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Department of Veterinary Clinic, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan;
Department of Pathology, Toxicology Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan; Departments of
Immunology and
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Dermatology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan; and
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Department of Veterinary Internal Medicine, Faculty of Agriculture, Tottori University, Tottori, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production of T
cells (5, 6, 7), IgE hyperproduction of B cells (8, 9), and defective
IL-12-induced IFN- production may be caused by hyperproduction of
IL-4 and IL-10 (10). Since AD is frequently associated with
elevated levels of serum IgE against many kinds of inhaled, ingested,
and/or epicutaneously encroached allergens (2), a possible involvement
of IgE Ab in the development of AD has been speculated. However,
the mechanisms of IgE hyperproduction and the pathogenesis of AD have
not been clarified.
NC/Nga mice were established as an inbred strain by Dr. K. Kondo in
1957 based on Japanese fancy mice (Nishiki-Nezumi) and has been
reported to have some biological characteristics: liver and kidney
esterase like a DBA/2 strain, high susceptibility to x-irradiation, and
high susceptibility to anaphylactic shock from OVA (11, 12). Recently,
we have demonstrated that inbred NC/Nga mice are available as an animal
model for human AD (13). When NC/Nga mice were raised in
air-uncontrolled conventional circumstances (conventional NC/Nga mice),
all the mice spontaneously suffered from AD-like skin lesions with
marked elevation in plasma levels of total IgE. In contrast, NC/Nga
mice grown in specific pathogen-free circumstances (SPF NC/Nga mice)
showed neither clinical signs nor IgE hyperproduction. The
immunohistochemical examination of the skin lesion in conventional
NC/Nga mice presented typical features of the affected skin observed in
patients with AD, such as an increased number of mast cells and
eosinophils with marked degranulation, infiltration of numerous
CD4+ T cells and macrophages, and infiltration of a few
CD8+ T cells. The activated mast cells and CD4+
T cells in the skin lesion expressed a positive reaction for IL-4 and
IL-5, but little or no reaction for IFN-. We also demonstrated a
close relationship between IgE levels and the onset or progression of
these skin lesions in NC/Nga mice, and a contribution of circumstances
to the pathogenesis of AD-like skin lesions in NC/Nga mice as well as
in human patients with AD (1, 2). Therefore, it is important for
clarifying the pathogenesis of AD to understand the mechanisms of IgE
hyperproduction.
For IgE synthesis by B cells in vivo, at least two stimuli are
necessary; IL-4 (14) and a cognate signal induced by the interaction of
CD40 on B cells and CD40 ligand (CD40L) on activated T cells (15, 16).
Defects in CD40L expression and mutations of a CD40L molecule induce
X-linked severe immuodeficiency with IgM hyperproduction in humans
(17, 18, 19, 20, 21), and the blockage of CD40/CD40L interaction with injection of
anti-CD40L mAb inhibits the Ig class switch in mice (22),
demonstrating that transient expression of CD40L on T cells may be
indispensable for the Ig class switch to IgE synthesis in vivo. In in
vitro culture systems, IL-4 (23) can induce IgE production by B cells
in the presence of CD40L (24, 25), soluble CD40L (15, 26),
anti-CD40 mAb (8, 27), anti-IgM Ab (15), LPS (15, 28, 29, 30, 31), or
hydrocortisone (32, 33). Namely, IL-4 is an essential factor for IgE
synthesis both in vivo and in vitro. IL-4 elicits its biological
effects through the specific receptor complex: the 
-chain that was
cloned as a high affinity receptor (34, 35) and the -chain that was
shared with receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (36, 37, 38, 39, 40, 41, 42).
Janus kinase 3 (JAK3), a member of Janus kinase family of nonreceptor
protein tyrosine kinases, is involved in IL-4 signalings (43). JAK3
associates with the -chain (42, 43) and is phosphorylated in the
tyrosine residue upon binding of IL-4 to the IL-4R complex on B cells
(42, 43, 44, 45). Ligation of CD40L to CD40 on B cells leads to tyrosine
phosphorylation of JAK3 as well (46).
In the present study we have demonstrated that B cells isolated from NC/Nga mice possess a higher potency for IgE synthesis, probably due to a higher sensitivity to CD40L and IL-4, compared with those of BALB/c mice that are a high responder strain in IgE production (30). The higher sensitivity of B cells of NC/Nga mice to CD40L and IL-4 was suggested to be due to enhanced activation of JAK3. Moreover, B cells of conventional NC/Nga mice with AD-like skin lesions showed constitutive phosphorylation of JAK3 comparable to those of AD patients with IgE hyperproduction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SPF NC/Nga mice (H-2d) were maintained in a filter-laminar flow enclosure in a bioclean room and were provided with autoclaved food and water ad libitum. Several mating pairs of SPF NC/Nga mice were moved to an air-uncontrolled conventional room. Skin lesions very similar to those in human AD spontaneously appeared from the age of 8 wk in all the progeny. SPF BALB/c mice were purchased from Clea Japan (Tokyo, Japan). Male mice were used at 4–12 wk of age.
Cytokines and Ab
Recombinant murine and human IL-4 were purchased from Genzyme (Cambridge, MA) and PharMingen (San Diego, CA), respectively. Murine soluble CD40L was provided by Dr. C. R. Maliszewski (Immunex, Seattle, WA) (15). Phycoerythrin (PE)-conjugated rat anti-mouse CD4 mAb, FITC-conjugated rat anti-mouse CD45R/B220 mAb, FITC-conjugated rat anti-mouse CD90 (Thy1.2) mAb, hamster anti-mouse CD40L mAb, and mouse anti-human CD40 mAb were obtained from PharMingen. Rat anti-mouse CD40 mAb and goat anti-rat IgG (H+L) F(ab')2 conjugated with magnetic beads were provided by Serotec (Oxford, U.K.) and Miltenyi Biotec (Sunnyvale, CA), respectively. FITC-conjugated goat anti-hamster IgG and PE-conjugated goat anti-rat IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Rabbit Ab against mouse JAK3 and human JAK3 were purchased from Upstate Biotechnology (Lake Placid, NY). Mouse anti-phosphotyrosine mAb and peroxidase-conjugated goat anti-rabbit IgG were provided by Sigma (St. Louis, MO) and BioMakor (Rehovot, Israel), respectively.
Immunization
Mice were injected s.c. with 0.2 ml of PBS containing 1 µg of OVA grade V (Sigma) conjugated with 4 mg of aluminum hydroxide (Boehringer Ingelheim, Heidelberg, Germany), and 2 wk later received an i.p. boost of 2.5 µg of OVA conjugated with 10 mg of aluminum hydroxide. Blood was collected from the retro-orbital plexus with glass capillary tubes and heparinized immediately. Plasma samples were stored at -20°C until quantitative analyses for total IgE.
Cell isolation
Thy1.2+ T cells, CD4+ T cells, and B220+ B cells were positively collected from spleens of NC/Nga and BALB/c mice by magnetic cell sorting according to the manufacturer’s instructions. Briefly, single cell suspensions were prepared from spleens in RPMI 1640 (Life Technologies, Grand Island, NY) containing 10% FCS (HyClone, Logan, UT), according to the method described previously (47). After treatment with ammonium chloride to deplete erythrocytes, spleen cells were resuspended in RPMI 1640 with 10% FCS at a concentration of 108 cells/ml. For isolation of CD90+ cells and B220+ cells, the cells were incubated with the specific mAb conjugated with FITC at a concentration of 25 µg/ml in PBS containing 0.5% BSA (Sigma), 5 mM EDTA, and 0.01% sodium azide (isolation buffer) for 15 min at 4°C. For CD4+ T cell isolation, before treatment with anti-CD4 mAb a T cell-rich fraction was collected from spleen cells using nylon wool columns, and then was incubated with PE-conjugated rat anti-mouse CD4 mAb at a concentration of 25 µg/ml for 15 min at 4°C. The cells treated with these primary mAb were resuspended in isolation buffer at a concentration of 108 cells/ml, mixed with a 1/5 dilution of goat anti-rat IgG (H+L) F(ab')2 conjugated with magnetic beads, and incubated for 15 min at 4°C. Labeled cells were loaded on separation columns with a magnet, and then the column was washed with ice-cold isolation buffer. The column was taken away from the magnet, and the cells carrying the relevant surface Ag were recovered by washing the column with ice-cold isolation buffer. Specimens of individual isolated cell populations were analyzed by an EPICS flow cytometer (Coulter, Hialeah, FL); the purity of the cells was >90%. There were no significant differences in numbers of Thy1.2+ T cells, CD4+ T cells, CD8+ T cells, or B220+ B cells in spleens between BALB/c and NC/Nga mice.
Flow cytometric analysis
To examine the expression of CD40L on CD4+ T cells and CD40 on the B220+ cells, double immunofluorescence staining was performed. CD4+ T cells were stimulated with PMA (10 ng/ml; Sigma) and ionomycin (0.25 µM; Sigma) for 4, 6, 8, and 24 h (48, 49) in RPMI 1640 supplemented with 10% FCS, 10-4 M 2-ME, 50 U/ml penicillin, and 50 µg/ml streptomycin at a concentration of 106 cells/ml in the 24-well culture plate (Nunc, Roskilde, Denmark). Freshly prepared or stimulated CD4+ T cells were harvested and incubated with hamster anti-mouse CD40L mAb (1 µg/ml) and PE-conjugated rat anti-mouse CD4 mAb (1 µg/ml) at a concentration of 107 cells/ml in 100 µl of PBS containing 2% FCS and 0.1% sodium azide for 1 h at 4°C. Cell preparations were then stained with FITC-conjugated goat anti-hamster IgG for 30 min at 4°C. To determine CD40 expression on the B220+ B cells, Thy1.2- cells were stimulated with IL-4 (200 U/ml) for 24, 48, and 72 h at a concentration of 106 cells/ml in the 24-well culture plate. Freshly prepared or stimulated B220+ B cells were treated with rat anti-mouse CD40 mAb (50 µg/ml) for 1 h at 4°C, and were then stained with PE-conjugated goat anti-rat IgG for 30 min at 4°C at a concentration of 107 cells/ml. After washing three times, the cells were reincubated with FITC-conjugated rat anti-mouse B220 mAb for 1 h at 4°C. The cell preparations were analyzed by a flow cytometer shortly after the double staining.
Cell culture
IgE synthesis of B cells was analyzed by an in vitro experimental system with T cell-B cell collaboration (25). CD4+ T cells activated with PMA and ionomycin (see above) were fixed in 1% paraformaldehyde for 10 min at 4°C. A total of 105 B220+ B cells isolated from spleens of SPF NC/Nga and BALB/c mice were cultured in 200 µl of RPMI 1640 supplemented with 10% FCS, 10-4 M 2-ME, 50 U/ml penicillin, and 50 µg/ml streptomycin together with 2 x 105 fixed CD4+ T cells in the presence of 200 U/ml murine IL-4 in the 96-well flat-bottom culture plate (Nunc) for 9 days at 37°C in a humidified atmosphere flushed with 5% CO2 in air. In experiments to determine a potency for IgE production and the sensitivity to IL-4, splenic B cells were incubated with various doses of IL-4 in the presence of soluble CD40L or LPS (from Salmonella typhimurium; Sigma). The culture supernatants were collected and stored at -20°C until quantitative analyses for total IgE. To assay cytokine productivity of T cells, 2 x 106 Thy1.2+ T cells were incubated in 1 ml of RPMI 1640 with 5 µg/ml Con A, FCS, 2-ME, and antibiotics in the 24-well culture plate at 37°C in a humidified atmosphere flushed with 5% CO2. Three days later the culture supernatants were collected and stored at -20°C until use.
Isolation of human B cells
Human B cells were isolated from PBMC from four patients with AD and four normal healthy volunteers after obtaining their informed consents. All the patients had elevated levels of serum total IgE (7,330–12,680 U/ml), and the healthy donors had no history of allergic diseases. PBMC were separated from heparinized venous blood by density gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway). CD19+ cells were isolated from PBMC using Dynabeads (Dynal, Oslo, Norway) according to the manufacturer’s instructions. To remove the magnetic beads from B cells, the cells with the beads were incubated with DETACHABeads (Dynal) for 60 min at room temperature. More than 98% cells were CD20 positive as assessed by flow cytometric analysis.
Measurement of total IgE and IgG1
IgE levels were measured by a sandwich ELISA using two kinds of rat anti-mouse IgE mAb (6HD5 and HMK12) according to a previously described method (50). 6HD5 mAb recognizes different epitopes of Fc fragments of IgE that are unrecognized by HMK12 mAb. Briefly, immunoplates (Maxi-sorp, Nunc) were coated with 50 µl of 6HD5 mAb (5 µg/ml) and blocked with PBS supplemented with 1% BSA. Collected samples or standard mouse IgE (SPE7, Seikagaku Kogyo, Tokyo, Japan) were added to the wells and incubated for 1 h at room temperature. After washing with PBS containing 0.05% Tween-20, 50 µl of biotinylated HMK12 mAb (1 µg/ml) was added, and the plates were incubated for 1 h, then 50 µl peroxidase-conjugated avidin (1/2000; Dakopatts, Glostrup, Denmark) was added. After incubation for 1 h, the reaction products were visualized with 0.4 mg/ml orthophenylenediamine and 0.012% H2O2. IgG1 levels were measured by a sandwich ELISA using two kinds of rat anti-mouse IgG1 mAb (PharMingen) according to the process stated above. The absorbance at 490 nm wavelength was measured by an ImmunoMini (NJ-2300, Nippon Intermed, Tokyo, Japan). The sensitivity of this assay was 2 ng/ml.
Cytokine assay
Murine IL-4 levels were measured by an ELISA kit provided by BioSource (Camarillo, CA) according to the manufacturer’s instructions.
Western blot analysis of tyrosine-phosphorylated JAK3
Tyrosine-phosphorylated JAK3 was detected as described previously (43). Briefly, 2 x 107 B220+ B cells isolated from NC/Nga and BALB/c mice were incubated with 100 ng/ml soluble CD40L and/or 200 U/ml murine IL-4 for 10 min at a concentration of 2 x 106 cells/ml. The cells were harvested and lysed in 1 ml of ice-cold lysis buffer (50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM NaCl, 100 mM sodium orthovanadate, 1 mM PMSF, and 1 mM EDTA) for 30 min. The lysates were centrifuged at 10,000 x g for 30 min at 4°C, and supernatants were immunoprecipitated with anti-phosphotyrosine mAb (1/200) and protein A-Sepharose CL-4B (50% suspension equilibrated with lysis buffer; Pharmacia, Uppsala, Sweden) for 12 h at 4°C under gentle rotation. The immunoprecipitates were washed four times with 1 ml of cold lysis buffer, resuspended in Laemmli’s sample buffer (50 mM Tris-HCl (pH 6.8), 10% glycerol, 1% SDS, 0.1% bromophenol blue, and 1 mM DTT), and boiled for 5 min. The samples were then subjected to 7.5% SDS-PAGE, and the resolved samples were transferred electrophoretically to Immobilon-P membranes (Millipore, Bedford, MA). The membranes were immunoblotted with rabbit anti-mouse JAK3 Ab (1/1000) for 1 h, washed, and incubated with peroxidase-conjugated anti-rabbit IgG for 30 min. Tyrosine-phosphorylated JAK3 was detected with an enhanced chemiluminescent detection reagent (Amersham, Arlington Heights, IL), and the images were analyzed by Gel Print 200i/VGA and Basic Quantifier (Genomic Solutions, Ann Arbor, MI) to determine their relative intensities. For analysis of JAK3 phosphorylation in human B cells, 5 x 106 B cells isolated from patients with AD and from healthy donors were incubated with 1 µg/ml anti-CD40 mAb and/or 200 U/ml human IL-4 for 15 min at a concentration of 2 x 106 cells/ml. The B cells were then treated as descried above, using rabbit anti-human JAK3 Ab.
Statistical analysis
Two-tailed Student’s t test was performed for statistical analysis of the data, and p < 0.05 was taken as the level of significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NC/Nga mice that were raised in nonsterile air-uncontrolled
conventional circumstances, started to scratch their faces, necks,
ears, and dorsal skins from the age of 8 wk, resulting in dermatitis
that consisted of eczema, erythema, hemorrhage, superficial erosion,
deep excoriation, scaling, and dryness (Fig. 1
). Grades of the dermatitis became more
severe with aging and reached a plateau at the age of 17 wk (13). This
kind of skin lesion was also observed in human patients with AD (1, 2);
histopathologic findings of the skin lesions were very similar to those
for human AD (13). Since >80% of patients with AD showed elevated
levels of serum IgE Ab (2), we determined total IgE levels in the
peripheral blood of conventional NC/Nga mice. Plasma levels of total
IgE in conventional NC/Nga mice began to be increased from the age of 8
wk with aging (Fig. 2), which had a
significant correlation with the onset and progression of dermatitis
(13). In contrast, SPF NC/Nga and BALB/c mice showed neither elevation
of IgE levels (Fig. 2) and IgG levels (data not shown) nor any clinical
sign of dermatitis (data not shown). These findings and our previous
studies suggested that IgE produced in NC/Nga mice might be involved in
both the onset and the development of AD skin lesions.
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To determine IgE production in vivo, mice were immunized with OVA
conjugated with aluminum hydroxide twice. The immunization with OVA and
aluminum hydroxide led to increased levels of total IgE in each strain
of mouse, but the levels in SPF NC/Nga mice were threefold higher than
those in BALB/c mice 2 wk after the first immunization (Table I). Two weeks after the second
immunization, levels of total IgE in SPF NC/Nga mice were markedly
increased compared with those in BALB/c mice.
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We attempted to demonstrate high productivity of B cells for IgE
in an in vitro experimental system with cognate interaction between T
and B cells. Splenic B220+ B cells of SPF NC/Nga and BALB/c
mice were incubated with 200 U/ml IL-4 and paraformaldehyde-fixed
CD4+ T cells expressing CD40L on the cell surface that had
been activated with PMA and ionomycin. As shown in Table II, both B cells of NC/Nga and BALB/c
mice incubated with the activated CD4+ T cells could
synthesize IgE in the presence of 200 U/ml IL-4. IgE levels in culture
supernatants of B cells of NC/Nga mice were higher than those in BALB/c
mice in the presence of CD4+ T cells from each mouse
strain. In contrast, no significant IgE production by B cells was
detected in the presence of the activated CD4+ T cells
alone, IL-4 alone, or nonactivated CD4+ T cells with IL-4
(data not shown). Thus, splenic B cells from SPF NC/Nga mice could
produce increased IgE regardless of the source of activated T cells.
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We examined CD40L expression on CD4+ T cells isolated
from spleens of SPF NC/Nga and BALB/c mice by flow cytometric analysis
using anti-CD40L mAb. Although CD40L was not detected on freshly
isolated CD4+ T cells of either SPF NC/Nga or BALB/c mice,
8-h incubation with PMA and ionomycin induced CD40L expression at a
maximal level, expressing very similar profiles of the fluorescence
intensity observed on CD4+ T cells from both NC/Nga and
BALB/c mice (Fig. 3A). After
8 h CD40 expression was gradually decreased with the same
reduction rate in both groups of mice. We, therefore, concluded that
the IgE hyperproduction by the B220+ B cells cocultured
with the activated CD4+ T cells could not be attributed to
the higher expression of CD40L on the activated CD4+ T
cells.
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We next examined CD40 expression on splenic B220+ B
cells of SPF NC/Nga and BALB/c mice by flow cytometric analysis using
rat anti-mouse CD40 mAb. The fluorescent intensity for CD40 on
freshly isolated B cells from NC/Nga mice was comparative to that on
cells from BALB/c mice (Fig. 3
B). Since CD40 expression on
PBMC is increased by stimulation with IL-4 (8), we investigated an
expression profile of CD40 on B cells following stimulation with IL-4.
When B220+ B cells isolated from spleens of SPF NC/Nga and
BALB/c mice were stimulated with 200 U/ml IL-4 for 24 h, the mean
fluorescent intensities (MFI) for CD40 on B cells from NC/Nga and
BALB/c mice were 11.4 and 11.0, respectively. Additional stimulation
for 24 h induced increased MFI for CD40, but there was no
significant difference between the mouse strains (Fig. 3B).
Thus, there was no difference in CD40 expression on splenic B cells
between NC/Nga and BALB/c mice even when the cells were stimulated with
IL-4.
Sensitivity of B cells to CD40L for IgE synthesis
To determine responsiveness of B cells to CD40L, splenic
B220+ B cells were incubated with various doses of soluble
CD40L (1–1000 ng/ml) and a fixed dose of IL-4 (200 U/ml) for 9 days.
When 100 ng/ml CD40L was added, significant levels of IgE were detected
in cultures of B cells from both groups of mice, but IgE levels in
cultures of B cells from NC/Nga mice were higher than those in cultures
of B cells from BALB/c mice (Fig. 4).
Costimulation with 1000 ng/ml CD40L and 200 U/ml IL-4 to B cells of
NC/Nga mice led to a marked increase in levels of IgE, whereas there
was no significant difference in a total number of viable B cells
during the incubation for 9 days between NC/Nga and BALB/c mice. Thus,
significant differences in the sensitivities of splenic B cells to
CD40L were observed between NC/Nga and BALB/c mice.
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To determine the sensitivities of B cells to IL-4 for IgE
synthesis, B220+ B cells isolated from spleens of SPF
NC/Nga and BALB/c mice were incubated with various doses of IL-4
(25–200 U/ml) and the fixed dose of soluble CD40L (100 ng/ml) for 9
days. As shown in Fig. 5, IL-4 induced
IgE production by splenic B cells of NC/Nga and BALB/c mice in a
dose-dependent manner. The minimal doses of IL-4 required for IgE
synthesis by B cells of NC/Nga and BALB/c mice were 50 and 100 U/ml,
respectively; B cells from NC/Nga mice produced higher levels of IgE at
individual doses of IL-4 than those of BALB/c mice (Fig. 5). Since IL-4
is also able to induce IgE synthesis by B cells with LPS (14, 22, 23, 24, 25, 26),
in the presence of 10 µg/ml LPS, B220+ B cells isolated
from spleens of SPF NC/Nga and BALB/c mice were incubated with various
doses of IL-4 (6.25–200 U/ml). IL-4 induced IgE production by B cells
of NC/Nga and BALB/c mice in a dose-dependent manner (Fig. 6). IgE levels in the culture
supernatants of B cells of NC/Nga mice were higher than those of BALB/c
mice at each dose of IL-4, and the minimal effective doses of IL-4 for
IgE synthesis by B cells of NC/Nga mice and BALB/c mice were 6.25 and
25 U/ml, respectively (Fig. 6). Thus, splenic B cells from NC/Nga mice
were more sensitive to IL-4 for IgE synthesis than those from BALB/c
mice, resulting in the elevated in vitro production of IgE by B cells
of NC/Nga mice.
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CD40L and IL-4 are sufficient to induce murine B cells to switch
Ig isotypes to IgG1 as well (15). Therefore, in the presence of
100 ng/ml CD40L and 200 U/ml IL-4, B cells were cultured for 9 days,
and IgG1 levels in the culture supernatants were measured.
Significantly high levels of IgG1 as well as IgE were detected in
supernatants from splenic B cells of NC/Nga mice compared with those
from cells of BALB/c mice (Table III).
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JAK3, a member of Janus kinase family of nonreceptor protein
tyrosine kinase, associates with CD40 (46) and with the -chain that
constitutes the IL-4R complex (40, 42) and mediates CD40L and IL-4
signalings through its phosphorylation in lymphoid cells (40, 43, 44, 46). Therefore, we attempted to determine phosphorylation of JAK3 in
murine splenic B cells and peripheral blood B cells from patients with
AD and in those from healthy nonatopic donors. No tyrosine
phosphorylation of JAK3 was detectable in unstimulated B cells of
spleens in SPF NC/Nga mice and BALB/c mice (Fig. 7, A and C). On the
other hand, splenic B cells isolated from conventional NC/Nga mice with
marked elevation of plasma IgE levels elicited constitutive tyrosine
phosphorylation of JAK3. CD40L (100 ng/ml) or IL-4 (200 U/ml) increased
its phosphorylation, and the additional effect was detected in B cells
costimulated with CD40L and IL-4 (Fig. 7, A andC).
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, B and D). In contrast, in B
cells from AD patients with elevated levels of serum IgE, JAK3 was
constitutively phosphorylated at the tyrosine residue, and
phosphorylation was enhanced by treatment with anti-CD40 mAb and/or
IL-4 (Fig. 7, B and D), very similar to a pattern
of the phosphorylation observed in splenic B cells from conventional
NC/Nga mice. IL-4 synthesis by Thy1.2+ T cells
IL-4 is an essential cytokine for IgE synthesis in mice.
Therefore, we investigated the productivity of IL-4 by
Thy1.2+ T cells. Thy1.2+ T cells isolated from
spleens of SPF NC/Nga and BALB/c mice were incubated with 5 µg/ml Con
A for 72 h, and IL-4 concentrations in the culture supernatants
were measured by an ELISA. The stimulation with an optimal dose of Con
A led to significant IL-4 production of Thy1.2+ T cells in
NC/Nga and BALB/c mice, but the level of IL-4 in the former was about
one-third that in the latter (Table IV).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Flow cytometric analysis with anti-CD40 mAb showed that the level
of CD40 expression and the proportion of CD40-positive cells in splenic
B cells from SPF NC/Nga mice were increased when the cells were
incubated with an optimal dose of IL-4, as were splenic B cells from
BALB/c mice. Renz et al. (8) investigated the role of CD40 molecule in
IgE production and showed the increased expression of CD40 on PBMC from
patients with AD and the increased production of IgE by treatment with
anti-CD40 mAb even in the absence of IL-4. They also found that
pretreatment of normal PBMC with IL-4 induced significant expression of
CD40, and subsequent costimulation with anti-CD40 mAb led to
increased IgE synthesis. These findings lead to two conclusions. One is
that IL-4 increases CD40 expression on B cells, possibly resulting in
high production of IgE in patients with AD and conventional NC/Nga mice
with dermatitis. The other is that B cells from patients with AD and
conventional NC/Nga mice may be activated with IL-4 constitutively
produced by CD4+ T cells and mast cells in the affected
skin (13, 52, 53, 54). Furthermore, our study strongly suggests that the
high sensitivity of B cells to CD40 cross-linking may be involved
in IgE hyperproduction. In fact, JAK3, which is involved in the signal
transduction of not only IL-4 but also IL-7, IL-9, and CD40L (40, 42, 43, 44, 45, 46, 55), was markedly phosphorylated in B cells from
conventional NC/Nga mice with dermatitis and in PBMC from
patients with AD and high IgE levels even in the absence of IL-4 and
CD40L/anti-CD40 mAb (Fig. 7).
Tyrosine phosphorylation of JAK3 was induced in splenic B cells from
SPF NC/Nga mice following treatment with IL-4 and CD40L, but not in the
absence of IL-4 and CD40L. This tyrosine phosphorylation in B cells
induced by IL-4 or CD40L/anti-CD40 mAb was comparable to that in B
cells of BALB/c mice and in PBMC of normal healthy donors. Since flow
cytometric analysis with anti-IL-4R -chain mAb showed the
expression level of IL-4R -chain on splenic B cells of SPF NC/Nga
mice was very similar to that on splenic B cells of BALB/c mice even
after treatment with LPS for 4 days (data not shown), we concluded that
B cells from NC/Nga mice were more sensitive to IL-4, resulting in
enhanced JAK3 tyrosine phosphorylation in the postreceptor process.
Taken together, elevated IgE levels in patients with AD and NC/Nga mice
with dermatitis may be attributable to tyrosine phosphorylation of JAK3
in B cells enhanced by the synergistic effect of increased IL-4 and
CD40 cross-linking.
Recently, Hershey et al. (56) demonstrated that a mutation in the
cytoplasmic domain of the IL-4R -chain, substitution of guanine for
adenine at nucleotide 1902 causing a change from glutamine to arginine
at position 576, occurs with higher frequently in patients with atopy
than in those without atopy. They suggested that its mutation was
associated with enhanced signaling of IL-4 through decreased binding of
SHP-1, previously known as PTP1C, SH-PTP1C, or HCP, the
nontransmembrane phosphotyrosine phosphatase containing Src homology 2
domain, to phosphorylated IL-4R -chain at tyrosine residue. Other
investigators also showed that SHP-1 was considered a candidate for a
modulator of the IL-4-induced signal (57). In the
erythropoietin-induced signals, binding of SH-PTP1C to its receptor
could cause dephosphorylation of JAK2, demonstrating that JAK2 was one
of substrates for SH-PTP1C, and it was suggested that SH-PTP1C binding
to the erythropoietin receptor was important for down-modulation of
intracellular signaling through the erythropoietin R (58). Schulte et
al. (59) speculated in mice that the altered ligand binding behavior of
the newly identified IL-4R allotype may influence IL-4 responsiveness.
Whether the molecules are up- or down-modulators of the JAK3 in B cells
has not been determined completely; therefore, further investigations
are crucial to clarify the signal transduction induced by IL-4 and
CD40L.
Although the exact mechanisms of IgE hyperproduction in AD patients have not been completely understood, our present study presented here demonstrating the constitutive tyrosine phosphorylation of JAK3 in B cells of patients with AD may provide further insight for clarifying its mechanisms.
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2 Address correspondence and reprint requests to Dr. Hiroshi Matsuda, Department of Veterinary Clinic, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183, Japan. E-mail address:
3 Abbreviations used in this paper: AD, atopic dermatitis; SPF, specific pathogen-free; CD40L, CD40 ligand; JAK3, Janus kinase 3; PE, phycoerythrin; MFI, mean fluorescent intensity; SH-PTP1C, phosphotyrosine phosphatase containing Src homology 2 domain.
Received for publication March 5, 1998. Accepted for publication October 7, 1998.
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