplasmic tail of 28 aa, followed by a single 24-aa transmem- regulating mature B cell survival (12) through a pathway leading to the activation of transcription mediated by the NF-kB p65/RelA *Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; and homodimer and its coactivator TAFII105 (13, 14). CD74 stim- †Department of Medicine, Yale University School of Medicine, New Haven, CT ulation by MIF activates the Syk and PI3K/Akt pathways, leading 06520 to NF-kB activation, enabling entry of the stimulated B cells Received for publication August 6, 2009. Accepted for publication June 3, 2010. into the S phase, an increase in DNA synthesis, cell division, This work was supported by the Israel Science Foundation (Morasha), the Israel Sci- and augmented expression of antiapoptotic in a CD44- ence Foundation, the Israel Cancer Association, and the Minerva Foundation. I.S. is dependent manner. These findings confirmed that surface CD74 the incumbent of the Dr. Morton and Ann Kleiman Professorial Chair. R.B. and L.L. are supported by National Institutes of Health Grants AR049610, AR050498, and functions as a survival receptor (15–17). AI042310 and the Alliance for Lupus Research. Interestingly, the , CD44, has also been Address correspondence and reprint requests to Dr. Idit Shachar, Department of implicated in the regulation of the signaling of the tyrosine kinase Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address: receptor c-Met (18–20), although the precise mechanism of this [email protected] interaction is unknown. The online version of this article contains supplemental material. c-Met is a unique disulfide-linked a–b heterodimeric receptor Abbreviations used in this paper: BAFF, B cell-activating factor; FLICA, fluorescent- labeled inhibitor of caspases; HGF, hepatocyte growth factor/scatter factor; HPRT, tyrosine kinase with a versatile role in regulating numerous biolog- hypoxanthine ribosyltransferase; MIF, macrophage migration inhibitory factor; MM, ical functions in response to its natural ligand hepatocyte growth multiple myeloma; MZ, marginal zone; PI, propidium iodide; T1, transitional 1; T2, factor/scatter factor (HGF). HGF is a multifunctional cytokine with transitional 2. a domain structure and proteolytic mechanism of activation similar Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 to that of the serine protease plasminogen. Activation of the HGF/

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902566 2 CD74–HGF/c-Met AXIS DEFINES A SURVIVAL PATHWAY IN B CELLS c-Met signaling pathway, which requires phosphorylation of var- 37˚C. For c-Met blocking, 1 3 107 B cells were incubated in 1 ml RPMI ious specific tyrosine residues on c-Met itself, leads to cellular 1640 medium containing 0.1% (v/v) FCS in the presence of 0.3 ng/ml responses, including increased motility, proliferation, morphogen- PHA-665752, a c-Met inhibitor (a gift from Pfizer, New York, NY), at 37˚C. esis, and cell survival (21–27). RNA isolation and reverse transcription Little is known about the role of HGF/c-Met in the regulation of lymphopoiesis in general and specifically that of B cells. HGF has Total RNA was isolated from cells using the TRIzol Reagent (MRC, Cincinnati, OH). Reverse transcription was carried out using Superscript II been reported to regulate hematopoiesis in mouse fetal liver and in reverse transcriptase (Life Technologies, Carlsbad, CA). Primers that were adult bone marrow (28, 29) and was found to have a stimulatory used included: c-Met, 59-GACTTCAGCCATCCCAATGT-39,39-GGTGAA- effect on Ig secretion in cultures of mouse splenocytes (30). CTTCTGCGTTTGC-59; HGF, 59-AGCGCTCTCCCTTCTTCTTT-39,39-T- 9 9 9 On the basis of the known interactions between c-Met and CD44, CACACAGAATCAGGCAAGA-5 ;Bcl-2,5-CAGGGCGATGTTGTCC-3 , 39-CTGGCATCTTCTCCTTCC-59; cyclin E, 59-GAAAATCAGACCAC- we wished to determine whether c-Met is expressed in naive B cells CCAGAGCC-39,39-GAAATGATACAAAGCAGAAGCAGCG-59; hypo- and whether it is involved in the regulation of their survival. In this xanthine ribosyltransferase (HPRT), 59-GAGGGTAGGCTGGCCTATGG- study, we demonstrate that naive murine B cells express both c-Met CT-39,39-GTTGGATACAGGCCAGACTTTGTTG-59. and its ligand HGF, which regulate peripheral B cell survival. In Real-time RT-PCR analysis addition, our study shows that c-Met participates in controlling MIF-induced signaling by forming a survival complex together Levels of mRNA of actin, c-Met, and Bcl-2 were analyzed by quantitative with CD74 and CD44 in B cells. These findings establish a key real-time RT-PCR using a LightCycler instrument (Roche Diagnostic Sys- and novel role for HGF/c-Met pair in the regulation of B cell sur- tems, Burgess Hill, West Sussex, U.K.). Total RNA was isolated from cells using the TRI Reagent kit. Reverse transcription was carried out using vival, demonstrating an additional level of control of the humoral Superscript II reverse transcriptase. The reaction volume (10 ml) contained Downloaded from immune response. 3 mM MgCl2, LightCycler HotStart DNA SYBR Green I mix (Roche Diagnostics), specific primer pairs, and 2.5 mg cDNA. Conditions for Materials and Methods PCR were as follows: 10 min at 95˚C followed by 40–50 cycles of 15 s Cells at 95˚C, 15 s at 60˚C, and 15 s at 72˚C. PCR was performed in duplicates, as previously described (38). Primer sequences were as follows: b-actin, Spleen cells were obtained from C57BL/6 CD442/2 (31) or CD742/2 (32) as previously described (39); c-Met, 59-GTGCCAAGCTACCAGT-39,59-CT- 9 9 9 9 mice. All of the animal procedures were approved by the Animal Research TCGTACAAGGCGTCT-3 ;Bcl-2,5-GCTACCGTCGTGACTT-3 ,5-GC- http://www.jimmunol.org/ Committee at the Weizmann Institute. CGGTTCAGGTACTC-39. b-Actin levels were used to normalize samples for calculation of the Cells and B cell separation relative expression levels of all of the genes (c-Met and Bcl-2). The results , Spleen cells were obtained from the indicated mice at 6–8 wk of age, as were evaluated for significance using the t test, and the p value was 0.05. previously described (33). B cells then were purified from each mouse strain using CD45R beads (BD Biosciences, San Jose, CA). The purity of the ELISA isolated cells (96–99%) was confirmed by FACS (using the B220+ marker) + B cells from C57BL/6 mice were incubated for 8 h in the presence or after each experiment. For isolation of AA4 cells, purified splenocytes absence of MIF (100 ng/ml). Cell supernatants were collected, and the HGF were stained with anti-AA4 Ab conjugated to PE, as previously described levels were determined by ELISA kit (R&D Systems), according to the (16). Separation of cells was performed using PE-conjugated beads (BD manufacturer’s instructions. by guest on September 28, 2021 Biosciences). The purity of the isolated cells (96–99%) was confirmed by FACS (using the IgD marker [eBioscience, San Diego, CA]) after each HGF intracellular staining experiment. For isolation of IgD+ cells, control IgD+ cells were separated from total Total splenocytes (107 cells per milliliter) from control mice were used splenocytes, as previously described (34). directly or stimulated with anti-CD74 (C-16; Santa Cruz Biotechnology) or Sorting of the transitional 1 (T1), transitional 2 (T2), marginal zone isotype control Ab (Santa Cruz Biotechnology) for 5 h. HGF intracellular (MZ), and mature populations was performed using anti-CD45R/B220, staining was performed using the Cell Fixation/Permeabilization Kit for anti-CD21 (CR2/CR), anti-CD24 (heat stable Ag), and anti-CD23 Abs Intracellular Cytokine Analysis according to the manufacturer’s instruc- (all from eBioscience), as previously described (35). tions (BD Cytofix/Cytoperm Kit; BD Biosciences).The cells were pre- T1 B cells were obtained from 1-wk-old mice, as previously described (36). stained with anti-CD45R/B220 and anti-IgD Abs (both from eBioscience). Then, the permeabilized cells were stained with goat anti-mouse HGF Ab MIF and HGF stimulation (R&D Systems) followed by donkey anti-goat Ab (Jackson ImmunoRe- search Laboratories, West Grove, PA). Recombinant murine MIF was prepared with its native sequence and pu- rified from an expression system, as previously described (37). Contami- nating endotoxin was removed by C8 affinity chromatography prior to Preparation of cell extracts protein renaturation, and the experimental preparations contained 1800 Stimulated cells were lysed in buffer containing: 25 mM Tris (pH 7.4), 7 EU/mg MIF. For MIF stimulation, 1 3 10 primary B cells were incubated 2 mM vanadate, 75 mM b-glycophosphate (pH 7.2), 2 mM EDTA, 2 mM in RPMI 1640 medium containing 0.1% (v/v) FCS at 37˚C for 3 h. EGTA, 10 mM NaPPi, and 0.5% Nonidet P-40 in the presence of the Next, cells were resuspended in medium containing 100 ng/ml rMIF and following protease inhibitors: 10 mg/ml leupeptin, 10 mg/ml aprotinin, 7 incubated at 37˚C for various periods. For HGF stimulation, 1 3 10 10 mg/ml pepstatin, 10 mg/ml chymostatin (Roche, Basel, Switzerland), primary B cells were incubated in RPMI 1640 medium containing 1% 1 mM PMSF (Sigma-Aldrich, St. Louis, MO), and 20 mM N-ethylmalei- (v/v) FCS at 37˚C for 3 h. Cells were then resuspended in medium con- mide (Sigma-Aldrich). taining 10 ng/ml rHGF (R&D Systems, Minneapolis, MN) and incubated at 37˚C for the indicated periods. Immunoprecipitation and Western blot CD74 stimulation Protein G-Sepharose beads (Pharmacia, Piscataway, NJ) were conjugated to c-Met mAb (B-2, SC-8057; Santa Cruz Biotechnology), CD44 (BD First, 1 3 107 primary B cells were suspended in 1 ml RPMI 1640 medium Biosciences), or Tyr(P) (Santa Cruz Biotechnology) mAb for 2 h at 4˚C, containing 10% (v/v) FCS. Next, 5 mg Ab specific for the extracellular followed by three washes in PBS. Beads were added to the cell lysates, domain of both murine and human CD74 (C-16; Santa Cruz Biotechnol- and c-Met, CD44, or Tyr(P) proteins were immunoprecipitated overnight. ogy, Santa Cruz, CA) or an isotype control (Santa Cruz Biotechnology) The protein G-bound material was washed three times with PBS contain- was added to the cells and incubated at 37˚C for various periods. ing 0.1% SDS and 0.5% Nonidet P-40. Immunoprecipitates were separated HGF and c-Met blocking by 10% (w/v) SDS-PAGE. The protein bands were transferred onto a ni- trocellulose membrane and probed with anti-CD74 (INI), anti–c-Met For HGF blocking, 1 3 107 B cells were incubated in 1 ml RPMI 1640 (AF527; R&D Systems), and anti-Syk (LR; Santa Cruz Biotechnology) medium containing 0.1% (v/v) FCS in the presence of 3 mg/ml anti-murine Abs, respectively, followed by HRP-conjugated anti-rabbit IgG or anti- HGF Ab (R&D Systems) and 3 mg/ml anti-isotype control Ab for 8 h at goat IgG (Jackson ImmunoResearch Laboratories). The Journal of Immunology 3

Blocking peptide room temperature. PI staining was analyzed by FACS. Doublet discrimination settings were used to eliminate cell clusters from consideration. Protein G-Sepharose beads (Pharmacia) were incubated in the presence of c-Met mAb (B-2, SC-8057; Santa Cruz Biotechnology) and the c-Met Cell death detection blocking peptide (B-2, SC-8057P; Santa Cruz Biotechnology), which rec- Magic Red apoptosis detection kit. Cells were incubated with Magic Red ognizes the c-Met cytoplasmic domain of murine origin. Immunoprecipi- (Immunochemistry Technology, Bloomington, MN), according to the tation and Western blotting were then performed, as described above. manufacturer’s instructions, at 37˚C for 1 h. Then, Magic Red staining was Western blot analysis quantified by FACS analysis. Fluorescent-labeled inhibitor of caspases apoptosis detection kit. Cells To detect changes in protein phosphorylation, lysates or immunoprecipitates were incubated with fluorescent-labeled inhibitor of caspases (FLICA; Im- were separated by 12% (w/v) SDS-PAGE. The proteins were transferred munochemistry Technology), according to the manufacturer’s instructions, onto a nitrocellulose membrane and probed with anti–Bcl-2 (C-2; Santa at 37˚C for 1 h. Cells were then washed three times with FLICA washing Cruz Biotechnology) or anti–c-Met (AF527; R&D Systems) followed by buffer. FLICA staining was measured by FACS analysis. The positive HRP-conjugated anti-mouse or anti-goat (Jackson ImmunoResearch Lab- population was identified by comparison of the staining to that of the oratories), respectively. The membranes then were stripped and reprobed negative population. with anti-tubulin Ab (Sigma-Aldrich), followed by peroxidase-conjugated anti-mouse Ab (Jackson ImmunoResearch Laboratories). Annexin/PI staining Spleens were collected from HGF- or PBS-injected mice. Cells were HGF injections washed and stained with annexin (BD Biosciences) and PI (Bender Med- C57BL/6 mice were i.p. injected daily with 4 mg HGF or PBS for 2 d. Systems, Vienna, Austria) for 15 min at room temperature. Annexin and PI Spleens were collected, and splenocytes were analyzed for their B cell staining were analyzed by FACS. The positive population was identified by comparison of the staining to that of the negative stained population. populations and survival. Downloaded from Injections with c-Met inhibitor Immunofluorescence and flow cytometry C57BL/6 mice were i.p. injected daily with 0.15 mg PHA-665752, a c-Met c-Met cell surface expression was analyzed using directly conjugated blocker (a kind gift from Pfizer), or DMSO twice a day for 5 d. Spleens anti-mouse c-Met (HGFR eBioclone 7; eBioscience) (40, 41) or goat anti- were collected, and splenocytes were analyzed for their B cell populations mouse HGFR (AF527; R&D Systems) Ab, followed by HRP-conjugated and survival. anti-goat Ab (Jackson ImmunoResearch Laboratories). CD74 cell surface staining was analyzed using goat anti-mouse CD74 (C-16; Santa Cruz Biotechnology), followed by HRP-conjugated anti-goat http://www.jimmunol.org/ Propidium iodide staining Ab (Jackson ImmunoResearch Laboratories). Spleens were collected from HGF- or PBS-injected mice as described. Pu- Characterization of B cells rified splenic B cells were cultured in six-well plates with 1 3 107 cells per well in RPMI 1640 medium supplemented with 1% FCS, 2 mM glutamate, Purified B cells from control and CD742/2 mice that were incubated for 100 U/ml penicillin, and 100 mg/ml streptomycin, with or without HGF 18 h in the presence or absence of HGF (10 ng/ml) and freshly isolated (10 ng/ml) for 18 h. Cells were collected by centrifugation, washed, and fixed splenocytes were stained for RA3-6B2 anti-CD45R/B220, anti-IgM, anti- in 70% cold ethanol and incubated in the presence of RNase (25 mg/ml). IgD, anti-AA4, anti-CD21 (CR2/CR), anti-CD24 (heat stable Ag), and Propidium iodide (PI; 25 mg/ml) (Sigma-Aldrich) was added for 20 min at anti-CD23 (all from eBioscience) and analyzed by FACS. by guest on September 28, 2021

FIGURE 1. CD74/CD44 forms a cell surface complex with c-Met in B cells. A, T1, T2, MZ, and mature B cells were purified as described in Materials and Methods. Total RNA was isolated, reverse transcription using primers for c-Met and HPRT was performed as described in Materials and Methods, and fold activation was calculated. The intensity of the c-Met band for each population was normalized by dividing by the measured intensity of the HPRT band from the same cell population. The activation fold ratio in the mature population was normalized to 1, and the ratio for the other populations was calculated as the intensity of the each sample relative to 1. B–D, Splenocytes from C57BL/6 mice were stained with (B) anti-B220 and anti-c-Met (eBioscience) or isotype control Abs, (C) anti-B220, anti-IgD, and anti–c-Met (eBioscience) Abs; or (D) anti-B220, anti-CD21, anti-CD24, and anti–c-Met (eBioscience) Abs. Histograms present cell surface expression of c-Met on B220+ cells (B), IgD+B220+ cells (C), and CD21low, CD24low B220+ cells (D). E, IgD+B220+ B cells or B220+ B cells were purified as described in Materials and Methods. Cells were lysed, and the level of c-Met was analyzed by Western blot analysis. F–H, Immunoprecipitations: control (F); CD442/2 (G); or CD742/2 (H). B220+ B cells were lysed. After anti–c-Met immunoprecipitation, proteins were separated by SDS-PAGE and transferred onto nitrocellulose. CD74 was detected by Western blot analysis. The results presented are representative of at least three different experiments. I, Control B220+ B cells were lysed. After anti-CD44 or isotype Ab immunoprecipitation, proteins were separated by SDS-PAGE and transferred onto nitrocellulose. c-Met was detected by Western blot analysis. 4 CD74–HGF/c-Met AXIS DEFINES A SURVIVAL PATHWAY IN B CELLS

FIGURE 2. c-Met is a MIF/CD74 target gene. A–C, B220+ B cells derived from control (A, B) or from CD742/2 or CD442/2 (C) mice were incubated in Downloaded from the presence or absence of MIF (100 ng/ml) for 10 h. RT-PCR (A, C), using primers for c-Met or HPRT, was performed as described in Materials and Methods, and fold activation was calculated. The intensity of the c-Met band after each treatment was normalized by dividing by the measured intensity of the HPRT band from the same treatment. The activation fold ratio in the absence of any treatment was normalized to 1, and the ratio for each treatment was calculated as the intensity of the treatment sample relative to 1. The results presented are representative of at least three different experiments. Quantitative real-time PCR (B) was performed using primers for c-Met and b-actin as described in Materials and Methods. b-Actin levels were used to normalize samples for calculation of the relative expression levels of c-Met. Results are expressed as a fold change in c-Met expression in stimulated cells compared +

with that in nonstimulated cells, which was defined as 1. Results shown are averages of three separate experiments. D, B220 B cells derived from control http://www.jimmunol.org/ mice were incubated in the presence or absence of MIF (100 ng/ml) for 18 h. Cells were lysed, and levels of c-Met and tubulin were analyzed by Western blot analysis. The results presented are representative of at least three separate experiments. E, B220+ cells derived from control or CD742/2 mice were incubated in the presence or absence of MIF (100 ng/ml) for 18 h. Cell surface expression of c-Met was analyzed by FACS. The cells were double-stained with anti-B220 and anti–c-Met (eBioscience) Abs. The histograms present cell surface expression of c-Met on B220+ cells. The results presented are representative of at least three different experiments.

Statistical analysis Results Comparisons between groups were evaluated by Student t test. Data are CD74/CD44 forms a cell surface complex with c-Met in B cells

6 by guest on September 28, 2021 expressed as mean SD and were considered statistically significant if p We first wished to determine whether CD74/CD44 might form values were #0.05. a cell surface complex with c-Met in B cells; to this end, we analyzed

FIGURE 3. HGF is a MIF/CD74 target gene. A, T1 and mature B cells were purified. Total RNA was isolated, reverse transcription using primers for HGF and HPRT was performed as described in Materials and Methods, and fold activation was calculated. B, Splenocytes derived from control mice were stained for B220 and IgD, and HGF intracellular protein levels were analyzed by intracellular staining as described in Materials and Methods. C, B220+ B cells derived from control or CD742/2 mice were incubated in the presence or absence of MIF (100 ng/ml) for 5 h. RT-PCR, using primers for HGF or HPRT, was performed as described in Materials and Methods, and fold activation was calculated. The results presented are representative of at least three different experiments. D, B220+ cells from control mice were stimulated with anti-CD74 or and isotype control Abs for 4 h. Intracellular HGF protein levels were analyzed by intracellular staining as described in Materials and Methods. The results presented are representative of at least three separate experi- ments. E and F, B220+ cells derived from control mice were incubated in the presence or absence of MIF (100 ng/ml) for 8 h. Conditioned medium was collected and analyzed by ELISA to determine HGF levels (E). HGF levels in nonstimulated cells were normalized to 1, and the relative ratio for each treatment was calculated (F). The results presented are an average of three independent experiments. The Journal of Immunology 5 c-Met expression in B cells. T1, T2, MZ, and mature B cell popula- form, showing that CD74 forms a complex with c-Met in B cells tions were sorted using cell surface markers specific for each popu- (Fig. 1F). This interaction was not observed in CD44- or lation (35), and then c-Met message was followed by RT-PCR. As CD74-deficient B cells (Fig. 1G,1H), suggesting that CD44 is seen in Fig. 1A, all of the splenic B cell populations trans- essential for the formation of the CD74/CD44/c-Met complex. cribed c-Met. To confirm that B cells express c-Met protein as well To further confirm that c-Met forms a cell surface complex with as mRNA, c-Met cell surface expression levels were analyzed in CD44, CD44 was immunoprecipitated, and the coimmunoprecipi- control and naive mature B cell populations, identified using various tation of c-Met was confirmed by Western blot analysis. As shown maturation markers. B cells, including the mature B cell population, in Fig. 1I, c-Met was specifically pulled down by anti-CD44 Ab, expressed c-Met on their cell surfaces (Fig. 1B–D, Supplemental further indicating that c-Met engages with CD44 in B cells. Fig. 1A,1B). c-Met protein expression was also detected by Western blot analysis in the mature (B220+IgD+) population (Fig. 1E). c-Met and HGF are MIF/CD74 target genes To identify complex formation between c-Met and CD74/CD44, To determine whether the survival cascade induced by CD74 control B cells were lysed, and c-Met was immunoprecipitated. is c-Met-dependent, we first investigated whether c-Met expression Immunoprecipitates were separated by SDS-PAGE and probed is modulated by CD74. B cells were stimulated with MIF for 8 h, with anti-CD74 Ab. Immunoprecipitation of c-Met (Supplemental and c-Met was analyzed by RT-PCR. As shown in Fig. 1C) specifically coprecipitated CD74, mainly the [31P] iso- Fig. 2A and 2B, c-Met mRNA levels were elevated after CD74 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. In vitro treatment with HGF induces survival and proliferation cascades in B cells. A, B220+ B cells derived from control mice were in- cubated in the presence or absence of HGF (10 ng/ml) for various time periods. Immediately after stimulation, cell pellets were washed and fast-frozenin liquid nitrogen. The cells then were lysed, and an aliquot was reserved for total Syk analysis. Phosphorylated proteins from the remaining lysate were immunoprecipitated with an anti-Tyr(P) Ab. Immunoprecipitates and total lysate proteins were separated by 10% (w/v) SDS-PAGE and blotted with an anti- Syk Ab. The results presented are representative of at least three different experiments. B and C, B220+ B cells derived from control mice were incubated in the presence or absence of HGF (10 ng/ml) for 8 h. Total RNA was isolated. RNA was reverse-transcribed using primers for Bcl-2 or HPRT. The results presented are representative of at least five different experiments (B). Quantitative real-time PCR was performed using primers for Bcl-2 and b-actin as described in Materials and Methods. b-Actin levels were used to normalize samples for calculation of the relative expression levels of Bcl-2. Results are expressed as a fold of change in Bcl-2 expression in stimulated cells compared with that in nonstimulated cells, which was defined as 1. Results shown are average of three separate experiments (C). D, B220+ B cells derived from control mice were incubated in the presence or absence of HGF (10 ng/ml) or MIF (100 ng/ml) for 8 h. Total RNA was isolated. Quantitative real-time PCR was performed using primers for Bcl-2 and b-actin as described in Materials and Methods. b-Actin levels were used to normalize samples for calculation of the relative expression levels of Bcl-2. Results are expressed as fold change in Bcl-2 expression in stimulated cells compared with that in nonstimulated cells, which was defined as 1. E, B220+ B cells derived from control mice were incubated in the presence or absence of HGF (10 ng/ml) for 18 h. Cells were lysed, and levels of Bcl-2 and tubulin were analyzed by Western blot analysis. The results presented are representative of at least three different experiments. F, B220+ B cells derived from control mice were incubated in the presence or absence of HGF (10 ng/ml) or MIF (100 ng/ml) for 16 h. Cell survival was then analyzed by annexin V and PI staining of intact cells. G, B220+ B cells derived from control mice were incubated in the presence or absence of HGF (10 ng/ml) for 18 h. Cell death was measured by Magic Red apoptosis detection kit. The graph shows the average of four independent experiments. H, B220+ B cells derived from control mice were incubated in the presence or absence of HGF (10 ng/ml) for 8 h. Total RNA was isolated and reverse-transcribed using primers for cyclin E or HPRT. I, B220+ B cells derived from control mice were incubated in the presence or absence of HGF (10 ng/ml) for 18 h and subjected to PI staining. The first gate includes cells in the G0/G1 phase, and the second one includes cells in the S/G2/M phases. The graph shows an average of four independent experiments. 6 CD74–HGF/c-Met AXIS DEFINES A SURVIVAL PATHWAY IN B CELLS activation. This elevation in c-Met mRNA levels was specific to ulation of the CD74/CD44 complex expressed on B cells aug- MIF stimulation and did not occur in CD74- or CD44-deficient ments both c-Met cell surface expression and HGF secretion. B cells (Fig. 2C). To follow c-Met protein levels in CD74- stimulated cells, c-Met protein and cell surface expression were HGF induces survival and proliferation cascades in B cells analyzed after MIF stimulation. c-Met protein levels were upregu- resulting in regulation of the peripheral B cell subsets lated after MIF stimulation, as detected by Western blot analysis Activation of the HGF/c-Met signaling pathway leads to cellular (Fig. 2D, Supplemental Fig. 2D). Similarly, a specific elevation responses, including cell survival (22, 24, 27, 42, 43). in c-Met cell surface expression after MIF stimulation was ob- To reveal whether HGF induces a survival cascade in B cells, we served in control B cells using different c-Met Abs (Fig. 2E, Sup- first followed the HGF-induced signaling cascade. After HGF plemental Fig. 2A–C), whereas there was no change in its levels stimulation, cells were lysed, and phosphorylated proteins were an- in CD74-deficient B cells (Fig. 2E). alyzed by Western blot analysis. We next investigated whether HGF expression is modulated by The BCR transduces Ag binding into alterations in the activity of CD74; to this end, we first measured HGF mRNA expression in intracellular signaling pathways through its ability to recruit and control B cells. The T1 and mature B cell populations were sorted activate the cytoplasmic protein-tyrosine kinase Syk. The recruit- as described in Materials and Methods, and HGF mRNA was ment of Syk to the receptor, its activation, and its subsequent inter- followed by RT-PCR. As shown in Fig. 3A, HGF expression was actions with downstream effectors regulate B cell survival (44). In detected in both T1 and mature subpopulations. HGF expression addition, we have recently shown that MIF induces Syk phosphor- in total B cells (B220+) and mature cells (B220+IgD+) was also ylation, also resulting in B cell survival (15, 16). Therefore, Syk confirmed by intracellular staining (Fig. 3B). We next examined phosphorylation after HGF stimulation was analyzed. B cells de- Downloaded from whether HGF mRNA expression is modulated by MIF stimulation. rived from control mice were incubated in the presence or absence As shown in Fig. 3C, a specific increase in HGF mRNA levels was of HGF for 0–5 min. The cells were then lysed, and Syk phos- detected in control B cells after CD74 stimulation, whereas no phorylation was analyzed. As can be seen in Fig. 4A, HGF stim- change was detected in CD74-deficient B cells. Moreover, CD74 ulation augmented Syk phosphorylation. stimulation elevated HGF intracellular expression (Fig. 3D) and Next, the role of HGF in B cell survival was examined. B cells

secretion into the conditioned medium, as analyzed by ELISA were incubated in the presence or absence of HGF, and the ex- http://www.jimmunol.org/ assay (Fig. 3E,3F). Together, these results demonstrate that stim- pression of the antiapoptotic protein Bcl-2 was analyzed. As shown by guest on September 28, 2021

FIGURE 5. The HGF survival pathway induced in vitro regulates peripheral B cell subsets. A and B, B220+ B cells derived from control mice were incubated in the presence or absence of HGF (10 ng/ml) for 18 h. The B cell subpopulations then were analyzed by FACS. Dot plots show IgD and AA4 expression on B220+ cells (A). Graph showing an average of four independent experiments (B). C, B220+ B cells derived from control mice were incubated in the presence or absence of HGF (10 ng/ml) for 18 h. Histograms show AA4 expression on B220+ cells (1). Cell survival of AA42 (2) or AA4+ (3) populations was analyzed by annexin V staining. D, Mature (sorted CD21lowCD24low [1] or AA42 [2]) and transitional (AA4+ [3]) B cells were incubated in the presence or absence of HGF (10 ng/ml) for 6 h. Cell death was measured by Magic Red apoptosis detection kit. E, T1 B cells derived from 1-wk-old control mice (1) and transitional (AA4+) B cells (2) were incubated in the presence or absence of HGF (10 ng/ml) for 18 h. Histograms show AA4 expression (1) and IgD expression (2) on B220+ cells. The Journal of Immunology 7 in Fig. 4B–E, HGF stimulation upregulated Bcl-2 mRNA (Fig. therefore incubated with or without HGF for 18 h. Then, the differ- 4B–D) and protein (Fig. 4E) levels. To further determine whether ent B cell populations were analyzed for the expression of various HGF regulates B cell survival, the B cell apoptotic populations maturation markers. As shown in Fig. 5A and 5B, accumulation of after HGF stimulation were analyzed by annexin/PI staining. As the mature B cell subpopulation (IgDhighAA42) was observed after shown in Fig. 4F, HGF reduced B cell apoptosis, resulting in an HGF stimulation, which resulted from elevated survival of the ma- elevation of the live population. Because B cells survive poorly ture (AA42, Fig. 5C2,5D2; CD21lowCD24low, Fig. 5D1) popula- in vitro and after incubation for 24 h a large proportion of the cells tion, whereas there was no change in the cell death of the immature are already dead, we followed cell survival over a shorter time (AA4+, Fig. 5C3,5D3) population. Thus, HGF preferentially pro- period. Therefore, cells were incubated in the presence or absence motes the viability of mature B cells. Next, to investigate whether of HGF for 8 h, and their caspase 3 and 7 activity was analyzed as HGF controls differentiation of the transitional B cell populations, a marker of apoptosis. As shown in Fig. 4G, a significant reduction purified T1 cells derived from a 1-wk-old mouse (Fig. 5E1; AA4+, in the apoptotic population was detected in control B cells stim- Fig. 5E2) B cells were incubated with or without HGF for 18 h and ulated with HGF. were analyzed for the expression of maturation markers. No change The possible regulation of B cell proliferation by HGF in vitro in the transitional and mature B populations was observed in was studied by analyzing cell entry into the S phase. Cell cycle HGF-stimulated cells. These results suggest that HGF regulates progression is regulated by cyclin-dependent kinases. We previously mature B cell survival and is not involved in the differentiation of showed that CD74 activation induces expression of cyclin E (15, 16), transitional B cells into mature cells. which regulates cell entry into the S phase. To determine whether To follow the in vivo effect of HGF on B cells, we injected HGF

HGF also regulates cell entry to the S phase, cyclin E mRNA levels (4 mg) into mice for 2 d and analyzed the apoptotic populations by Downloaded from were followed. As demonstrated in Fig. 4H, HGF elevated the in- annexin/PI staining of B cells recovered from the spleens. As tracellular level of cyclin E mRNA. To further evaluate cell cycle demonstrated in Fig. 6A, a high proportion of live B splenocytes entry and DNA synthesis after HGF stimulation, PI staining was was detected in both HGF- and PBS-injected mice. However, HGF performed. As shown in Fig. 4I, HGF stimulation induced the entry significantly reduced the apoptosis of B cells. To further evaluate of B cells into the S phase. The HGF-induced survival pathway may cell division, cell cycle entry, and DNA synthesis after HGF in-

control peripheral B cell subsets; B cells from control mice were jection, PI staining was performed. As shown in Fig. 6B, the http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. The HGF-induced survival pathway regulates peripheral B cell subsets in vivo. Control mice were injected with HGF (4 mg) or PBS for 2 d. A, Cell survival was analyzed by annexin Vand PI staining of intact B220+ cells. B, Cell cycle entry in B220+ cells was analyzed by PI staining after cell fixation.

The first gate includes cells in the G0/G1 phase, and the second one includes cells in the S/G2/M phases. The results presented are representative of at least three separate experiments. C–E, Control mice were injected with HGF (4 mg) or PBS for 2 d, and the B cell subpopulations then were analyzed for IgD and IgM (C); AA4 (D); and CD21, CD24, and CD23 (E) expression on B220+ cells. The graph shows an average of four independent experiments. F and G, Control mice were injected with the c-Met inhibitor PHA-665752 (0.15 mg) twice per day for 5 d. B cell survival was analyzed by annexin Vand PI staining of intact cells (F). B cell subpopulations were analyzed for IgD and AA4 expression on B220+ cells (G). The results presented are representative of four separate experiments. 8 CD74–HGF/c-Met AXIS DEFINES A SURVIVAL PATHWAY IN B CELLS proportion of cells that entered the S and M phases was elevated in HGF-treated mice. These results show that HGF induces survival and proliferation cascades in vivo. To directly follow the in vivo role of HGF in B cell survival, the splenic B cell populations were analyzed in mice treated with HGF or with c-Met inhibitor. C57BL/6 mice were injected with HGF (4 mg) for 2 d, and the B cells obtained from the HGF-treated and nontreated mice were analyzed using maturation markers for the peripheral differentiation of B cells. As shown in Fig. 6C–E, HGF treatment resulted in a reduction of the transitional 1 (IgMhigh IgDlowAA4+CD21lowCD24high) and transitional 2 (CD21highCD24high CD23high) populations and an elevation in the proportion of the mat- ure B cells (IgMlowIgDhighAA42CD21lowCD24low). To directly show that c-Met is involved in the survival of peripheral mature B cells, C57BL/6 mice were injected with PHA-665752, a specific c-Met in- hibitor. PHA-665752 is a selective, small-molecule, active-site inhib- itor of the catalytic activity of c-Met kinase (Ki = 4 nM) that competes with its ATP binding (45). As shown in Fig. 6F and 6G, block- ing c-Met in vivo induced apoptosis of B cells (Fig. 6F), resulting in Downloaded from the reduction of the mature population in the spleen (Fig. 6G). To- gether, these results show that activation of c-Met induces cell division and suppression of apoptosis, which enlarge the mature B cell com- partment, whereas its blockade results in mature B cell death. CD74 induces B cell survival in an HGF/c-Met-dependent FIGURE 7. HGF transmits a survival signal in CD74-deficient cells, and http://www.jimmunol.org/ manner stimulation with exogenous HGF restores the mature population missing in + 2/2 To determine whether the c-Met-induced survival cascade is a CD74 cells lacking CD74. A, B220 B cells derived from CD74 mice were downstream event, we first tested whether exogenous HGF could incubated in the presence or absence of HGF (10 ng/ml) for 8 h. Total bypass reduced survival in CD74-deficient cells. CD742/2 B cells RNA was isolated, and reverse transcription using primers for Bcl-2 or HPRT was carried out. The results presented are representative of at least were treated in the presence or absence of HGF, and Bcl-2 mRNA + 2/2 five different experiments. B, B220 B cells derived from CD74 mice and protein levels were evaluated. As shown in Fig. 7, HGF stimu- were incubated in the presence or absence of HGF (10 ng/ml) for 18 h. lation upregulated Bcl-2 mRNA (Fig. 7A) and protein (Fig. 7B) Cells were lysed, and levels of Bcl-2 and tubulin were analyzed by Western 2/2 levels in cells lacking CD74. Next, CD74 B cells were incubated blot analysis. The results presented are representative of at least three in the presence or absence of HGF for 18 h, and cell death was separate experiments. C, B220+ B cells derived from CD742/2 mice were by guest on September 28, 2021 analyzed. As shown in Fig. 7C, a reduced apoptotic population incubated in the presence or absence of HGF (10 ng/ml) for 18 h. Cell was observed in HGF-stimulated cells. To directly determine death was measured by Magic Red apoptosis detection kit. The graph whether HGF can shape B peripheral cell subsets by regulating shows an average of the results obtained from four independent experi- + 2/2 the survival of the mature population, CD742/2 B cells were in- ments. D, B220 B cells derived from CD74 mice were incubated in cubated in the presence or absence of HGF, and the various B cell the presence or absence of HGF (10 ng/ml) for 18 h. Next, B cell sub- populations were analyzed by FACS. The graph shows an average of populations were analyzed 18 h later by FACS. A specific elevation results obtained from four independent experiments. in the mature population and reduction in the transitional B cell population were observed in HGF-stimulated cells (Fig. 7D). To- gether, these results show that HGF induces a survival cascade in were stimulated with MIF in the presence or absence of an CD74-deficient cells, and stimulation with exogenous HGF restores anti-HGF blocking Ab or an isotype control Ab, and Bcl-2 mRNA the mature population missing in cells lacking CD74. and protein levels were analyzed. As shown in Fig. 8F and 8G, Finally, we tested whether the formation of the CD74/CD44/ although stimulation with MIF augmented Bcl-2 mRNA and pro- c-Met cell surface complex is MIF-dependent. As shown in Fig. tein levels, blocking of HGF significantly reduced this elevation. 8A, stimulation of B cells with MIF induced the coprecipitation of Downregulation of Bcl-2 transcription and expression was specific CD74 with c-Met, showing that MIF regulates the formation of the and did not occur in the cells incubated in the presence of an CD74/CD44/c-Met cell surface complex. Next, B cells were in- isotype control Ab. In addition, blocking of the secreted HGF cubated in the presence or absence of the c-Met inhibitor PHA- by the anti-HGF blocking Ab abolished the MIF-induced elevation 665752 (0.3 ng/ml) for 3 h. After incubation with PHA-665752, of survival. Thus, the MIF-dependent survival of mature cells is the cells were stimulated with MIF for 0–5 min. Then, the cells regulated by secreted HGF in an autocrine manner. were lysed, and Syk phosphorylation was analyzed. As shown in Together, these results show that blocking the HGF/c-Met path- Fig. 8B, the c-Met inhibitor abolished MIF-induced Syk phosphor- way results in induction of cell death and inhibition of the MIF/ ylation. Thus, c-Met activation is required for the MIF/CD74 sig- CD74-induced survival cascade. Thus, HGF/c-Met axis functions naling cascade. as a CD74 downstream pathway, regulating B cell survival in an We then analyzed the CD74-induced survival cascade in the autocrine manner. presence of MIF and the c-Met inhibitor PHA-665752, as above. Bcl-2 transcription levels were analyzed by means of RT-PCR. Discussion As shown in Fig. 8C and 8D, addition of the c-Met inhibitor In B cells, as in every other tissue, the balance between cell survival significantly downregulated the MIF-induced Bcl-2 mRNA levels. and apoptosis is essential for homoeostasis. Adaptive immunity In addition, as shown in Fig. 8E, the MIF-induced support of cell depends on the production and maintenance of a pool of mature survival was abolished in PHA-665752-treated cells. Finally, cells peripheral lymphocytes throughout life. Most of these cells circu- The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. c-Met regulates the CD74-induced survival cascade. A, B220+ B cells derived from control mice were incubated in the presence or absence of MIF (100 ng/ml) for various periods. Immediately after stimulation, cells were washed and fast-frozen in liquid nitrogen. Next, the cells were lysed; proteins were immunoprecipitated with anti–c-Met, separated by SDS-PAGE, and transferred to nitrocellulose. CD74 was detected by Western blot analysis. B, B220+ B cells derived from control mice were incubated in the presence or absence of the c-Met inhibitor PHA-665752 (0.3 ng/ml) for 3 h and were then stimulated with MIF (100 ng/ml) for various periods. Immediately after stimulation, cells were washed and fast-frozen in liquid nitrogen. The cells then were lysed, and an aliquot was reserved for total Syk analysis. Phosphorylated proteins from the remaining lysate were immunoprecipitated with an anti-Tyr(P) Ab. Immunoprecipitates and total lysate proteins were separated by 10% (w/v) SDS-PAGE and blotted with an anti-Syk Ab. C, B220+ B cells derived from control mice were incubated in the presence or absence of MIF (100 ng/ml) or the c-Met inhibitor PHA-665752 (0.3 ng/ml) for 8 h. Total RNA was isolated, and reverse transcription using primers for Bcl-2 or HPRT was carried out. The results presented are representative of at least three different experiments. D, Quantitative real-time PCR was performed using primers for Bcl-2 and b-actin as described in Materials and Methods. b-Actin levels were used to normalize samples for calculation of the relative expression levels of c-Met. Results are expressed as fold change in Bcl-2 expression in stimulated cells compared with that in nonstimulated cells, which was defined as 1. Results shown are a summary of three separate experiments. E, B220+ B cells derived from control mice were incubated in the presence or absence of MIF (100 ng/ml) or the c-Met inhibitor PHA-665752 (0.3 ng/ml) for 5 h. Cell death was analyzed by Magic Red and FLICA apoptosis detection kits. Graphs summarize results of three different experiments. F–H, B220+ B cells derived from control mice were incubated in the presence or absence of MIF (100 ng/ml), anti-HGF (3 mg), or an isotype control Ab. After 8 h, total RNA was isolated and reverse-transcribed using primers for Bcl-2 or HPRT. The results presented are representative of at least three different experiments (F). After 12 h, cells were lysed, and levels of Bcl-2 and tubulin (loading control) were analyzed by Western blot analysis. The results presented are representative of at least three different experiments (G). After 12 h, cell death was analyzed by Magic Red apoptosis detection kits. The graph summarizes results of three different experiments (H). late in the periphery in a quiescent state, without actively con- ment and maintenance (1, 3, 4). Our previous studies demonstrated tributing to a given acute immune response. This vast number that survival of mature B cells is controlled by MIF. of resting cells must be maintained to preserve a diverse B cell MIF is a ubiquitous protein that has a broad tissue distribution repertoire. Long-term B cell persistence in the periphery is depen- and is found in virtually all cells (46); thus, it undergoes tonic dent on survival signals that are transduced by cell surface recep- production in the spleen as well. Engagement of the CD74/CD44 tors. Conversely, resistance to apoptosis, leading to enhanced complex by MIF triggers the antiapoptotic Syk–PI3K/Akt sig- survival, is associated with initiation and progression of B cell naling pathway and thus promotes B cell survival (15, 16, 47). In malignancies. this study, we identified two novel components of the CD74/CD44 It was previously shown that the regulation of B cell homeostasis B cell survival complex, the c-Met receptor and its ligand HGF. depends on tonic and induced BCR signaling and on receptors The c-Met tyrosine kinase receptor and its cognate ligand HGF sensitive to trophic factors, such as BAFFR (BR3), during develop- are known to efficiently induce the survival of many cell types 10 CD74–HGF/c-Met AXIS DEFINES A SURVIVAL PATHWAY IN B CELLS subjected to various apoptotic inducers (48). HGF is a paracrine Our model suggests that both c-Met and HGF are CD74/CD44 factor that is produced by stromal and mesenchymal cells (21, 49). target genes. After MIF stimulation, c-Met engages with CD74 We found that HGF can be produced by B cells, indicating a pos- and CD44 on the cell membrane and, together with HGF, triggers sible autocrine mechanism of c-Met activation. Although para- an additional signaling pathway, which is necessary to initiate the crine HGF appears to be sufficient for survival of CD742/2 MIF-induced survival signaling cascade. It is interesting to note that B cells, we believe that because in CD742/2 mice B cell survival both MIF/CD74 and HGF/c-Met pathways have been implicated in is nevertheless impaired, the autocrine pathway induced by MIF/ tumor progression. It has been reported that MIF is overexpressed in CD74 plays an essential role in upregulating HGF secretion in- solid tumors (56, 57) and that its expression is associated with the ducing optimal B cell survival. growth of malignant cells (58). In addition, anti-MIF Ig therapy has Our results show that HGF does not affect differentiation of been shown to suppress tumor growth (59). Many studies have transitional cells in the spleen but rather induces a signaling cas- demonstrated the overexpression of CD74 in various cancers cade involving Syk, leading to B cell entry into the S phase and to cell (60–65), and CD74 has been suggested to serve as a prognostic survival of mature B cells. In addition, our studies show that the factor in several tumor types, with higher relative expression of HGF/c-Met–induced survival cascade is regulated by the CD74/ CD74 behaving as a marker of tumor progression (66). Moreover, CD44 cell surface complex, because exogenous HGF can bypass a humanized anti-CD74 mAb (hLL1) was shown to have therapeu- the absent survival signal and rescue the mature population missing tic activity in multiple myeloma, perhaps due to the high level of in cells lacking CD74. Moreover, blocking HGF or c-Met activity expression of CD74 in this plasma cell malignancy (67). Finally, abolishes MIF-induced Syk phosphorylation and Bcl-2 elevation, we recently showed that activation of cell surface CD74, expressed thereby inhibiting cell survival. at high levels from an early stage of B-chronic lymphocytic leuke- Downloaded from The precise mechanism by which MIF activates c-Met is still mia by MIF initiates a signaling cascade that contributes to tumor unclear. It is possible that MIF binding to the CD74/CD44 complex progression (68). Uncontrolled activation of c-Met is oncogenic can induce c-Met activation in an HGF-independent manner, as was and has been implicated in the growth, invasion, and metastasis shown in tumor cells (50, 51) or after cellular adherence (52). of a variety of tumors in both mice and humans (69). It has been However, because HGF was shown in our studies to be sufficient shown that receptor overexpression is by far the most frequent to support survival of mature B cells and its blocking inhibits the alteration of c-Met in human tumors (70). c-Met overexpression http://www.jimmunol.org/ MIF-induced survival pathway, we believe that HGF is involved in has been described in a variety of human cancers, including B cell malignancies, such as multiple myeloma (MM) (42, 71). Further- the MIF-induced survival cascade. We suggest that MIF activation more, in MM, Hodgkin lymphoma, and diffuse large B cell lym- induces transient CD74/CD44/c-Met complex formation. This can phoma, elevated serum HGF levels correlate with unfavorable lead to induction of a signaling cascade leading to Syk phosphor- prognosis (72, 73). Moreover, it was shown that HGF induces ylation and augmented transcription of genes, including the sur- a potent proliferative and antiapoptotic response in MM cell lines vival genes and c-Met and HGF. Subsequently, c-Met cell surface and primary MMs (42, 43). Thus, CD74 and c-Met might operate expression becomes elevated together with the augmented levels together in supporting tumor cell survival.

of HGF. by guest on September 28, 2021 These studies establish the function of the CD74/CD44/c-Met There is limited information regarding the expression and func- complex in delivering signals important for B cell survival and tion of c-Met and HGF in naive mature B cells. Previous studies may provide a basis for therapy of B cell malignancies with en- showed that human tonsillar and human peripheral B cells do not hanced specificity. express c-Met (53–55) and HGF (55), whereas germinal center activated cells and plasma cells exhibit c-Met expression. These results suggest that B cell activation significantly elevates c-Met Acknowledgments We thank Pfizer for accepting the proposal for this study and for providing expression, whereas its expression in resting mouse and human the PHA-665752 (c-Met inhibitor) reagent. We also thank Prof. Tsvee Lap- splenic B cells might be lower. Our study shows that naive murine idot and members of the Shachar laboratory team for helpful discussions splenic B cells express c-Met and its ligand HGF. Thus, upon and review of this manuscript. activation, it is likely that c-Met expression is significantly upregu- lated. Further studies will be required to compare c-Met expression Disclosures in activated B cells versus that in naive murine or human cells. The authors have no financial conflicts of interest. One of the signaling proteins that plays a crucial role in B cell development is the Syk protein-tyrosine kinase. Syk belongs to the Syk/ZAP70 family of protein-tyrosine kinases and participates in References B cell fate decisions and Ag processing. Recent findings indicate that 1. Lam, K. P., R. Ku¨hn, and K. Rajewsky. 1997. In vivo ablation of surface im- munoglobulin on mature B cells by inducible gene targeting results in rapid cell expression of Syk in nonhematopoietic cells is involved in a wide death. Cell 90: 1073–1083. variety of cellular functions and in the pathogenesis of malignant 2. Kraus, M., M. B. Alimzhanov, N. Rajewsky, and K. Rajewsky. 2004. Survival of tumors. Syk was previously shown to be required for the activation resting mature B lymphocytes depends on BCR signaling via the Igalpha/beta heterodimer. Cell 117: 787–800. of Akt in a PI3K-dependent manner. Syk is a critical component of 3. Crowley, J. E., L. S. Treml, J. E. Stadanlick, E. Carpenter, and M. P. Cancro. this signaling machinery. Studies in knockout mice and cell lines in- 2005. Homeostatic niche specification among naı¨ve and activated B cells: dicate that Syk is essential for most of the biochemical responses to a growing role for the BLyS family of receptors and ligands. Semin. Immunol. 17: 193–199. BCR engagement, including regulation of cell survival (44). Pre- 4. Woodland, R. T., M. R. Schmidt, and C. B. Thompson. 2006. BLyS and B cell viously, we showed that MIF initiates a signaling cascade in mature homeostasis. Semin. Immunol. 18: 318–326. B cells that involves Syk and Akt phosphorylation and is dependent 5. Schiemann, B., J. L. Gommerman, K. Vora, T. G. Cachero, S. Shulga-Morskaya, M. Dobles, E. Frew, and M. L. Scott. 2001. An essential role for BAFF in the on PI3K (16). In this study, we demonstrate that in B cells HGF normal development of B cells through a BCMA-independent pathway. Science stimulation leads to Syk phosphorylation. Blocking c-Met activity 293: 2111–2114. abolishes MIF-induced Syk phosphorylation. Therefore, c-Met 6. Schneider,P.,H.Takatsuka,A.Wilson,F. Mackay, A. Tardivel, S. Lens, T. G. Cachero, D. Finke, F. Beermann, and J. Tschopp. 2001. Maturation of marginal zone and fol- appears to be necessary for the signaling mechanism induced by licular B cells requires B cell activating factor of the tumor necrosis factor family and is MIF in a Syk-dependent manner. independent of B cell maturation antigen. J. Exp. Med. 194: 1691–1697. The Journal of Immunology 11

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