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The Journal of Immunology, 2007, 178, 7042 -7053
Copyright © 2007 by The American Association of Immunologists, Inc.
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Impaired Mast Cell Maturation and Degranulation and Attenuated Allergic Responses in Ndrg1-Deficient Mice1

Yoshitaka Taketomi*,{dagger}, Kohei Sunaga{dagger}, Satoshi Tanaka2,¶, Masanori Nakamura{ddagger}, Satoru Arata*, Tomohiko Okuda3,#, Tae-Chul Moon||, Hyeun-Wook Chang||, Yukihiko Sugimoto, Koichi Kokame#, Toshiyuki Miyata#, Makoto Murakami{dagger},§,** and Ichiro Kudo4,{dagger}

* Center for Biotechnology, {dagger} Department of Health Chemistry, School of Pharmaceutical Sciences, and {ddagger} Department of Oral Anatomy and Developmental Biology, School of Dentistry, Showa University, Tokyo, Japan; § Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; || College of Pharmacy, Yeungnam University, Gyonsan, Korea; # National Cardiovascular Center Research Institute, Osaka, Japan; and ** Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have previously reported that N-myc downstream regulated gene-1 (NDRG1) is an early inducible protein during the maturation of mouse bone marrow-derived mast cells (BMMCs) toward a connective tissue mast cell-like phenotype. To clarify the function of NDRG1 in mast cells and allergic responses, we herein analyzed mast cell-associated phenotypes of mice lacking the Ndrg1 gene. Allergic responses including IgE-mediated passive systemic and cutaneous anaphylactic reactions were markedly attenuated in Ndrg1-deficient mice as compared with those in wild-type mice. In Ndrg1-deficient mice, dermal and peritoneal mast cells were decreased in number and morphologically abnormal with impaired degranulating ability. Ex vivo, Ndrg1-deficient BMMCs cocultured with Swiss 3T3 fibroblasts in the presence of stem cell factor, a condition that facilitates the maturation of BMMCs toward a CTMC-like phenotype, displayed less exocytosis than replicate wild-type cells after the cross-linking of Fc{epsilon}RI or stimulation with compound 48/80, even though the exocytotic response of IL-3-maintained, immature BMMCs from both genotypes was comparable. Unlike degranulation, the production of leukotriene and cytokines by cocultured BMMCs was unaffected by NDRG1 deficiency. Taken together, the altered phenotypes of Ndrg1-deficient mast cells both in vivo and ex vivo suggest that NDRG1 has roles in the terminal maturation and effector function (degranulation) of mast cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells have long been considered to serve primarily as important effector cells for acute IgE-associated allergic reactions such as anaphylaxis, rhinitis, and asthma. Mast cells are tissue-resident cells of hemopoietic origin, representing an important source of a variety of inflammatory mediators such as vasoactive amines, proteases, eicosanoids, cytokines, and chemokines. They orchestrate various aspects of the IgE-associated and even IgE-independent immune responses not only through the release of these mediators but also through cell-cell interaction by which they regulate the function of other cells.

It has been elucidated that mast cells originate from hemopoietic stem cells in vivo (1) or multipotential progenitors in vitro (2). Mast cell precursors circulate in the blood and migrate into connective or mucous tissues where they differentiate into mature mast cell phenotypes depending on the microenvironment of the tissue (3, 4, 5, 6). Stem cell factor (SCF)5 and its receptor c-kit are prerequisites for the homing and subsequent differentiation of mast cells in the whole tissue (7, 8), and the {alpha}4beta7 integrin (9) and the chemokine receptor CXCR2 (10) have additional and profound influences on the basal homing, establishment, and maintenance of mast cells in the small intestine. However, the precise mechanism underlying the tissue-based maturation of mast cells is still a challenging area in this field of research.

Coculture of IL-3-maintained immature mouse bone marrow-derived mast cells (BMMCs), a progenitor population of mast cells, with fibroblasts is a useful system for analyzing certain aspects of change into a connective tissue mast cell (CTMC)-like phenotype (11, 12, 13, 14). We have developed a unique mast cell maturation system by which BMMCs cocultured with Swiss 3T3 fibroblasts in the presence of soluble SCF alter their morphological and functional properties from an immature to a mature CTMC-like phenotype after only 4–6 days of coculture (14). Following cDNA subtraction between BMMCs before and after such coculture, we have identified N-myc downstream regulated gene-1 (NDRG1) as the most frequently induced gene during the maturation of BMMCs into a mature CTMC-like phenotype under these conditions (15, 16). Moreover, the overexpression of NDRG1 in the rat mastocytoma RBL-2H3 augments exocytotic degranulation in response to IgE-dependent and -independent stimuli (15), suggesting that NDRG1 is involved in the divergent signaling pathways leading to exocytosis at their point of convergence or beyond or that it allows immature mast cells to differentiate into a mature phenotype that degranulates more efficiently in response to various secretagogues.

NDRG1, an intracellular protein with an {alpha}beta hydrolase fold (17) and three unique tandem repeats of 10 hydrophilic amino acids near the COOH-terminal end is a member of the emerging NDRG family that also contains NDRG2, NDRG3, and NDRG4 (18, 19, 20). NDRG1 has been independently identified as a molecule whose expression is markedly altered in several cell types under various conditions such as cellular stress response, hypoxia, and cell differentiation (15, 21, 22, 23, 24, 25, 26, 27). Significantly, the forcible expression of NDRG1 in cancer cells decreases their growth rate and metastasis by inducing cell differentiation and reversing their propensity to metastasize (28, 29), suggesting that NDRG1 is a cell differentiation regulator. Recently, a nonsense mutation in the Ndrg1 gene has been reported to be responsible for hereditary motor and sensory neuropathy-Lom, a severe autosomal recessive peripheral neuropathy known as Charcot-Marie-Tooth disease type 4D (CMT4D) (30, 31). Furthermore, mice lacking the Ndrg1 gene exhibit a peripheral neuropathy characterized by demyelination, a symptom similar to that observed in patients with CMT4D (32). These observations suggest that NDRG1 is essential for axon survival and appropriate differentiation, although the molecular machinery responsible for the neuronal function of NDRG1 still awaits further study.

To gain further insights into the functional role of this unique protein in mast cells, we have herein analyzed the mast cell-related phenotypes of Ndrg1-deficient mice. We found that the Ndrg1-deficient mice had mitigated passive systemic and local anaphylactic responses and that the mast cells from these mice were morphologically and functionally abnormal in terms of their aberrant granule structure and reduced exocytotic capacity. Thus, our findings provide unequivocal evidence that NDRG1 is a critical modulator of the maturation, and thereby the function, of mast cells.


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

The construction of the Ndrg1-deficient mice was described previously in detail (32). These Ndrg1-deficient mice were further backcrossed 10 generations onto a C57BL/6 background. All mice were bred in our animal facility under specific pathogen-free conditions. Mast cell-deficient WBB6F1-W/Wv (W/Wv) and littermate control WBB6F1-+/+ mice were purchased from Japan SLC. We used 8- to 12-wk-old mice for all experiments. The genotypes of Ndrg1+/+ and Ndrg1–/– were confirmed by PCR analysis of tail biopsies as described (32).

Passive systemic anaphylaxis

The anaphylaxis method used was described previously (33). Briefly, mice were administered 3 µg of anti-DNP mouse monoclonal IgE (SPE-7; Sigma-Aldrich) in 200 µl of saline i.v. through the tail vein. Then, 24 h later, the mice were challenged i.v. with 500 µg of DNP-conjugated human serum albumin (HSA) (DNP-HSA; Sigma-Aldrich) in 200 µl of saline. After Ag challenge, body temperature was monitored at various intervals using a rectal probe coupled to a digital thermometer (BAT-12R and RET-3; Physitemp Instruments). Blood samples were collected by puncturing the hearts of the sacrificed mice 1.5 min after Ag challenge. The sera were prepared and treated with 3% perchloric acid for the removal of proteins. The resulting supernatants were subjected to measurement of histamine. Histamine was separated by HPLC on a cation-exchange WCX-1 column (Shimadzu) and then measured fluorometrically by the o-phthalaldehyde method (34).

Passive cutaneous anaphylaxis (PCA)

The left and right ears of the mice were treated intradermally with 25 ng of anti-DNP IgE in 25 µl of saline. Then, 24 h later the mice were challenged i.v. through the tail vein with various doses of DNP-HSA together with 1 mg of Evans blue (Wako Pure Chemical) in 200 µl of saline. At various intervals after the Ag challenge, extravasation was visualized by blue staining of the ear skin. The ears were removed and incubated at 37°C in 1 ml of 3 N KOH. On the following day the mixtures were extracted with 1.24 M phosphoric acid and acetone. Absorbance of the resulting supernatants was measured at 620 nm. The relationship between Evans blue concentration and absorbance was linear, indicating that the absorbance represented the quantity of Evans blue extravasation. Ear thickness was recorded 30 min after Ag challenge using a dial thickness gauge (Mitutoyo Corporation) with a minimum sensitivity of 1 µm. Changes in ear thickness were determined as the difference before and after Ag challenge. For IgE-independent, compound 48/80-induced anaphylaxis, the ears were treated intradermally with various doses of compound 48/80 (Sigma-Aldrich) in 25 µl of saline followed by i.v. injection of 1 mg of Evans blue in 200 µl of saline. After 30 min, Evans blue extravasation was measured in a similar way.

Histological analysis

In a series of IgE-mediated, Ag-dependent PCA experiments, Ndrg1+/+ and Ndrg1–/– mice were sacrificed before and after Ag challenge. The left and right ears were removed, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections (5-µm thick) were cut and then stained with 0.05% acidic toluidine blue (pH 1.0). Intact and degranulated tissue mast cells were counted in the skin sections under an optical microscope (Axioskop 2 FS plus; Carl Zeiss MicroImaging) at x100 magnification. Degranulated tissue mast cells were defined as those showing the release of >10% cellular granules.

For transmission electron microscopy, ears were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2), postfixed with 2% OsO4, dehydrated by a graded ethanol series, passed through propylene oxide, and then embedded in Epon 812. Ultrathin sections (0.08-µm thick) were stained with uranyl acetate and lead citrate and then examined using an electron microscope (H-7600; Hitachi).

For immunohistochemistry, cytospin preparations were incubated with 5% normal rabbit serum in PBS containing 5% BSA and 0.025% Triton X-100 for 1 h and then with goat anti-NDRG1 polyclonal Ab (N-19; Santa Cruz Biotechnology) at 1/100 dilution in the same buffer at 4°C overnight. These preparations were incubated with biotinylated rabbit anti-goat IgG (Vector Laboratories) at 1/100 dilution in PBS containing 5% BSA, 0.025% Triton X-100, and 10% mouse serum for 30 min followed by incubation with avidin DH and biotinylated HRP (Vectastain ABC kit; Vector Laboratories). After 30 min these preparations were stained with 0.5 mg/ml 3,3'-diaminobenzidine and 0.1% hydrogen peroxide solution.

Preparation and activation of peritoneal mast cells (PMCs)

To harvest peritoneal cells, 5 ml of HBSS was injected into the mouse peritoneal cavity and the abdomen was massaged gently. After the fluid containing peritoneal cells had been collected and centrifuged, the pellets were resuspended in PIPES-buffered saline. The cells were cytospun onto glass slides for 5 min and then incubated for 30 min with 1% Alcian blue (pH 2.5) and counterstained for 3 min with 0.1% safranin O.

For degranulation analysis, 106 peritoneal cells were incubated for 1 h in culture medium containing 100 ng/ml SCF and 10 µg/ml anti-DNP IgE and then treated for 30 min with 100 ng/ml DNP-BSA as an Ag and 4 µM 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoryl-L-serine (lyso-PS; Avanti Polar Lipids) as a cofactor in the same buffer. The contents of beta-hexosaminidase (beta-HEX) in both supernatants and cell pellets were then measured by triplicate. The percentage releases were calculated using the formula [S/(S + P)] x 100, where S and P are the beta-HEX contents of the supernatants and cell pellets, respectively, from each sample. beta-HEX assay was performed as described previously (35).

Analyses of protease activities

The chromogenic peptide substrates S-2586 (3-carbomethoxypropionyl-L-arginyl-L-propyl-L-tyrosine-p-nitroaniline) and S-2288 (H-D-isoleucyl-L-propyl-arginine-p-nitroaniline) were purchased from Chromogenix, and M-2245 (N-(4-methoxyphenylazoformyl)-Phe-OH) from Bachem.

Ear extracts were obtained by the addition of 1 ml of PBS containing 2 M NaCl per ear, followed by homogenization using a PT3100 Polytron device (Kinematica). After homogenization, Triton X-100 was added to give a final concentration of 0.5%. The extracts were centrifuged at 10,000 x g, and 10-µl aliquots of the resultant supernatants were diluted with 90 µl of H2O followed by incubation with 20 µl of 1.8 mM solution (in H2O) of the chromogenic substrates S-2586 (for chymotrypsin-like proteases), S-2288 (for trypsin-like proteases), and M-2245 (for carboxypeptidase A (CPA)) at 37°C. Changes in absorbance at 405 nm were measured as described previously (36).

Culture of primary and maturated BMMCs

Bone marrow cells were obtained from the femurs and tibias of mice and cultured in IL-3-containing BMMC-complete medium comprising DMEM, 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 100 mM nonessential amino acids, and 5 ng/ml mouse rIL-3. Nonadherent cells were transferred to fresh IL-3-containing BMMC-complete medium at least once a week. After 4–5 wk of culture, the majority of the floating cells were confirmed to be immature mast cells as assessed by Alcian blue-positive and safranin-negative staining of their granules.

The maturation of immature BMMCs toward CTMC-like cells was described previously in detail (14). Briefly, 5 x 106 BMMCs were seeded on the subconfluent Swiss 3T3 fibroblast monolayer in 100-mm culture dishes and cocultured for 4–6 days in the presence of 50 ng/ml SCF with replacement of the medium every 2 days. The cells were trypsinized and replated, and nonadherent cells (>95% were mast cells) were collected and used for analyses. The maturation of BMMCs into CTMC-like cells was verified by staining of their granules with Alcian blue and counterstaining with safranin O.

Western blotting

BMMCs (105) were lysed in SDS-PAGE sample buffer (63 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 0.08% bromophenol blue) containing 5% 2-ME and subjected to SDS-PAGE. Proteins were subsequently blotted onto nitrocellulose membranes, followed by blocking with 5% milk powder in PBS containing 0.05% Tween 20. The membranes were incubated for 1 h with rabbit anti-mouse NDRG1 polyclonal Ab (15) followed by reprobing with mouse anti-{alpha}-tubulin mAb (Zymed Laboratories) at 1/5000 dilution in PBS with 0.05% Tween 20. Tyrosine phosphorylation of phospholipase C (PLC) {gamma}1 and {gamma}2 was determined by immunoblotting with rabbit anti-human phospho-PLC{gamma}1 (Tyr783) and {gamma}2 (Tyr1217) polyclonal Abs followed by reprobing with rabbit anti-human PLC{gamma}1 and {gamma}2 polyclonal Abs (Cell Signaling Technology) at 1/1000 dilution in PBS with 0.05% Tween 20 (37). After washing with PBS and 0.05% Tween 20, the membranes were incubated with a secondary anti-rabbit Ig Ab conjugated with HRP (Zymed) at 1/5000 dilution in PBS plus 0.05% Tween 20. After 1 h of incubation the membranes were washed extensively with PBS plus 0.05% Tween 20 followed by washing with PBS without detergent. The membranes were developed with the ECL system (PerkinElmer Life Sciences) according to the protocol provided by the manufacturer.

RT-PCR

Total RNA was extracted from BMMCs with TRIzol reagent (Invitrogen Life Technologies). First-strand cDNA synthesis was conducted using the SuperScript III reverse transcriptase kit (Invitrogen Life Technologies). Five micrograms of total RNA was used in reactions primed with oligo(dT) (12–18-mer) primer (Invitrogen Life Technologies) to obtain cDNA. Then, 1 µl of the synthesized cDNA was used as the template for the mRNA amplification reactions. The PCR amplification was performed using a GeneAmp PCR System 9600 (PerkinElmer) using a standard PCR protocol. The RT-PCR product was analyzed on a 1.5% agarose gel and visualized using ethidium bromide staining.

The primer pairs for NDRG1, NDRG2, NDRG3, and NDRG4 were described previously (32). The primer pairs were 5'-ACCACATTCTCGCCCTTACAT-3' and 5'-TCTCAGTTTCACCTCCCTCAG-3' for mouse mast cell protease (mMCP)-4; 5'-ATAACAGTCCTCCTAGGAGCC-3' and 5'-GATCCAGGGCCTGTAATGGGA-3' for mMCP-5; 5'-GCACATCAAAAGCCCACAGC-3' and 5'-TAGACAGGGGAGACAGAGGAC-3' for mMCP-6; 5'-CAGGCAGGCACAGTTATGCAA-3' and 5'-AACCCAGTCTAAGGAAGACC-3' for mMC-CPA3; 5'-ATGGAGACCCCATTGCTCTGA-3' and 5'-ATGATCTCCATTGAGGCTGCC-3' for N-deacetylase/N-sulfotransferase-2; 5'-CAGCTAGTTGTCAATCCTGCTCTTC-3' and 5'-GGTGCAGCTTATCGATGAATCCAG-3' for IL-4; 5'-CAGAGGATACCACTCCCAACAGAC-3' and 5'-CCTTAGCCACTCCTTCTGTGACTC-3' for IL-6; 5'-GAAAGCATGATCCGCGACGTGGAA-3' and 5'-GCTGACGGTGTGGGTGAGGAGCAC-3' for TNF-{alpha}; and 5'-TCGTGGATCTGACGTGCCGCCTG-3' and 5'-CACCACCCTGTTGCTGTAGCCGTAT-3' for GAPDH.

Flow cytometry

Suspensions of 106 BMMCs were treated with rat anti-mouse CD16/CD32 (Fc{gamma}RII/RIII) mAb (clone 2.4G2, BD Biosciences Pharmingen) (final concentration, 10 µg/ml) in 2% FBS-PBS for 10 min on ice to block cell surface Fc{gamma}Rs, followed by incubation for 45 min with FITC-labeled rat anti-mouse CD117 (c-kit) mAb (clone 2B8, BD Biosciences) at 1/50 dilution in the same buffer. To assess Fc{epsilon}RI expression, the cells were treated for 3 h with 10 µg/ml mouse IgE (SPE-7). Fc{gamma}Rs were blocked as described above and the cells were subsequently incubated with PE-labeled rat anti-mouse IgE mAb (clone 23G3; eBioscience) at 1/50 dilution in 2% FBS-PBS. Flow cytometry was conducted on a FACSCalibur instrument (BD Biosciences).

Activation of BMMCs

For stimulation with IgE plus polyvalent Ag (DNP-BSA), BMMCs (107 cells/ml) before and after coculture with fibroblasts were sensitized with 100 ng/ml anti-DNP IgE for 1 h at 37°C. After washing with medium, the cells were stimulated for appropriate periods with various doses of DNP-BSA at 37°C. For stimulation with compound 48/80, the cells were treated for the appropriate periods with various doses of compound 48/80 at 37°C. The resulting supernatants and cell pellets were then taken for a beta-HEX assay. The cysteinyl leukotriene (LT) C4 (LTC4) production was determined using enzyme immunoassay kits according to the manufacturer’s instructions (Cayman Chemical). Replicate cells were subjected to RNA extraction followed by RT-PCR for several cytokines, as noted above.

Statistical analysis

Results from in vivo and ex vivo experiments (mean ± SEM) were assessed with Student’s unpaired, two-tailed t test. Differences between replicate Ndrg1+/+ and Ndrg1–/– groups were regarded as significant at p < 0.05, unless otherwise stated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Impaired mast cell-associated anaphylactic reactions in Ndrg1-deficient mice

Anaphylaxis represents an extreme form of mast cell-associated allergic reaction consisting of a sensitization phase in which allergen-specific IgE is produced and binds to mast cell surfaces and a subsequent acute effector phase in which allergen-induced activation of mast cells leads to the release of copious amounts of vasoactive amines and other inflammatory mediators (38). To clarify the in vivo role of NDRG1 in mast cell biology, we first examined the mast cell-dependent, IgE-mediated passive systemic anaphylactic reaction in Ndrg1-deficient mice.

Ndrg1+/+ and Ndrg1–/– mice were sensitized with IgE directed against DNP and challenged 24 h later with DNP-HSA as an Ag, and their rectal temperatures were monitored at regular intervals. As shown in Fig. 1A, a temporary decrease in rectal temperature was observed in Ndrg1+/+ mice 10–20 min after antigenic challenge, whereas such a decrease was virtually absent from mast cell-deficient W/Wv mice. Notably, although a significant decrease in rectal temperature following antigenic challenge was observed in Ndrg1–/– mice, it was modest in comparison with that in Ndrg1+/+ mice. Serum samples were taken from these mice 1.5 min after Ag challenge for the determination of histamine concentration. As shown in Fig. 1B, Ndrg1+/+ mice showed a marked increase in the serum histamine level upon the cross-linking of Fc{epsilon}RI, whereas no appreciable increase was evident in W/Wv mice. Although there was a significant increase of the serum histamine level in Ag-challenged Ndrg1–/– mice compared with that in W/Wv mice, it reached a mean value of only 37% relative to that in replicate Ndrg1+/+ mice (21.1 ± 1.6 vs 7.9 ± 1.6 nM in Ndrg1+/+ and Ndrg1–/– mice, respectively; p < 0.01, n = 11).


Figure 1
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  		FIGURE 1. Effects of NDRG1 deficiency on IgE-mediated, Ag-dependent passive systemic anaphylaxis as assessed by rectal temperature change and serum histamine concentration. A, Ndrg1+/+ (circles; n = 13), Ndrg1–/– (squares; n = 13), and WBB6F1-W/Wv (W/Wv, triangles; n = 3) mice were sensitized with anti-DNP IgE and challenged with DNP-HSA to induce systemic anaphylaxis as described in Materials and Methods. Passive systemic anaphylactic response was monitored by measuring rectal temperatures at the indicated times after antigenic challenge. B, The histamine concentrations in sera from individual Ndrg1+/+ (+/+, n = 11), Ndrg1–/– (–/–, n = 11), and WBB6F1-W/Wv (W/Wv, n = 3) mice after Ag challenge for 30 min (filled symbols) or no stimulation (open symbols) were plotted, with the mean value for each group indicated by a line. *, p < 0.01 vs Ndrg1+/+; and **, p < 0.01 vs Ndrg1–/– mice.

 
To further assess the role of NDRG1 in anaphylaxis, we next investigated the capacity of Ndrg1-deficient mice to respond to immune challenge in PCA, where local extravasation and tissue swelling are induced by the local application of Ag-specific IgE followed by i.v. challenge of Ag. The ears of Ndrg1+/+ and Ndrg–/– mice were injected intradermally with DNP-specific IgE and challenged 24 h later with an i.v. injection of DNP-HSA together with Evans blue dye. Mediators released upon mast cell activation increase vascular permeability, which allows the dye to leak from blood vessels into the surrounding tissue, causing edema and providing a measure of mast cell activation.

As shown in Fig. 2A, marked extravasation of Evans blue was seen in the ears of Ndrg1+/+ mice 30 min after Ag challenge, whereas the ears of replicate Ndrg1–/– mice exhibited markedly less extravasation of the dye. Consistently, when tissue swelling of the left and right ears was measured with a dial thickness gauge, the change in ear thickness of Ndrg1–/– mice was as little as 40% compared with that of Ndrg1+/+ mice (61.7 ± 6.7 vs 24.7 ± 4.3 µm in Ndrg1+/+ and Ndrg1–/– mice, respectively; p < 0.01, n = 15) (Fig. 2B). To evaluate the extravasation of Evans blue quantitatively, the ears were removed and the OD of the extracted dye was measured as a function of Ag dose (Fig. 2C) or time (Fig. 2D). We observed that Evans blue extravasation was lower in Ndrg1–/– mice than in replicate Ndrg1+/+ mice at all Ag doses (Fig. 2C) and all time points (Fig. 2D), even though dye extravasation was still evident in Ndrg1–/– mice. Furthermore, when these mice were intradermally administered compound 48/80, a polycationic IgE-independent mast cell secretogogue, a result similar to the IgE-associated passive immune response was obtained. Thus, the extravasation of Evans blue was only modest in Ndrg1–/– mice treated with compound 48/80 relative to that in replicate Ndrg1+/+ mice (Fig. 2E). In mast cell-deficient W/Wv mice there was minimal dye extravasation under all conditions tested (Fig. 2, B–E). Collectively, these results indicate that NDRG1 deficiency causes a significant reduction of mast cell-associated passive systemic and local cutaneous anaphylactic reactions.


Figure 2
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  		FIGURE 2. Effects of NDRG1 deficiency on PCA as assessed by ear swelling and Evans blue extravasation. Ndrg1+/+ (+/+), Ndrg1–/– (–/–), and WBB6F1-W/Wv (W/Wv) mice were sensitized with anti-DNP IgE and challenged 24 h later with DNP-HSA (A–D) or directly treated with compound 48/80 (E) together with Evans blue to induce PCA as described in Materials and Methods. A, Dye extravasation following administration of 60 µg of Ag was visualized by blue staining at the injection sites in the ears of Ndrg1+/+ and Ndrg1–/– mice. Photographs of the mice were taken 30 min after Ag challenge. One representative of 11 mice for each genotype is shown. B, Ear thickness of Ndrg1+/+ (n = 15), Ndrg1–/– (n = 15), and W/Wv (n = 6) mice was measured before and 30 min after Ag challenge using a dial thickness gauge. Ear thickness change, which represents the degree of ear swelling, was determined as the difference before and after Ag challenge into the right and left ears of each mouse. **, p < 0.01; and *, p < 0.05. C, Dose-dependent extravasation of Evans blue into the ears of mice following IgE-mediated, Ag-dependent PCA. Ears of the IgE-sensitized Ndrg1+/+ (open bars), Ndrg1–/– (filled bars), and W/Wv (gray bars) mice were challenged with 0 (n = 3), 0.6 (n = 9), 6 (n = 10), and 60 (n = 18) µg of DNP-HSA for 30 min. Extravasation of Evans blue was quantified as described in Materials and Methods. **, p < 0.01 and *, p < 0.05. D, Kinetics of extravasation of Evans blue into the ears of mice following IgE-mediated, Ag-dependent PCA. Ears of the IgE-sensitized Ndrg1+/+ (circles, n = 18, respectively), Ndrg1–/– (squares; n = 13, respectively), and W/Wv (triangles; n = 4, respectively) mice were challenged with 60 µg of DNP-HSA for the indicated periods. *, p < 0.01; and **, p < 0.05 vs Ndrg1+/+ mice. ***, p < 0.05 vs Ndrg1–/– mice. E, Dose-dependent extravasation of Evans blue into the ears of mice following compound 48/80-induced PCA. The left and right ears of Ndrg1+/+ (open bars), Ndrg1–/– (filled bars), and W/Wv (gray bars) mice were treated with 0 (n = 3), 2.5 (n = 10), 25 (n = 10), and 250 (n = 10) ng of compound 48/80 for 30 min. **, p < 0.01, and *, p < 0.05.

 
Histochemical and functional analyses of mast cells in Ndrg1-deficient mice

Because NDRG1 is widely expressed in many tissues/cells, it was still not apparent whether the diminished anaphylaxis reactions could be due to a defect in mast cells or other cell types, such as endothelial cells (39). To investigate the underlying mechanism(s) responsible for the reduced allergic reactions in Ndrg1-deficient mice, we next examined mast cells in the ear skin of wild-type and Ndrg1-deficient mice histochemically during the IgE-mediated, Ag-dependent PCA. Ear sections of Ndrg1+/+ and Ndrg1–/– mice with or without Ag challenge were stained with toluidine blue to quantify degranulated tissue mast cells by light microscopy. In the absence of Ag challenge (no stimulation), the ear skin of Ndrg1–/– mice contained 37.8% fewer intact toluidine blue-positive mast cells than that of Ndrg1+/+ mice (Fig. 3A, a and b, and Table I). After challenge with Ag for 2 min, the ear skin sections of Ndrg1+/+ and Ndrg1–/– mice contained 18.8 and 2.8% degranulated mast cells relative to their respective total mast cells, revealing ~85% less degranulation in the null mice than in the control mice (Fig. 3A, c and d, and Table I). These observations suggest that the number of ear skin mast cells is reduced and that the degranulation efficacy of these cells is markedly impaired as a result of NDRG1 deficiency.


Figure 3
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  		FIGURE 3. Histological and ultrastructural features of mast cells in the ear skin of wild-type and Ndrg1-deficient mice before and after Ag challenge. A, Ndrg1+/+ (+/+) and Ndrg1–/– (–/–) mice were subjected to IgE-mediated, Ag-dependent PCA as described in Materials and Methods. Before and 2 min after Ag challenge the ear sections were stained with toluidine blue and examined by light microscopy. Photographs of Ndrg1+/+ (a and c) and Ndrg1–/– (b and d) tissue sections before (a and b; n = 20 for each) and after (c and d; n = 10 for each) Ag challenge are shown. Yellow and red arrows indicate intact and degranulated tissue mast cells, respectively. Black arrows indicate chromocytes. Magnified views of a single mast cell for each are shown in the insets (a–d). Bar, 10 µm. B, Transmission electron microscopy (lead citrate staining) of ear skin mast cells in Ndrg1+/+ (a, b, and e) and Ndrg1–/– (c, d, and f–h) mice before (a–d) and after (e–h) Ag challenge. Bar, 2 µm.

 

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  		Table I. Quantification of ear skin mast cells in Ndrg1+/+ and Ndrg1–/– micea

 
To further corroborate the abnormal histochemical aspects of ear skin mast cells, we next examined their ultrastructural features by transmission electron microscopy after 2 min of stimulation with Ag in comparison with those of unstimulated cells. As shown in Fig. 3B, there were obvious and significant differences in secretory granule and cell surface morphology between Ndrg1+/+ and Ndrg1–/– ear mast cells. Intact mast cells in Ndrg1+/+ mice were oval with regular short processes and had many secretory granules filled with electron-lucent and dense contents (Fig. 3B, a and b). Relative to the mast cells in Ndrg1+/+ mice, those in Ndrg1–/– mice had unusual granules that were small and irregular in size (Fig. 3B, c and d), suggesting immaturity. Two minutes after antigenic challenge the ear skin mast cells in Ndrg1+/+ mice possessed swollen granules that exhibited a loss of crystalline materials and decreased electron density (Fig. 3Be). The fusion of swollen granules had resulted in the formation of large vacuolar degranulation channels continuous with the plasma membrane and, thereby, the appearance of large surface pores. Massive exocytosis, demonstrated by the extrusion of flocculent matrix materials through the surface pores into the extracellular space, was regularly observed in Ndrg1+/+ mast cells (Fig. 3Be). In contrast, these Ag-induced morphological changes were scarcely observed in Ndrg1–/– mast cells (Fig. 3B, f, g, and h). Thus, it is likely that the reduced PCA response in Ndrg1-deficient mice (Fig. 2) resulted from abnormalities in the maturation and exocytosis of mast cells in the ear skin.

We then examined the morphology and ex vivo function of PMCs isolated from wild-type and Ndrg1-deficient mice. The immunostaining of cytospun PMCs with the anti-NDRG1 Ab showed intense immunoreactivity in Ndrg1+/+ but not Ndrg1–/– PMCs (Fig. 4A, a and b), confirming the specificity of the Ab. Interestingly, NDRG1 immunoreactivity in Ndrg1+/+ PMCs was largely associated with compact granular structures (Fig. 4Aa), a staining pattern similar to that of replicate cells treated with safranin O (Fig. 4Ac), which stains heparin-containing serglycin proteoglycan in secretory granules. Considering that NDRG1 is a cytosolic protein, this result may indicate that NDRG1 is located in close contact with the cytosolic surface of secretory granules. The fact that the granules in Ndrg1+/+ and Ndrg1–/– PMCs were equally safranin-positive (Fig. 4A, c and d) suggests that NDRG1 deficiency does not affect heparin synthesis. Notably, safranin staining of cytospin preparations of peritoneal cells demonstrated that Ndrg1–/– mice contained 53.4% fewer safranin-positive PMCs than Ndrg1+/+ mice (4885 ± 492 vs 2280 ± 225 per 106 peritoneal cells in Ndrg1+/+ and Ndrg1–/– mice, respectively (p < 0.01), in 15 independent cytospin preparations). Hence, Ndrg1-deficient mice contain significantly less CTMC-type mast cells (both in the ear (Fig. 3) and the peritoneum (Fig. 4)) than do wild-type mice. We then compared IgE/Ag-dependent degranulation of PMCs from Ndrg1+/+ and Ndrg1–/– mice by measuring the release level of the extracellular activity of beta-HEX, a marker enzyme for histamine-containing granules. Because IgE/Ag-dependent activation of rodent PMCs is greatly augmented by lyso-PS (40, 41, 42), PMCs sensitized with anti-DNP IgE were stimulated by Fc{epsilon}RI cross-linking with DNP-BSA as an Ag in the presence of lyso-PS as a cofactor. As shown in Fig. 4B, PMCs from Ndrg1–/– mice displayed significantly less release of beta-HEX than those from Ndrg1+/+ mice in response to IgE/Ag plus lyso-PS, whereas the basal release levels of these mediators were indistinguishable between the two genotypes.


Figure 4
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  		FIGURE 4. Morphology and exocytotic response of PMCs from wild-type and Ndrg1-deficient mice. A, Immunohistochemistry of NDRG1 in PMCs from Ndrg1+/+ (a, +/+) and Ndrg1–/– (b, –/–) mice. In each case the cells were incubated with an anti-NDRG1 Ab and then with a HRP-conjugated anti-goat Ig. Other sets of PMC preparations from Ndrg1+/+ (c) and Ndrg1–/– (d) mice were stained with safranin O. Representative data from five independent experiments are shown. B, The exocytotic response was determined by measuring the release of beta-HEX. PMCs (106) from Ndrg1+/+ (open bars) and Ndrg1–/– (filled bars) mice were sensitized with anti-DNP IgE and stimulated with DNP-BSA plus lyso-PS as described in Materials and Methods. beta-HEX enzymatic activity (n = 6) was measured in supernatants and cell pellets solubilized with 0.5% Triton-X-100 in PIPES-buffered saline. *, p < 0.01. C, Transmission electron microscopy of PMCs prepared from Ndrg1+/+ (a, b, e, and f) and Ndrg1–/– (c, d, g, and h) mice before (a–d) and after (e–h) exo vivo stimulation. Bar, 1 µm.

 
Ultrastructural analyses under electron microscopy revealed that Ndrg1–/– mice contained a population of PMCs that looked similar to Ndrg1+/+ PMCs (Fig. 4C, a and b for Ndrg1+/+ and c for Ndrg1–/–) and another population with fewer and irregular granules (Fig. 4Cd). After IgE/Ag (plus lyso-PS) stimulation, Ndrg1+/+ PMCs were well degranulated (Fig. 4C, e and f), whereas a large portion of intact granules still remained in Ndrg1–/– PMCs (Fig. 4C, g and h). Thus, these functional and morphological studies imply that NDRG1 deficiency causes a reduced Fc{epsilon}RI-mediated exocytotic response of CTMCs both in vivo (skin mast cells; Fig. 3) and ex vivo (PMCs; Fig. 4).

To further assess the differences in secretory granules between Ndrg1+/+ and Ndrg1–/– mast cells, we measured the content of histamine and the activity of mast cell-associated proteases in homogenates of ears or peritoneal cells from both genotypes. Histamine levels and chymase-, typtase-, and CPA-like protease activities were pretty low in the ears (Fig. 5, Aa and B, a–c) and peritoneal cells (Fig. 5Ab) of mast cell-deficient W/Wv mice, confirming that mast cells are the main source of these granule components. As shown in Fig. 5A, histamine levels in the ears (a) and peritoneal cells (b) of Ndrg1+/+ and Ndrg1–/– mice were comparable. Given that the number of mast cells was decreased by half in both ears and peritoneal cavities by Ndrg1 deficiency (see above), histamine content per PMC was estimated to nearly double in Ndrg1–/– over Ndrg1+/+ mice (Fig. 5A, c and d). Measurement of chymase-like (Fig. 5Ba), tryptase-like (Fig. 5Bb), and CPA (Fig. 5Bc) activities in the ears showed that the tryptase-like, but not chymase-like and CPA, activity was significantly reduced in Ndrg1–/– mice compared with that in Ndrg1+/+ mice. These results suggest that the absence of Ndrg1 alters the features of secretory granules in CTMCs.


Figure 5
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  		FIGURE 5. The storage of granule histamine and proteases in tissue mast cells of wild-type and Ndrg1-deficient mice. A, The histamine contents in ear extracts (a and c) and peritoneal cells (b and d) from Ndrg1+/+ (+/+, n = 13), Ndrg1–/– (–/–, n = 12), control WBB6F1-+/+ (Cont.W, n = 3), and WBB6F1-W/Wv (Wv, n = 3) mice. Data were shown as histamine (ng) per protein of ear tissue (a) or 106 peritoneal cells (b) and per protein of ear skin mast cells per mm2 (c) or 106 PMCs (d). **, p < 0.01; and N.S., not significant. B, Protease activities in ear tissue extracts from Ndrg1+/+ (n = 7), Ndrg1–/– (n = 8), and W/Wv (n = 5) mice. The extracts prepared from ear tissue were assayed for trypsin-like (a), chymotrypsin-like (b), and CPA (c) activities as described in Materials and Methods. Data were shown as protease activities per protein of ear tissue. *, p < 0.05; and N.S., not significant.

 
Impaired maturation and reduced exocytotic degranulation of BMMCs derived from Ndrg1-deficient mice

To further evaluate the abnormal aspects of mast cells observed in Ndrg1-deficient mice, we analyzed BMMCs of Ndrg1+/+ and Ndrg1–/– mice that were obtained after bone marrow cells were cultured for 4–5 wk in IL-3-containing medium. Nearly 95% of the cells in these cultures from both genotypes were mast cells, as they contained metachromatic granules after staining with Alcian blue (Fig. 6A, a and e) or toluidine blue (data not shown). The kinetics of cell growth and the expression of mast cell surface markers (c-kit+CD34+ or c-kit+Sca-1+ as determined by flow cytometric analysis) were also similar between the BMMCs from both sources (data not shown). Electron microscopy studies of various preparations of BMMCs from Ndrg1+/+ and Ndrg1–/– mice revealed similar cell morphology and membrane projections and the presence of morphologically distinct cytoplasmic granules, including those with internal vacuoles (type I), those with an electron-dense core surrounded by membrane vacuoles (type II), and those completely filled with the electron-dense core (type III) (Fig. 6A, c and g) (43). These results indicate that NDRG1 is not essential for IL-3-dependent development of BMMCs from bone marrow progenitor cells.


Figure 6
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  		FIGURE 6. Histological and ultrastructural features of wild-type and Ndrg1-deficient BMMCs before and after coculture with fibroblasts. A, The cytospin preparations of Ndrg1+/+ (a and b) and Ndrg1–/– (e and f) BMMCs before (a and e) and after (b and f) coculture with fibroblasts in the presence of SCF were stained by Alcian blue and safranin O. Ndrg1+/+ (c and d) and Ndrg1–/– (g and h) BMMCs before (c and g) and after (d and h) coculture with fibroblasts were further analyzed by electron microscopy to reveal their ultrastructures. Typical types I, II, and III granules in BMMCs are shown by arrows. B, Expression of NDRG1 protein in Ndrg1+/+ (+/+) and Ndrg1–/– (–/–) BMMCs before and after coculture with fibroblasts as assessed by Western blotting with anti-NDRG1 Ab. Blotting with {alpha}-tubulin was also performed to confirm the equal sample loading. Representative results for BMMCs from Ndrg1+/+ and Ndrg1–/– mice are shown. C, mRNA expression of NDRG family members. RT-PCR analysis was performed on total RNA samples from Ndrg1+/+ and Ndrg1–/– BMMCs before and after coculture for the indicated periods to detect transcripts for NDRG1 (23 cycles of amplification), NDRG2 (32 cycles), NDRG3 (28 cycles), and NDRG4 (35 cycles). Expression of GAPDH was examined as an internal control (23 cycles).

 
We next examined whether the loss of NDRG1 would have some impact on the maturation of immature BMMCs toward CTMC-like mast cells. As reported previously, a coculture of BMMCs with Swiss 3T3 fibroblasts in the presence of SCF facilitates morphological and functional maturation toward a CTMC-like phenotype (14), and NDRG1 is an early inducible protein in this process (15). Therefore, as expected, NDRG1 protein was minimally expressed before coculture and was markedly induced after coculture in Ndrg1+/+ BMMCs, whereas it was undetectable in Ndrg1–/– BMMCs irrespective of coculture (Fig. 6B). When Ndrg1+/+ and Ndrg1–/– BMMCs after coculture with fibroblasts were counterstained with safranin O, the latter appeared less granulated than the former (Fig. 6A, b and f). Indeed, electron microscopy showed that, in contrast with cocultured Ndrg1+/+ BMMCs in which type III secretory granules were well organized (Fig. 6Ad), replicate Ndrg1–/– BMMCs had mainly type I and type II secretory granules that were small and irregular in size and were partially unfilled with electron-lucent and dense contents (Fig. 6Ah). Moreover, the number of Ndrg1+/+ BMMCs increased ~3-fold after 4 days of coculture, whereas replicate Ndrg1–/– BMMCs grew slower (2.9 ± 0.2 vs 2.2 ± 0.1 -fold for Ndrg1+/+ and Ndrg1–/– BMMCs, respectively; p < 0.01; n = 13), suggesting that Ndrg1 deficiency has a propensity to reduce the expansion of mast cells interacting with fibroblasts.

We next performed RT-PCR using RNA samples from BMMCs of Ndrg1+/+ and Ndrg1–/– mice to compare the expression patterns of all NDRG members. Consistent with our previous report (15), NDRG1 mRNA was weakly expressed before coculture and was highly induced as early as 1 h after the start of coculture in wild-type BMMCs, whereas it was not detected at all in Ndrg1-deficient BMMCs (Fig. 6C). We found that NDRG2, NDRG3, and NDRG4 mRNAs were also expressed in both Ndrg1+/+ and Ndrg1–/– BMMCs (Fig. 6C). In contrast to the marked inducibility of NDRG1, expressions of NDRG2, NDRG3, and NDRG4 were nearly constant throughout the experimental period. Judging from the optimized PCR cycles, the expression level of NDRG1 was >30-fold higher than that of NDRG3 and even more than that of NDRG2 and NDRG4 in wild-type BMMCs after coculture.

Next, we compared the granule release from Ndrg1+/+ and Ndrg1–/– BMMCs before and after coculture with fibroblasts by measuring the extracellular activity of beta-HEX. BMMCs were incubated with DNP-specific IgE and subsequently stimulated by Fc{epsilon}RI cross-linking with DNP-BSA. In dose-related (Fig. 7, A and C) and kinetic (Fig. 7, B and D) granule release responses, Ndrg1–/– BMMCs after coculture displayed significantly less exocytosis than did replicate Ndrg1+/+ BMMCs (Fig. 7, C and D), whereas the responses of IL-3-maintained immature BMMCs derived from both genotypes were comparable (Fig. 7, A and B). It was noteworthy that Ndrg1+/+ BMMCs after coculture released more beta-HEX than before coculture, whereas this increased exocytosis after coculture was almost absent from Ndrg1–/– BMMCs. The total amount of beta-HEX per cell did not differ between Ndrg1+/+ and Ndrg1–/– BMMCs before and after coculture, and treatment with IgE alone did not induce granule release from either genotype (data not shown).


Figure 7
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  		FIGURE 7. The exocytotic response of wild-type and Ndrg1-deficient BMMCs before and after coculture with fibroblasts as assessed by release of beta-HEX. Ndrg1+/+ (+/+, open symbols) and Ndrg1–/– (–/–, filled symbols) BMMCs before (A and B) and after (C and D) coculture with fibroblasts were preloaded with anti-DNP IgE and stimulated with the indicated concentrations of DNP-BSA (A and C) and with 10 ng/ml DNP-BSA for the indicated periods (B and D). Alternatively, Ndrg1+/+ and Ndrg1–/– BMMCs after coculture with fibroblasts were stimulated with the indicated concentrations of compound 48/80 (E) and 10 µg/ml compound 48/80 for the indicated periods (F). Data shown are the mean ± SEM of at least six independent experiments with triplicate samples at each point. *, p < 0.05; and **, p < 0.01 vs Ndrg1+/+ BMMCs.

 
Because the coculture of BMMCs with fibroblasts led to the acquisition of responsiveness to compound 48/80 (14), we next compared the responses of Ndrg1+/+ and Ndrg1–/– BMMCs to this Gi-coupled polycationic secretagogue after coculture with fibroblasts. The stimulation of cocultured Ndrg1+/+ BMMCs with compound 48/80 resulted in marked exocytosis, whereas that of replicate Ndrg1–/– BMMCs provided partial albeit significant (~50% reduction as compared with Ndrg1+/+ BMMCs) responses at all doses (Fig. 7E) and times (Fig. 7F) tested. Furthermore, beta-HEX release in response to the Ca2+ ionophore (ionomycin) was also substantially reduced in Ndrg1–/– BMMCs relative to Ndrg1+/+ BMMCs after coculture (61.0 and 41.7% release in Ndrg1+/+ and Ndrg1–/– BMMCs, respectively, at 1 µM ionomycin). Collectively, these observations suggest that Ndrg1-deficient BMMCs after coculture with fibroblasts in the presence of SCF are functionally less mature than replicate wild-type BMMCs.

To gain further insights into the attenuated exocytotic response of Ndrg1–/– BMMCs after coculture with fibroblasts, we next examined several parameters of mast cell activation following Fc{epsilon}RI signaling. When the expression of mast cell surface markers in Ndrg1+/+ and Ndrg1–/– BMMCs before coculture was monitored by flow cytometric analysis, markers of BMMCs, including c-kit (Fig. 8A, a and b) and IgE-bound Fc{epsilon}RI (Fig. 8A, e and f) were equally expressed in cells of both genotypes. After coculture with fibroblasts, Ndrg1+/+ and Ndrg1–/– BMMCs still expressed similar levels of c-kit (Fig. 8A, c and d). Remarkably, the expression of Fc{epsilon}RI was elevated in cocultured Ndrg1+/+ BMMCs (Fig. 8Ag), whereas this increase was negligible in replicate Ndrg1–/– BMMCs (Fig. 8Ah). Accordingly, Ndrg1–/– BMMCs expressed a lower level of Fc{epsilon}RI than did Ndrg1+/+ BMMCs after coculture (Fig. 8A, f and h). Tyrosine phosphorylation of the {gamma} isoforms of PLC, which hydrolyze phosphatidylinositol bisphosphate to produce the second messengers inositol triphosphate and diacylglycerol, is an early post-Fc{epsilon}RI event that is subsequently linked to intracellular Ca2+ mobilization and protein kinase C activation (44, 45, 46). As shown in Fig. 8B, the phosphorylation of PLC{gamma}1 and PLC{gamma}2, which occurred within a few minutes after Ag challenge as revealed by immunoblotting with Abs specific for phosphorylated PLC{gamma} isoforms, was partially reduced in cocultured Ndrg1–/– BMMCs compared with replicate wild-type BMMCs, although the total amount of each PLC{gamma} isoform was indistinguishable between the both genotypes. These results raised the possibility that the reduced Fc{epsilon}RI-mediated exocytotic response in cocultured Ndrg1–/– BMMCs (Fig. 7) might be at least partly attributable to a defective maturation-associated elevation of Fc{epsilon}RI expression (Fig. 8A) and, thereby, reduced activation of PLC{gamma} (Fig. 8B).


Figure 8
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  		FIGURE 8. Fc{epsilon}RI signaling of wild-type and Ndrg1-deficient BMMCs after coculture with fibroblasts. A, Surface expression of c-kit and Fc{epsilon}RI in BMMCs before and after coculture with fibroblasts. Ndrg1+/+ (a, c, e, and g) and Ndrg1–/– (b, d, f, and h) BMMCs before (a, b, e, and f) and after (c, d, g, and h) coculture with fibroblasts were incubated with or without 10 µg/ml mouse IgE, and then stained with PE-labeled anti-mouse IgE Ab (e–h). c-kit expression was detected with FITC-labeled anti-mouse c-Kit Ab (a–d). Representative histograms of BMMCs from three independent experiments are shown. B, Tyrosine phosphorylation of PLC{gamma}1 or PLC{gamma}2 in BMMCs after coculture with fibroblasts. IgE-sensitized BMMCs from Ndrg1+/+ (+/+) and Ndrg1–/– (–/–) mice were stimulated with DNP-BSA for indicated times. Lysates from these cells were subjected SDS-PAGE, transferred to nitrocellulose membrane filters, and then immunoblotted with Abs against tyrosine-phosphorylated forms of PLC{gamma}1 and PLC{gamma}2 (P-PLC{gamma}1 and P-PLC{gamma}2, respectively). After stripping, the filters were reprobed with anti-PLC{gamma}1 or anti-PLC{gamma}2 Ab to determine their protein levels. C, LTC4 production by Ndrg1+/+ (open bars) and Ndrg1–/– (filled bars) BMMCs after coculture with fibroblasts. The IgE-sensitized cells were stimulated with the indicated doses of Ab for 30 min. LTC4 levels in the supernatants were measured by ELISA. N.S., not significant. D, Expression of cytokine mRNAs in BMMCs after coculture with fibroblasts. Total RNAs were extracted from cocultured Ndrg1+/+ and Ndrg1–/– following stimulation for the indicated periods with IgE and Ag and then subjected to RT-PCR for IL-4 (30 cycles), IL-6 (25 cycles), and TNF-{alpha} (25 cycles) as well as GAPDH (control; 23 cycles) using the respective specific primers.

 
However, the production of cysteinyl LTC4, an arachidonate-metabolizing product, which depends entirely on an increased intracellular Ca2+ level and MAPK (47, 48), was unaffected in Ndrg1–/– BMMCs after coculture (Fig. 8C). In addition, Fc{epsilon}RI-induced expression of several cytokines including IL-4, IL-6 and TNF-{alpha}, an event that depends on the activation of multiple signaling pathways such as protein kinase C, MAPK, NF-{kappa}B, NF-AT, or PI3K (49, 50, 51, 52, 53, 54), occurred normally in Ndrg1–/– BMMCs even after coculture (Fig. 8D). Consistently, the increase in intracellular Ca2+, an upstream event for LT and cytokine generation after Fc{epsilon}RI cross-linking, was comparable between cocultured Ndrg1+/+ and Ndrg1–/– BMMCs (data not shown). These results argue that the residual activation of PLC{gamma} is still sufficient for downstream signaling pathways, leading to full eicosanoid synthesis and cytokine expression in cocultured Ndrg1–/– BMMCs. It appears, therefore, that the reduced degranulation in Ndrg1–/– BMMCs may have resulted from a regulatory step(s) other than the receptor-proximal events.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Recent reports that overexpression or knockdown of NDRG1 in cultured neoplastic cells alters their proliferation, differentiation, metastasis, and apoptosis statuses (25, 28, 29, 55, 56, 57, 58) and that genetic mutations in the Ndrg1 gene cause Schwann cell dysfunction leading to peripheral neuropathy in both human and mouse (30, 31, 32) imply that this inducible intracellular protein plays roles in diverse processes linked to these cellular events. Despite its widespread distribution, however, the regulatory expression and functions of NDRG1, particularly in the immune system, have been poorly understood. We previously found that NDRG1 is markedly induced during ex vivo differentiation of IL-3-dependent BMMCs, a relatively immature population of mast cells, into more mature CTMC-like cells that contain safranin-positive secretory granules, produce large amounts of PGD2, and show sensitivity to G protein-coupled polycationic secretagogues such as compound 48/80 and substance P (14, 15). Although the forcible transfection of NDRG1 into a mast cell-line augmented the exocytotic response (15), an event suggesting the potential ability of this protein to promote the functional maturation of mast cells, the physiological relevance of these observations has still remained elusive. In an effort to gain further insight into the functional roles of NDRG1 in mast cell biology, in the present study we examined mast cell-associated phenotypes of Ndrg1-deficent mice. Our results provided evidence that NDRG1 plays a pivotal role in the terminal maturation and effector function (degranulation) of mast cells in vivo and ex vivo.

Ndrg1–/– mice were partially resistant to passive systemic anaphylaxis, displaying only modest changes in rectal temperature and plasma histamine level in comparison with the replicate Ndrg1+/+ littermate control (Fig. 1). Likewise, in the passive cutaneous anaphylactic response Ndrg1–/– mice exhibited attenuated extravasation (an event triggered by mast cell-derived mediators such as histamine and cysteinyl LTs) at the sites of stimuli (Fig. 2), whereas skin mast cells in the null mice showed only minimal degranulation (Fig. 3). The latter observation is supported by the ex vivo experiments showing that PMCs from Ndrg1–/– mice were less sensitive to Fc{epsilon}RI cross-linking than those from Ndrg1+/+ mice (Fig. 4B). In addition to these functional defects, dermal and serosal CTMCs of Ndrg1–/– mice contained fewer and unusual secretory granules (Figs. 3–5), suggesting their immaturity.

Studies using the culture system in which BMMCs differentiate into CTMC-like cells revealed that, although immature BMMCs maintained in IL-3 were virtually identical between the Ndrg1+/+ and Ndrg1–/– genotypes, after maturation into CTMC-like cells by coculture with fibroblasts in the presence of SCF the BMMCs showed several notable differences in terms of ultrastructure and function. Thus, as compared with the Ndrg1+/+ control, Ndrg1–/– BMMCs after coculture contained aberrant secretory granules that were small and irregular with a paucity of electron-lucent and dense contents (Fig. 6), in agreement with the altered morphology of dermal and serosal CTMCs in Ndrg1–/– mice as mentioned above (Figs. 3–5). In addition, the retarded proliferation of Ndrg1–/– BMMCs in coculture may be a reflection of reduced mast cell number in vivo. The coculture was accompanied by augmented Fc{epsilon}RI-mediated exocytosis in Ndrg1+/+ BMMCs, whereas this event did not occur appreciably in replicate Ndrg1–/– BMMCs (Fig. 7). The surface expression levels of c-kit, CD34, and Sca-1 on BMMCs were similar between the two genotypes irrespective of coculture, indicating that NDRG1 does not affect the expression of these early mast cell surface markers (data not shown). Interestingly, the expression of Fc{epsilon}RI was elevated in Ndrg1+/+ but not in Ndrg1–/– BMMCs after coculture (Fig. 8A), and this change appears to be associated with reduced PLC{gamma} phosphorylation, an Fc{epsilon}RI-proximal event, in Ndrg1–/– BMMCs (Fig. 8B). However, almost normal LT synthesis and cytokine expression in cocultured Ndrg1–/– BMMCs (Fig. 8, C and D) argues against the contribution of the moderate changes in these receptor-proximal events to the reduced exocytosis. It is also notable that the exocytotic response to compound 48/80, a Gi-coupled secretagogue to which response became apparent after coculture, as well as the response to ionomycin, a Ca2+ ionophore, was also partially reduced in cocultured Ndrg1–/– BMMCs compared with replicate Ndrg1+/+ cells (Fig. 7, E and F). These results are in agreement with our previous observation that the overexpression of NDRG1 in RBL-2H3 mastocytoma led to a marked enhancement of degranulation but not eicosanoid synthesis following various stimuli (15). Thus, it is speculated that NDRG1 plays a role in the divergent signaling at the point of convergence or beyond leading to exocytosis.

Whereas it has been reported that NDRG1 shows cytoplasmic, nuclear, and even mitochondrial localization and often shuttles between the cytoplasm and the nucleus according to cell type, stimulus, and cell cycle stage (15, 25, 39, 59, 60), in the present study we found that in mast cells NDRG1 exhibits a unique punctate distribution in the cytoplasm, particularly around secretory granules (Fig. 4). A likely explanation for this location is that NDRG1 binds to certain proteins or lipid components that are enriched in the mast cell granule membranes. Interestingly, by means of yeast two-hybrid screening we and others have recently found that NDRG1 has the potential capacity to associate with several cellular proteins, such as HSC70, PICK-1, p47, Pra1, RTN-1C, and Aip-1, all of which are known to participate in cellular events related to membrane transport and fusion (59, 60). The interrelated protein trafficking of NDRG1 binding partners points to its possible involvement in the complex network of vesicular transport. In relation to this, the blood level of high-density lipoprotein is reduced in CMT4D patients harboring the R148X mutation in the Ndrg1 gene (60), and several genetic disorders of lipid vesicular transport cause the CMT-like peripheral neuropathy that accompanies demyelination (61, 62). These facts raise the possibility that the neuropathic phenotype of Ndrg1 mutation might be due to a perturbation of lipid trafficking and membrane transport in Schwann cells. Moreover, high steady-state expression of NDRG1 is found in renal proximal tubular and intestinal epithelial cells, which actively transport vesicles with polarity from apical to basolateral membranes (39). Thus, apart from the regulatory role of NDRG1 in the terminal maturation of mast cells, the reduced exocytosis of Ndrg1-null mast cells may be indicative of an additional role of this protein in the regulation of the secretory process. Supporting this idea, our recent GeneChip analysis of Ndrg1+/+ vs Ndrg1–/– BMMCs after coculture has revealed that Ndrg1 deficiency leads to a marked reduction in the expression of a panel of genes related to cytoskeletal organization and rearrangement (data not shown) that could have a deep impact on cellular shape, cell division, membrane integrity and fusion, vesicular transport, and even exocytotic function. A possible functional link of NDRG1 with each component of those GeneChip-identified genes is now under investigation at molecular levels.

The finding that there are fewer electron-dense secretory granules in CTMCs in Ndrg1–/– mice than in Ndrg1+/+ mice is suggestive of substantial changes in the granule contents. Because >50% of the weight of a PMC consists of protease/proteoglycan complexes that are packaged in the cell granules, we evaluated for gross changes in total protease levels by using colorimetric substrates and found that tryptase-like activity was markedly diminished in Ndrg1–/– PMCs compared with littermate control PMCs (Fig. 5B). In contrast, the chymase-like and CPA activities, as well as heparin content as judged from safranin-positive staining (Figs. 4A and 6A) and N-deacetylase/N-sulfotransferase-2 expression (data not shown), appeared to be unaffected by the absence of NDRG1. Because transcripts for the four major CTMC-associated proteases, mMCP-4, mMCP-5, mMCP-6, and mMC-CPA3, did not differ significantly between Ndrg1+/+ and Ndrg1–/– mice (data not shown), selective reduction of the tryptase-like activity in Ndrg1–/– mice might be caused at posttranslational rather than transcriptional levels. Unexpectedly, Ndrg1–/– CTMCs contained more histamine than did Ndrg1+/+ CTMCs (Fig. 5A, c and d). We speculate that the reduction in physiological histamine secretion from CTMCs upon microenvironmental stimuli in Ndrg1–/– mice might eventually result in the accumulated storage of granule histamine. Nevertheless, although the regulation of individual granule components by NDRG1 needs further elucidation, at this stage we can say that NDRG1 deficiency influences several if not all granule materials, possibly as a result of the alterations in granule moiety and heterogeneity.

Although the molecular mechanisms whereby NDRG1 regulates several processes in mast cells (and possibly in other cell types) are still a subject of debate, it is intriguing to note that the NDRG1 protein has several intriguing motifs, including the phosphopantetheine-binding site, the esterase/lipase/thioesterase domain, and the three Ser/Thr-rich tandem repeats, which might be responsible for protein-protein interaction, subcellular localization, and posttranslational modification such as phosphorylation. We have recently shown that deletion of the C-terminal Ser/Thr-rich repeats abrogates the degranulation-enhancing potential of NDRG1 (15) and that NDRG1 undergoes phosphorylation of Ser and Thr residues in mast cells (63). Interestingly, a recent study has shown that Ser/Thr-rich repeats provide sites for the sequential phosphorylation by serum- and glucocorticoid-induced kinase 1 (SGK1) and then by glycogen synthase kinase 3 (GSK3) (64, 65). Thus, SGK1/GSK3-directed phosphorylation within these repeats might constitute a regulatory pathway for NDRG1-mediated signals.

In summary, this study is the first to demonstrate that Ndrg1 deficiency profoundly affects the terminal maturation and a particular effector function (degranulation), thereby affecting mast cell-associated pathological outcomes, i.e., anaphylactic responses. Unexplained is why Ndrg1–/– BMMCs before coculture already have a normal degranulation response. In this regard, it is possible that other NDRG members (NDRG2–4) that are constitutively expressed in BMMCs regardless of the coculture (Fig. 6C) might play a redundant role. Nonetheless, it is important to clarify the relevance of this study to human mast cell development and associated diseases. Interestingly, human mast cells express NDRG1 as well as NDRG2 as evidenced by Affymetrix GeneChip transcript data (66). We have also confirmed in our preliminary study that human cord blood-derived mast cells also express NDRG1 (data not shown), although the proper culture condition under which NDRG1 expression could be modified in these cells has not yet been defined. Determining the regulatory expression and function of NDRG1 (as well as other NDRG members) in human mast cells would shed further light on the importance of this family of proteins in the context of the pathology of allergy and other mast cell-associated biological events. Moreover, continued analyses of Ndrg1-null mice in combination with ongoing GeneChip analysis will expand our understanding of the unexplored roles of this unique protein in the homeostasis and disorders of the mast cell-related and -unrelated immune systems as well as in other pathogenic events including neuronal degeneration and cancer.


   Acknowledgment
 
We thank Dr. Atsushi Iwama (Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Tokyo, Japan) for help with flow cytometric analyses.


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


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

1 This work was supported by a Showa University special grant-in-aid for innovative collaborative research projects and a special research grant-in-aid for development of characteristic education from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. M.M. was supported by Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency. Back

2 Current address: Department of Immunobiology, School of Pharmaceutical Sciences, Mukogawa Women’s University, Hyogo, Japan. Back

3 Current address: COE Formation for Genomic Analysis of Disease Model Animals with Multiple Genetic Alterations, Graduate School of Medicine, Kyoto University, Kyoto, Japan. Back

4 Address correspondence and reprint requests to Dr. Ichiro Kudo, Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, Japan. E-mail address: ichi-ku{at}pharm.showa-u.ac.jp Back

5 Abbreviations used in this paper: SCF, stem cell factor; NDRG1, N-myc downstream regulated gene-1; BMMC, bone marrow-derived mast cell; CMT4D, Charcot-Marie-Tooth disease type 4D; CPA, carboxypeptidase A; CTMC, connective tissue mast cell; HSA, human serum albumin; beta-HEX, beta-hexosaminidase; LT, leukotriene; lyso-PS, lysophosphatidyl-L-serine; mMCP, mouse mast cell protease; PCA, passive cutaneous anaphylaxis; PLC, phospholipase C; PMC, peritoneal mast cell. Back

Received for publication August 9, 2006. Accepted for publication February 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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