HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bastien, Y. Articles by Mazer, B. D. Search for Related Content PubMed PubMed Citation Articles by Bastien, Y. Articles by Mazer, B. D.

Detection of Functional Platelet-Activating Factor Receptors on Human Tonsillar B Lymphocytes¹

Yolande Bastien^*,, Baruch J. Toledano^*,, Noha Mehio, Lisa Cameron, Bouchaib Lamoukhaid, Poalo Renzi, Qutayba Hamid and Bruce D. Mazer^2,*,

^* Division of Allergy and Immunology, Montreal Children’s Hospital, McGill University/Montreal Children’s Hospital Research Institute, and Meakins Christie Laboratories, McGill University, Montreal, Quebec, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although platelet-activating factor (PAF) receptors have been found on B lymphoblastoid cell lines, the action of PAF on freshly isolated human B cells has not been clearly demonstrated. Using a sensitive semiquantitative reverse-transcriptase PCR, we have found PAF receptor mRNA expressed by tonsillar B lymphocytes, but little in T lymphocytes. Examination of Percoll-fractionated tonsillar B cells indicated that the low density (primarily germinal center cells) and medium density fractions had approximately twofold more PAF receptor mRNA relative to the high density fraction. PAF (10^-7 M) stimulated increases in intracellular Ca²⁺ that were consistently higher in the low and medium density B lymphocytes compared with high density cells. The PAF receptor antagonist Web 2170 inhibited this. Addition of PAF, but not lyso- or enantio-PAF, induced four- to sixfold greater synthesis of IgM and IgG in low and medium density cells compared with unstimulated controls, but had little effect on Ig production by high density cells. To investigate how PAF may influence Ig synthesis, PAF-stimulated B cells were examined for production of the Th2-type cytokines IL-4 and IL-13. PAF induced IL-4 and IL-13 mRNA expression in 17% of CD20⁺ cells, and IL-4 was detected in cell supernatants after 48–72 h of culture. Together, these data strongly suggest that functional PAF receptors are expressed on B cells in tonsils.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Platelet-activating factor (PAF),³ a potent lipid mediator released during inflammatory and immune responses, has a wide range of physiologic actions. PAF induces bronchoconstriction and vasodilatation in animals, and has been implicated in the pathogenesis of asthma and septic shock (1). PAF is a chemoattractant for neutrophils, basophils, and eosinophils, activating cells involved in the response to inflammatory and infectious stimuli (2). Animals that overexpress the PAF receptor (PAFR) have marked bronchial hyperreactivity, and enhanced susceptibility to endotoxin infection (3).

There is emerging data that suggest that PAF may be an important intracellular communication molecule for lymphocytes. Stimulation of B lymphoblastoid cell lines with PAF induces a cascade of early activation events, including increases in intracellular calcium ([Ca²⁺]_i) (4), tyrosine phosphorylation (5), mitogen-activated protein kinase activation (5), and cell cycle-related gene expression (4, 6, 7). Recently, we determined that PAF rescues Ramos cells from apoptosis, by decreasing the production of reactive oxygen substances following ligation of the B cell receptor (8). However, it has been unclear whether the observations in B cell lines could be extended to fresh B lymphocytes. Simon et al. (9) examined peripheral blood cells from asthmatic subjects for expression of PAFR and did not find any PAFR mRNA in T and B cells, while finding high levels of mRNA expression in neutrophils and monocytes. In contrast, Deryckx et al. (10) have demonstrated that PAF may modulate IL-4-stimulated IgE production, and PAFR mRNA expression and radiolabeled PAF binding have been detected in B cells isolated from tonsils (11, 12).

Tonsillar B lymphocytes represent a heterogeneous population, unlike peripheral blood neutrophils or monocytes. They are made up of mantle zone (MZ) cells, cells in the marginal zone, and germinal center (GC) cells (13). Because these cells represent different stages of B cell maturation, the presence of the PAFR may differ between subsets of peripheral B lymphocytes. To understand the discordance in the literature, well-characterized B lymphocyte populations must be studied.

In this study, we have examined fractionated tonsillar B lymphocytes representing different stages of B lymphocyte maturation. Using semiquantitative RT-PCR, we demonstrate that B lymphocytes with the GC phenotype appear to express greater amounts of PAFR mRNA compared with MZ cells. PAFR expressing B lymphocytes are responsive to stimulation with PAF, demonstrating increases in [Ca²⁺]_i, Ig synthesis, and importantly, production of the cytokine IL-4. These data confirm the findings in B cell lines regarding presence of functioning PAFR on B cells, and suggest that PAF may have a crucial role in GC B cell maturation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell culture

B lymphocytes were isolated from human tonsils discarded following surgery. The tonsils were thoroughly minced; resuspended in wash medium consisting of RPMI 1640 supplemented with 2% FCS, and 500 U/ml penicillin, 500 µg/ml streptomycin, and amphotericin B (1/500 w/v) from Life Technologies (Burlington, ON, Canada); and then layered onto a Ficoll-Paque (Phamacia Biotech, Uppsala, Sweden) gradient. Tonsillar lymphocytes were separated by rosetting with neuraminidase-treated SRBC and Ficoll-Paque density centrifugation. Monocytes were removed from the E-rosette-negative fraction by adherence depletion; the remaining B cells were routinely demonstrated to be >98% pure on flow cytometry by CD19 staining, with <1% CD14⁺ and <2% CD3⁺.

The E-rosette-positive fraction was subjected to further purification by negative selection of T cells using a high affinity T cell enrichment column purchased from R&D Systems (Minneapolis, MN). The T cell yield was 65%, and the cells were 99% CD3⁺ via flow cytometry.

Percoll gradient centrifugation

Adherence-depleted B cells were layered onto Percoll gradients (Phamacia Biotech). These were prepared by diluting 100% Percoll with PBS. Dilutions of 100%, 60%, 50%, and 30% were layered in 15 ml Falcon tubes (Becton Dickinson, La Jolla, CA), according to the methods of Liu et al. (14) and Suzuki et al. (15). The cell suspension was mixed 1:1 with 20% Percoll and applied to the top of the gradient. Following centrifugation (20 min, 4°C, 2400 rpm), the fractions were aspirated; cells recovered below the 60% layer were considered high density cells; between the 50 and 60% layers, medium density and low density cells were recovered from the 30–50% layers (14).

Synthesis of competitive RNA template (cRNA)

A competitive RNA template (cRNA) was created from a synthetic PAFR DNA (682 bp) synthesized by RT-PCR using the primers 5'-CGGACATGCTCTTCTTGATCA-3' (sense) and 5'-GTCTAAGACACAGTTGGTGCTA-3' (antisense) (11). The insert was prepared by restriction enzyme digestion with (Escherichia coli) Klenow fragment (Life Technologies). Vector DNA Bluescript (KS^-) (Promega, Madison, WI) was linearized by digestion with EcoRV (BRL Technologies, Burlington, ON, Canada). After ligation, screening of the plasmids containing the 682-bp insert was performed by restriction enzyme digestion, and PCR amplification was performed using the same primers. Transformation was conducted using DH5 competent cells (Life Technologies), and the appropriate plasmids were multiplied and purified. The construct was subsequently digested with BsgI (New England Biolabs, Mississaugua, ON, Canada), which yielded a 100-bp deletion in the insert. After ligation and multiplication of the appropriate plasmid, the orientation of the insertion was confirmed by digestion with RsaI (Life Technologies). The resultant DNA construct was 582 bp.

The plasmid was linearized by BamHI or EcoRI digestion (Life Technologies), purified, and precipitated with ethanol. In vitro transcription of the synthetic RNA was performed using T3 RNA polymerase (Promega), in accordance with the manufacturers’ recommendations. The cRNA was purified by phenol chloroform extraction and ethanol precipitation after digestion with RNase-free DNase I (Life Technologies). Quantification of the cRNA was performed by spectrophotometry, and its integrity was verified by 1% agarose gel electrophoresis.

Semiquantitative PCR

Total cellular RNA was extracted from 15 x 10⁶ cells with Trizol (Life Technologies) using the modifications for RT-PCR. RNA was dissolved in DEPC H₂O and stored until use at -80°C. First-strand cDNA was synthesized in a 25 µl reaction volume containing 150 ng of total RNA and/or 5–15 pg of cRNA following standard methodologies with 100 U MMuLV reverse transcriptase and 200 ng of random hexamer (Life Technologies), and incubated at 37°C for 1 h. The enzyme was heat inactivated for 5 min at 94°C, and samples were stored at -20°C until use. cDNAs were preheated for 5 s at 65°C to ensure solubility. A total of 3 µl of the original 25 µl cDNA mixture was then amplified in a volume of 50 µl containing 1.5 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 200 µM of each dNTP including 0.7 µCi [-³²P]dCTP (ICN Biomedicals Canada, Toronto, ON, Canada), 0.5 µM of primers, and 1.5 U Taq polymerase (Life Technologies). All amplifications were performed in a Hybaid OmniGene Thermal Cycler for 28 cycles using the following programs: an initial cycle of 94°C for 5 s, 62°C for 3 s, and 72°C for 2 min, followed by 27 cycles of 94°C for 60 s, 60°C for 90 s, and 72°C for 90 s. This cycle number ensured that the PCR remained within the exponential range of amplification. A total of 10 µl of the PCR samples was applied on 2% agarose, and following electrophoresis the gel was dried under vacuum for 35 s at room temperature, and then for 35 s at 60°C. Mean density of the radioactive bands was analyzed by phosphor imaging (Fuji Bioimaging Analyzer; Fuji, Nokoyama, Japan). Calculated averages were performed on three to eight identical experiments.

[³H]Thymidine incorporation

Fractionated B lymphocytes were resuspended in serum-free medium (16) at a concentration of 2 x 10⁶/ml, and were incubated with or without PAF at indicated concentrations for 30 min. Following this, the cells were resuspended in an equal volume of complete medium containing 20% FCS, with or without 1/1000 w/v SAC (Staphylococcus aureus Cowan strain I; Calbiochem, San Diego, CA), and plated on 96-well plates. Following 90–114 h of incubation at 37°C, in 5% CO₂ [³H]thymidine was added (1 µCu/well) and the cells were incubated an additional 6 h. The cells were harvested by water lysis (PHD Cell Harvester, Cambridge, MA), and [³H]thymidine incorporation was measured by liquid scintillation counting (Wallac, Gaithersburg, MD).

Measurement of Ig synthesis

Freshly isolated fractionated B lymphocytes were incubated with or without PAF, as described above, and then plated at 10⁶ cells/ml in complete medium in 24-well plates. After 7 days of culture, supernatants were harvested and frozen at -20°C until use. Measurement of IgG, A, and M by ELISA was performed as described by Mazer et al. (17), using specific goat anti-human IgG, A, or M Abs (Biosource, Camarillo, CA). Cell culture Ig values were compared with a standard curve generated from serial dilution of standardized sera (The Binding Site, San Diego, CA).

Measurement of [Ca²⁺]_i

Changes in [Ca²⁺]_i were measured as previously described (4). Briefly, fractionated B lymphocytes (10⁷/ml) were resuspended in complete medium, incubated with the fluorescent calcium indicator Indo-1-AM for 45 min at 37°C, then washed and resuspended in serum-free medium. Aliquots of 4 x 10⁶/ml cells were transferred to microfuge tubes, spun to pellet, and resuspended in 2 ml of buffer containing 2 mM HEPES, 140 mM NaCl, 10 mM glucose, 2 mM KCl, 1 mM MgCl₂, and 1 mM CaCl₂. The cell suspension was transferred to clear plastic cuvettes. Changes in [Ca²⁺]_i were measured using a RF-5000 Spectrofluorimeter (Shimadzu, Gaithersburg, MD) at 37°C with constant stirring, employing an emission wavelength of 405 nm and an excitation wavelength of 454 nm. Changes in [Ca²⁺]_i were calibrated and calculated as described (4).

In situ hybridization for IL-4 and IL-13 mRNA

In situ hybridization with ³⁵S-labeled complementary RNA probes coding for IL-4 and IL-13 mRNA was performed. Permeabilization of the cell membranes was achieved through incubation with Triton X-100 and proteinase K solution (1 mg/ml) in 0.1 M Tris containing 50 mM EDTA for 20 min at 37°C. Slides were then immersed in 0.1 M triethanolamine and 0.5% acetic anhydride for 10 min, and then in N-ethylalamide (1.25 mg/ml) and iodoacetamide (1.85 mg/ml) for 20 min at 37°C to prevent the nonspecific binding of the cRNA probes. Prehybridization of the samples was completed with 50% formamide in 2x SSC for 15 min at 37°C. Cytospins were incubated overnight with a hybridization mixture containing either the IL-4 or IL-13 cRNA probe (0.75 x 10⁶ cpm/slide). Posthybridization involved high stringency washing of the samples in decreasing concentrations of SSC at 42°C. To remove any unbound RNA probe, samples were washed with an RNase solution (20 mg/ml) for 20 min at 42°C. The samples were then dehydrated with increasing concentrations of ethanol and left to air dry. Following this, cytospins were dipped in Amersham LM-2 emulsion and exposed for a period of 9 days. The autoradiographs were developed in Kodak D-19 developer, fixed, and counterstained in Mayer’s hematoxylin for 60 s. The samples were mounted with a coverslip and examined under a graduated microscope for positive signals. CD20 was detected by immunohistochemistry using the alkaline phosphatase antialkaline phosphatase (APAAP) technique, as described by Hamid et al. (18). Slides prepared using radioactive in situ hybridization to detect IL-4 mRNA (white) and APAAP for detection of CD20 (red) were read and photographed using dark field illumination at x400.

Measurement of supernatant IL-4

A total of 100 µl/well of anti-human IL-4 mAb (4 µg/ml; R&D Systems) was added to 96-well plates (Costar, Cambridge, MA; Fisher Scientific, Pittsburgh, PA) and incubated overnight in a humidified box at 25°C. The wells were washed three times with 0.05% Tween-20 in PBS. A total of 300 µl/well of blocking buffer (1% BSA, 5% sucrose, 0.05% NaN₃ in PBS) was added into the wells and incubated for 1 h at room temperature, and the wells were washed three times. A total of 100 µl/well of human rIL-4 (R&D Systems) was added in duplicate using a range of concentrations (0–2000 pg/ml) diluted with 0.1% BSA, 0.05% Tween-20, and PBS. Cell culture supernatants (100 µl/well) were also added in duplicate. The plate was incubated and covered in a humidified box for 2 h. The plate was washed, and 100 µl/well of biotinylated anti-human IL-4 Ab (100 ng/ml; R&D Systems) was added and incubated an additional 2 h. Following washing, 100 µl/well of streptavidin HRP (62.5 ng/ml; Zymed, San Francisco, CA) was added and the plate was further incubated for 20 min. Subsequently, 100 µl/well of substrate solution (1:1 H₂O₂ and tetramethylbenzidine; Genzyme Diagnostics, Cambridge, MA) was added, and the plate was incubated in the darkness at room temperature for 20–30 min. A total of 50 µl/well of stop solution (0.5 M H₂SO₄; Sigma) was added. OD was read using a microplate reader (Model 3550; Bio-Rad, San Diego, CA) within 30 min at 450 nm and 570 nm for background correction.

Statistical analysis

Graphic analysis and Student’s t tests were performed using Prism software (Graphpad, San Diego, CA). For Ig studies, means of treated conditions were compared, whereas for in situ studies, positive staining cells were hand counted and the mean of the control conditions was compared with PAF-stimulated conditions.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Detection of PAFR mRNA by semiquantitative radioactive RT-PCR

We have demonstrated previously in purified human lymphocytes that PAFR mRNA is expressed primarily in B lymphocytes, with minimal PAFR mRNA detected in freshly isolated human T cells or T cell lines (11). To better understand the heterogeneity of PAFR expression in lymphocyte populations, we established a semiquantitative RT-PCR using primers developed for our previous studies of PAFR mRNA (11). The internal standard control (cRNA) was produced by creating a 100-bp deletion via BsgI digestion of wild-type RNA, allowing for clear differentiation of the wild-type fragment from the cRNA by agarose gel electrophoresis (Fig. 1, A and B). Consistent band resolution and linear synthesis of the PCR products were determined to be in the range of 24–30 cycles. After 30 cycles, synthesis plateaued; under 22 cycles, PCR products were not easily detectable. We subsequently utilized 28 cycles for the studies of PAFR in lymphocytes. Fig. 1A demonstrates the semiquantitative RT-PCR using standardized conditions (28 cycles, 150 ng/ml sample) and varying the concentration of the cRNA construct. Sample RNA from mixed B lymphocytes freshly isolated from tonsils (Fig. 1A), or cell lines such as Ramos (data not shown) gave similar results. Further demonstration that the assay is linear within the number of cycles we employed is confirmed in Fig. 1B. Using standardized conditions (5 pg cRNA, 28 cycles), the amount of sample RNA was varied, and the radioactive PCR product was quantified by phosphor imaging. The ratio of sample/cRNA increased proportionally to the amount of sample RNA (Fig. 1B). From standard curves using varying doses of cRNA, we have estimated that the amount of PAFR mRNA in freshly isolated mixed human B lymphocytes is approximately 20.8 pg/10⁶ cells, and in Ramos cells approximately 55.6 pg/10⁶ cells.

View larger version (38K): [in this window] [in a new window]   FIGURE 1. Standardization of semiquantitative RT-PCR for PAFR mRNA. RNA was extracted from mixed tonsillar B lymphocytes, and RT-PCR was conducted as in Materials and Methods. A, Upper panel, The RT-PCR was conducted using incremental increases in the internal standard, and unvarying concentrations of total sample RNA (150 ng/sample). After 28 cycles of amplification, radiolabeled RT-PCR products were applied to 2% agarose gel, and following gel electrophoresis, the gel was exposed to a Fuji Bioanalyzing screen, and the resulting bands were quantitated by phosphor imaging analysis. Representative of three identical experiments. 5/0 represents 5 ng of cRNA and no added sample. Lower panel, Increasing amounts of cRNA lead to a decrease in the specific PAFR mRNA/cRNA ratio, which is graphically represented in the lower panel. B, Upper panel, The RT-PCR was conducted using incremental increases in the total RNA with cRNA fixed at 5 pg/sample. Analysis is conducted as above. This is representative of three identical experiments. Lower panel, Graphic representation of the above experiment, showing near linearity of the amplification as mRNA is increased.

We next analyzed expression of PAFR message in tonsils. Lymph nodes are populated by both B and T cells; B cells are found in the GC, MZ, and the marginal zones in between, whereas T cells are found predominantly in the MZ and marginal zones (13, 19). Following separation of B and T lymphocytes by E-rosetting and column depletion, we assessed their relative amounts of PAFR mRNA by semiquantitative RT-PCR. Mixed populations of tonsillar B lymphocytes have a high level of expression of PAFR mRNA, in comparison with T lymphocytes (Fig. 2). Expression of PAFR in T cells was either absent or exceedingly low in all samples tested (n = 8).

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Comparison of PAFR mRNA expression in tonsillar T and B cell populations. T and B cells were purified from human tonsils, as indicated in Materials and Methods; after resting overnight, RNA was extracted and semiquantitative RT-PCR was performed. Right panel, Representative autoradiography showing strong expression of PAFR mRNA in B lymphocytes with little detected in T lymphocytes. No RT indicates RT-PCR performed with tonsillar B lymphocyte mRNA with the reverse-transcriptase enzyme omitted from the reaction. Left panel, Graphic representation of eight experiments comparing ratio of PAF mRNA/cRNA in T cells and B cells, demonstrating a higher level of expression in the B lymphocyte fraction.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Expression of PAFR mRNA in fractionated B lymphocyte populations. Tonsillar B cells were purified by Percoll centrifugation, and RNA was extracted for detection of PAFR mRNA by semiquantitative RT-PCR. Right panel, Representative autoradiogram of PAFR mRNA detected in the high, medium, and low density Percoll fractions. Left panel, Average mRNA/cRNA ratios calculated from phosphor imaging analysis of six experiments. The difference between PAFR mRNA in the low and medium density fractions is significantly greater than the high density fraction (*, p < 0.03).

Having detected mRNA expression in tonsillar B cells, we undertook to determine whether there was functional PAFR present in these populations. Signal transduction through the PAFR is mediated through G proteins that are linked to calcium channels (20). Thus, increases in [Ca²⁺]_i following stimulation by PAF are indicative of a functional PAFR on cells. The three purified B cell populations were individually loaded with Indo-1-AM, and [Ca²⁺]_i was measured. Fig. 4 illustrates responses of each fraction following the addition of PAF (10^-7 M). PAF increased [Ca²⁺]_i in each fraction, with the low density fraction increasing [Ca²⁺]_i by 247 ± 29 nM (n = 3), the medium fraction 186 ± 25 nM, and the high density fraction exhibiting an increase of 114 ± 18 nM. The increases in [Ca²⁺]_i were inhibited by the specific PAFR antagonist WEB 2170 (Fig. 4).

View larger version (11K): [in this window] [in a new window]   FIGURE 4. PAF induced elevations in [Ca2+]i in tonsillar B cells. Tonsillar B cells were purified by Percoll centrifugation and then loaded with Indo-1-AM for 45 min, as described above. This shows representative tracings of PAF-stimulated increases in [Ca2+]i in the low (A), medium (B), and high (C) density fractions. D, Low density cells preincubated for 5 min with WEB 2170, which inhibits the rise in [Ca2+]i induced by PAF.

We and others (17, 21) have demonstrated that PAF can augment secretion of Ig in immortalized B cell lines, and therefore we examined the effect of PAF on the Percoll-generated populations of B cells that we had isolated. B cells from each fraction were cultured with or without PAF, and 7-day cell culture supernatants were measured for the production of Igs. PAF increased IgG production in the low density population in a dose-dependent manner (Fig. 5A). In both the low and medium density, but not the high density cultures, addition of the optimal dose of PAF (10^-8 M) significantly augmented IgG and IgM production compared with control cells (Fig. 5, B and C), a process that was inhibited by Web 2170. The addition of the polyclonal stimulus SAC to PAF did not significantly alter the pattern of IgG and M production that was observed with PAF alone (Table II). SAC-stimulated low density B cells only produced Ig when in the presence of PAF. Although SAC induced IgG and IgM production in the medium and high density cells, addition of PAF to SAC-treated medium density cells significantly increased the amount of IgM that was produced. PAF had little influence on IgM or IgG production by high density B lymphocytes (Table II). Neither the PAF sterioisomer enantio-PAF nor lyso-PAF significantly increased Ig synthesis (data not shown). The production of IgA, measured from the same culture supernatants, was not influenced significantly by the addition of PAF to the tonsil cells (data not shown). Cells cultured with PAF had equal or better viability than cells cultured in medium alone, intimating that the augmented Ig production was not a function of increased cell death or lysis. Cell counts at the end of the full 7-day culture period were also not greatly different between PAF-treated and untreated groups (data not shown).

View larger version (14K): [in this window] [in a new window]   FIGURE 5. PAF increases production of IgG and IgM in B cells from low and medium density fractions. Tonsillar B cells were purified by Percoll centrifugation and then cultured with or without PAF for 7 days. Supernatants from cell culture were measured for the presence of IgG or IgM (ng/ml) by ELISA. A, Dose response of low density B cells to PAF. IgG secretion is increased significantly by PAF at a range of doses between 10-7 and 10-9 M. Solid bars represent mean ± SEM for IgG production; n = 3. Similar data were obtained for IgM (data not shown). B, Using optimal doses of PAF (10-8 M) (solid bars), IgG is increased significantly in the low and medium, but not the high density fraction compared with unstimulated cells (shaded bars) (n = 7; *, p < 0.05). C, Using optimal doses of PAF (10-8 M) (solid bars), IgM is increased significantly in the low and medium, but not the high density fraction compared with unstimulated cells (shaded bars) (n = 7; *, p < 0.05).

PAF can up-regulate cytokine production in cell lines, including TNF (22, 23). To explain the increase in Ig synthesis induced by PAF, we examined whether the lipid mediator was capable of inducing the production of cytokines. Specifically, Th2-like cytokines such as IL-4 and IL-13 are capable of inducing Ig secretion in B cells without inducing proliferation (24). B cells stimulated with PAF (10^-7 M) and/or PAF and Web 2170 (10^-6 M) were examined by in situ hybridization for the presence of cytokine-specific mRNA. This dose of PAF was optimal for calcium mobilization, and therefore was used for all cytokine studies. Control cells had very low background production of IL-4 mRNA (<2%, Fig. 6A), whereas 17% of PAF-stimulated B lymphocytes had IL-4 mRNA (Figs. 6, B and D, and 7A) and 19% had IL-13 mRNA (Fig. 7). No increase in cytokine mRNA was detected in the presence of the PAF antagonist Web 2170 (Fig. 6C). Although the cells plated for in situ hybridization were >98% CD20⁺ cells by flow cytometry, we ensured that the cells that exhibited IL-4 and IL-13 mRNA were indeed B lymphocytes by simultaneous immunohistochemical staining with anti-CD20 Abs. This confirmed that the cells were B lymphocytes and not CD3-positive T cells or monocytes (Fig. 6D).

View larger version (88K): [in this window] [in a new window]   FIGURE 6. In situ hybridization for the detection of IL-4 mRNA. Isolated tonsillar B lymphocytes were incubated with 10-7 M PAF for 24 h, and the cells were then cytospun and IL-4 mRNA was detected by in situ hybridization, as described in Materials and Methods. This is representative of three experiments. A, Unstimulated B cells; B, B cells stimulated with 10-7 M PAF; C, B cells preincubated for 10 min with Web 2170 (10-6 M) and then stimulated with 10-7 M PAF. D, Colocalization of IL-4 mRNA and CD20 staining to indicate B lymphocytes. Cells were simultaneously stained for the presence of IL-4 mRNA by radioactive in situ hybridization (white) and for expression of CD20 (red) by immunohistochemistry using the APAAP technique. Slides were read and photographed using dark field illumination at x400.

View larger version (15K): [in this window] [in a new window]   FIGURE 7. Detection of Th2-type cytokines in PAF-stimulated B cells. A, Results of three identical in situ hybridization experiments probing for IL-4 and IL-13 mRNA. Tonsillar B cells were purified and then cultured with or without 10-7 M PAF for 24 h, then probed for IL-4 and IL-13 mRNA by in situ hybridization. Positive signals were hand counted by microscopy. **, p < 0.005. B, Results of IL-4 protein detected by ELISA from culture supernatants. Tonsillar B cells were purified and then cultured with or without 10-7 M PAF and/or 10-6 M Web 2170 for 72 h. Results are the mean ± SEM from four experiments. **, p < 0.005.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
B lymphocyte growth and maturation in peripheral tissues are dependent on stimulation by Ag and an obligatory second signal such as adhesion molecules, cytokines, and mediators. We have shown that B lymphoblastoid cell lines express mRNA and receptors for platelet-activating factor (4, 7, 11, 25). Additionally, we have demonstrated that this potent and ubiquitous lipid mediator can provide a critical second signal to B cells, in that PAF rescues the Ramos B cell line from apoptosis induced by cross-linking of the B cell receptor by anti-IgM Abs (8). In this work, we present data that confirm that human B lymphocytes isolated from tonsils express mRNA for PAFR. Moreover, addition of PAF to defined populations of tonsillar B lymphocytes leads to intracellular signals such as increases in [Ca²⁺]_i. Importantly, we have evidence that PAF induces the secretion of the key cytokine IL-4, which may influence the production of Ig.

The PAFR is a GTP-binding protein-linked receptor with seven transmembrane domains (2). The PAFR gene is on chromosome 1 and has no introns in the coding region (26). Using a sensitive, radioactive semiquantitative RT-PCR, we have delineated that tonsillar B lymphocytes express mRNA for PAFR. Moreover, we have shown a clear difference in PAFR mRNA expression between B cells and resting T cells. Minimal expression of PAFR mRNA has been noted now both with fresh T lymphocytes and T cell lines (9, 11). Our results are similar to those of Yang et al. (27), who examined PAFR expression in Ramos cells using a similar quantitative PCR and to those of Nguer et al. (12) using Northern blot analysis of unfractionated tonsil preparations. In addition, we found differences in expression between the various fractions of tonsillar B cells, with increased mRNA from cells in the low and medium density fractions, which includes GC B cells.

Simon et al. (9) reported that B and T lymphocytes from the blood of asthmatics do not express PAFR mRNA, compared with the high levels of mRNA expressed by neutrophils and monocytes. There are several reasons that their results may differ from ours or those of Nguer (12). As B cells have fewer PAF binding sites than neutrophils (28), detection by Northern blot analysis may be more difficult. Thus, for lymphocytes, the semiquantitative RT-PCR technique may be better suited for PAFR mRNA detection. Additionally, Simon et al. studied B cells from peripheral blood (9), which are mostly derived from the mantle zone of lymphoid tissue and are represented by the high density cells that we have isolated. This population had the lowest expression of PAFR mRNA in our studies. Because of the heterogeneity of B cell populations, different results may therefore be obtained with mixed tonsillar B cells (11, 12) or fractionated B cells. We are currently examining the GC population to better phenotype the PAF-responsive cell populations.

Following stimulation of PAFR in B cell lines, there are measurable changes in phosphorylation of mitogen-activated protein kinase (5), inositol phosphate turnover (4), increases in [Ca²⁺]_i (4, 6, 7), and induction of cell cycle active genes (4, 29). These events are all inhibited by specific PAFR antagonists (4). In the present study of characterized B cell populations, addition of PAF immediately increases [Ca²⁺]_i. This response compares favorably with B cell lines such as HSCE^- and U266, in which PAF (10^-7 M) increases [Ca²⁺]_i levels by 200–300 nM (4), and the IgM-secreting cell line LA350 with increases in [Ca²⁺]_i of 80–100 nM (4, 25). Low density or GC-like cells had the greatest response (247 ± 29 nM), and MZ-like cells from the high density region had the lowest response (114 ± 18 nM). There is clearly a degree of correlation between receptor mRNA expression and the [Ca²⁺]_i response of the receptor. It is difficult to compare our [Ca²⁺]_i results with other studies using unfractionated B cells such as those of Nguer et al. (12). However, as in the studies by Siffert et al. (30), differences in level of maturation or activation may play a role in the G protein response to PAF.

In our previous work, we determined that PAF increased Ig production in IgG- and IgE-secreting B cell lines (17), as well as in the Ramos IgM-secreting B cell line (31). This increase ranged from 100% in Ramos cells (31) to 300% for the IgE-producing line U266 (17). Using immortalized B cells from patients with hypertension, Rosskopf et al. found that PAF (10^-7 M) also increased IgM and IgG production (21). PAF induced marked increases in IgM and IgG in the low density, and to a lesser extent the medium density cell populations. Low density cells have been shown to be unresponsive to the polyclonal B cell mitogen SAC (15), yet were stimulated to produce Ig by PAF. Although this is a striking finding, it is unclear as to the relative importance of PAF signals compared with other, T cell, or Ag-derived signals. We did not examine IgE production, as there was no source of CD40 in our cultures, an obligatory molecule for class switch to IgE.

These results may be explained by the finding that PAF can induce the production of IL-4 in B lymphocytes. This novel finding adds to an emerging literature regarding B cells and cytokines (32). Recently, the production of granulocyte-macrophage CSF was demonstrated in tonsillar B cells (13), IL-10, and TGF-ß1 in plasma cells (33), and IFN- production was detected in B cells stimulated with IL-12 and IL-18 (34). In addition, B cell lines have been found to express mRNA for several cytokines (13), including IL-4 (35). We have also shown that phorbol esters and ionomycin can induce the expression of IL-4 and IL-13 mRNA, and that IL-4 is detected in highly purified B cells cultured with phorbol esters and ionomycin (36). The IL-4 gene is regulated via several promoter regions, including an nuclear factor-B responsive element. PAF can induce nuclear factor-B transcription (37), which most likely contributes to IL-4 production. The finding in this work that PAF can induce IL-4 mRNA and protein and therefore could stimulate Ig secretion in the low and the medium density populations is indeed intriguing. PAF has been previously shown to cause cells to produce IL-4, but the experiments presumed the action was on T cells by a monocyte-dependent mechanism (38). It also may up-regulate IL-4 production in thymocytes (39). PAF induces cytokine production in monocytes (40), macrophages (41), synovium (23), fibroblasts (42), endothelial cells (43), and B cell lines (22). It can induce the synthesis of IL-6, which also may play a role in Ig secretion (43). The production of IL-4 by B lymphocytes may help clarify some of the underlying mechanisms by which T cells are driven to the Th2 phenotype, helping to magnify humoral responses in stimulated B cells in the GC (44, 45).

The most PAF-responsive B cells in these studies are found in the low and medium density Percoll fractions. In this population, there are B cell blasts, centrocytes, and centroblasts (19, 13). The GC cells are found exclusively in these fractions, as they are CD10⁺, IgD^-, and express higher levels of CD38 compared with the high density fraction (13, 46) (Table II). The majority of the rapidly proliferating centroblasts are in the medium density fraction (13). These cells most resemble Ramos B lymphoblastoid cells (CD38⁺, CD10⁺, IgD^-). By semiquantitative RT-PCR, it appears that there is an increased amount of PAFR mRNA in Percoll-separated low density and medium density B cells. The cells of these two fractions interact with FDCs, which present Ag and provide other second signals to the B cells (47). PAF can act as a second signal that prevents apoptosis in Ag receptor-ligated Ramos cells. Although it is unclear what specific second signals are transmitted by the FDCs, they express CD40, CD23, as well as adhesion molecules such as ICAM-1 and VLA-4 (47, 48). Because they express markers suggestive of myelomonocytic origin (49), it is conceivable that FDCs produce lipid mediators in a manner similar to monocytes or tissue macrophages. We are currently exploring this possibility.

What may be the role for PAF in B cell development? Ag-stimulated B cells are recruited to lymphoid tissue through specialized high endothelial venules, and in the specialized areas of lymph nodes known as GC, FDCs directly interact with maturing B cells. Endothelial cells are known to produce large quantities of PAF, and like other monocyte-derived cells (50), FDCs are potential sources of lipid mediators. Thus, B cells most likely encounter bioactive lipids such as PAF in lymphoid tissues. Subsequently, PAF signals may induce B cells to produce IL-4, which would drive T cell differentiation to the Th2 phenotype. This would lead to an enhanced humoral response as Ag-stimulated B cells interact with T cells in the GC. Demonstration of PAFR on B lymphocytes within the GC adds to the complex array of signals that contribute to lymphocyte development.

	Acknowledgments

 
We thank Dr. Felicia Ghibu, Elsa Shottman, and Ali Samer Al Assaad for excellent technical support in the completion of these studies.

	Footnotes

 
¹ This work was supported by the Medical Research Council of Canada, the Fonds de la Recherche en Sante du Quebec, the Costello Foundation, and the McGill University/Montreal Children’s Hospital Research Institute. B.J.T. was the recipient of a fellowship from the McGill University/Montreal Children’s Hospital Research Institute.

² Address correspondence and reprint requests to Dr. Mazer, Meakins Christie Laboratories, 3626 St. Urbain Street, Montreal Quebec, Canada H2X 2P2. E-mail address:

³ Abbreviations used in this paper: PAF, platelet-activating factor; APAAP, alkaline phosphatase antialkaline phosphatase; [Ca²⁺]_i, intracellular calcium; cRNA, competitive RNA; FDC, follicular dendritic cell; GC, germinal center; MZ, mantle zone; PAFR, platelet-activating factor receptor; SAC, Staphylococcus aureus Cowan strain I; sIg, surface immunoglobulin.

Received for publication July 6, 1998. Accepted for publication February 8, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Venable, M. E., G. A. Zimmerman, T. M. McIntyre, S. M. Prescott. 1993. Platelet-activating factor: a phospholipid autocoid with diverse actions. J. Lipid Res. 34:691.[Medline]
2. Izumi, T., T. Shimizu. 1995. Platelet-activating factor receptor: gene expression and signal transduction. Biochim. Biophys. Acta 1259:317.[Medline]
3. Ishii, S., T. Nagase, F. Tashiro, K. Ikuta, S. Sato, I. Waga, K. Kume, J. Miyazaki, T. Shimizu. 1997. Bronchial hyperreactivity, increased endotoxin lethality and melanocytic tumorigenesis in transgenic mice overexpressing platelet-activating factor receptor. EMBO J. 16:133.[Medline]
4. Mazer, B., J. Domenico, H. Sawami, E. W. Gelfand. 1991. Platelet-activating factor induces an increase in intracellular calcium and expression of regulatory genes in human B lymphoblastoid cells. J. Immunol. 146:1914.[Abstract]
5. Franklin, R. A., B. Mazer, H. Sawami, G. B. Mills, N. Terada, J. J. Lucas, E. W. Gelfand. 1993. Platelet-activating factor triggers the phosphorylation and activation of MAP-2 kinase and S6 peptide kinase activity in human B cell lines. J. Immunol. 151:1802.[Abstract]
6. Schulam, P. G., A. Kuruvilla, G. Putcha, L. Mangus, J. Franklin-Johnson, W. T. Shearer. 1991. Platelet-activating factor induces phospholipid turnover, calcium flux, arachadonic acid liberation, eicosanoid generation and oncogene expression in a human B cell line. J. Immunol. 146:1642.[Abstract]
7. Mazer, B. D., J. Domenico, A. Szepesi, J. J. Lucas, E. W. Gelfand. 1994. Role for Ca²⁺ in expression of cell cycle regulated genes in PAF-stimulated cells. J. Lipid Mediat. Cell Signal. 10:269.[Medline]
8. Toledano, B. J., Y. Bastien, F. Noya, S. Baruchel, B. Mazer. 1997. Platelet-activating factor abrogates apoptosis induced by cross-linking of the surface IgM receptor in a human B lymphoblastoid cell line. J. Immunol. 158:3705.[Abstract]
9. Simon, H. U., P. W. Tsao, K. A. Siminovitch, G. B. Mills, K. Blaser. 1994. Functional platelet-activating factor receptors are expressed by monocytes and granulocytes but not by resting or activated T and B lymphocytes from normal individuals or patients with asthma. J. Immunol. 153:364.[Abstract]
10. Deryckx, S., R. de Waal Malefyt, J. F. Gauchat, E. Vivier, Y. Thomas, J. E. de Vries. 1992. Immunoregulatory functions of paf-acether. VIII. Inhibition of IL-4-induced human IgE synthesis in vitro. J. Immunol. 148:1465.[Abstract]
11. Bastien, Y., B. D. Mazer. 1994. Detection of the human platelet-activating factor receptor mRNA in human B lymphocytes by polymerase chain reaction on reverse transcripts (RT-PCR). Biochem. Biophys. Res. Commun. 202:1373.[Medline]
12. Nguer, C. M., D. Treton, M. Rola-Pleszczynski, Z. Mishal, Y. Thomas, P. Galanaud, Y. Richard. 1996. Regulation of PAF receptor expression in human B cells and B cell lines. Lipids 10:1051.
13. Pistola, V., A. Corcione. 1995. Relationships between B cell cytokine production in secondary lymphoid follicles and apoptosis of germinal center B lymphocytes. Nat. Immun. Cell Growth Regul. 13:487.
14. Liu, Y.-J., D. E. Joshua, G. Williams, C. A. Smith, J. Gordon, I. C. M. MacLennan. 1989. Mechanism of antigen driven selection in germinal centers. Nature 342:929.[Medline]
15. Suzuki, N., Y. Ueda, T. Sakane. 1988. Differential mechanism for differentiation into immunoglobulin secreting cells in human resting B lymphocyte subsets isolated on the basis of cell density. J. Clin. Invest. 81:261.
16. Kovar, J., F. Franek. 1986. Serum free medium for hybridoma and parental myeloma cell cultivation. Methods Enzymol. 121:277.[Medline]
17. Mazer, B., K. L. Clay, H. Renz, E. W. Gelfand. 1990. Platelet-activating factor enhances Ig production in B lymphoblastoid cell lines. J. Immunol. 145:2602.[Abstract]
18. Hamid, Q., J. Barkans, Q. Meng, S. Ying, J. S. Abrams, A. B. Kay, R. Moqbel. 1992. Human eosinophils synthesize and secrete interleukin-6, in vitro. Blood 80:1496.[Abstract/Free Full Text]
19. Liu, Y. J., C. Arpin. 1997. Germinal center development. Immunol. Rev. 156:111.[Medline]
20. Amatruda, T. T. I., D. A. Steele, V. Z. Slepak, M. I. Simon. 1991. G16, a G protein subunit specifically expressed in hematopoietic cells. Proc. Natl. Acad. Sci. USA 88:5587.[Abstract/Free Full Text]
21. Rosskopf, D., K. Hartung, J. Hense, W. Siffert. 1995. Enhanced immunoglobulin formation of immortalized B cells from hypertensive patients. Hypertension 26:432.[Abstract/Free Full Text]
22. Smith, C. S., L. Parker, W. T. Shearer. 1994. Cytokine regulation by platelet-activating factor in a human B cell line. J. Immunol. 153:3997.[Abstract]
23. Rocha, F. A., L. E. Andrade, M. Russo, S. Jancar. 1997. PAF modulates eicosanoids and TNF release in immune-complex arthritis in rats. J. Lipid Mediat. Cell Signal. 16:1.[Medline]
24. Brown, M. A., J. Hural. 1997. Functions of IL-4 and control of its expression. Crit. Rev. Immunol. 17:1.[Medline]
25. Mazer, B. D., H. Sawami, A. Tordai, E. W. Gelfand. 1992. Platelet-activating factor-mediated transmembrane signaling in human B lymphocytes is regulated through a pertussis- and cholera toxin-sensitive pathway. J. Clin. Invest. 90:759.
26. Seyfried, C. E., V. L. Schweickart, R. Gdiska, P. W. Gray. 1992. The human platelet-activating factor receptor gene (PAFR) contains no introns and maps to chromosome 1. Genomics 13:832.[Medline]
27. Yang, H. H., J. H. Pang, R. Y. Hung, L. Y. Chau. 1997. Transcriptional reglation of platelet-activating factor receptor gene expression in B lymphoblastoid Ramos cells by TGF-ß. J. Immunol. 158:2771.[Abstract]
28. Thivierge, M., N. Alami, E. Muller, A. J. de Brum-Fernandes, M. Rola-Pleszczynski. 1993. Transcriptional modulation of platelet-activating factor receptor gene expression by cyclic AMP. J. Biol. Chem. 268:17457.[Abstract/Free Full Text]
29. Kuruvilla, A., C. Pielop, W. T. Shearer. 1994. Platelet-activating factor induces the tyrosine phosphorylation and activation of phospholipase C-1, Fyn and Lyn kinases, and phosphatidylinositol 3-kinase in a human B cell line. J. Immunol. 153:5433.[Abstract]
30. Siffert, W., D. Rosskopf, A. Moritz, T. Wieland, S. Kaldenberg-Stasch, N. Kettler, K. Hartung, S. Beckmann, K. H. Jakobs. 1995. Enhanced G protein activation in immortalized lymphoblasts from patients with essential hypertension. J. Clin. Invest. 96:759.
31. Mazer, B. D., B. J. Toledano, M. Saririan, Y. Bastien. 1998. Dose dependent agonist and antagonist effects of the platelet-activating factor analogue 1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine on B lymphocytes. J. Allergy Clin. Immunol. 102:231.[Medline]
32. Pistoia, V.. 1997. Production of cytokines by human B cells in health and disease. Immunol. Today 18:343.[Medline]
33. Matthes, T., C. Werner-Favre, R. H. Zubler. 1995. Cytokine expression and regulation of human plasma cells: disappearance of interleukin-10 and persistence of transforming growth factor-ß1. Eur. J. Immunol. 25:508.[Medline]
34. Yoshimoto, T., H. Okamura, Y.-I. Tagawa, Y. Iwakura, K. Nakanishi. 1997. Interleukin 18 together with interleukin 12 inhibits IgE production by induction of interferon- production from activated B cells. Proc. Natl. Acad. Sci. USA 94:3948.[Abstract/Free Full Text]
35. Tang, H., T. Matthes, N. Carballido-Perig, R. H. Zubler, V. Kindler. 1993. Differential induction of T cell cytokine mRNA in Epstein Barr virus transformed B cell clones: constitutive and inducible expression of interleukin-4 mRNA. Eur. J. Immunol. 23:899.[Medline]
36. Bastien, Y., N. Mehio, Q. Hamid, B. Mazer. 1997. Potential for human B lymphocytes to produce IL4 and IL13 following stimulation by platelet activation factor and phorbol ester and ionomycin. J. Allergy Clin. Immunol. 98:S632.
37. Kravchenko, V. V., Z. Pan, J. Han, J. M. Herbert, R. J. Ulevitch, R. D. Ye. 1995. Platelet-activating factor induces NF-B activation through a G protein-coupled pathway. J. Biol. Chem. 270:14928.[Abstract/Free Full Text]
38. Huang, Y. H., L. Schafer-Elinder, H. Owman, J. C. Lorentzen, J. Ronnelid, J. Frostegard. 1996. Induction of IL-4 by platelet-activating factor. Clin. Exp. Immunol. 106:143.[Medline]
39. Barthelson, R. A.. 1993. Quantitation of IL-4 expression in small numbers of cells from mice. J. Immunol. Methods 161:67.[Medline]
40. Rola-Pleszczynski, M., M. Thivierge, N. Gagnon, C. Lacasse, J. Stankova. 1993. Differential regulation of cytokine and cytokine receptor genes by PAF, LTB4 and PGE2. J. Lipid Mediat. 6:175.[Medline]
41. Zhang, F., K. Decker. 1994. Platelet-activating factor antagonists suppress the generation of tumor necrosis factor- and superoxide induced by lipopolysaccharide or phorbol ester in rat liver macrophages. Eur. Cytokine Netw. 5:311.[Medline]
42. Roth, M., M. Nauck, S. Yousefi, M. Tamm, K. Blaser, A. P. Perruchoud, H. U. Simon. 1996. Platelet-activating factor exerts mitogenic activity and stimulates expression of interleukin 6 and interleukin 8 in human lung fibroblasts via binding to its functional receptor. J. Exp. Med. 184:191.[Abstract/Free Full Text]
43. Lacasse, C., S. Turcotte, D. Gingras, J. Stankova, M. Rola-Pleszczynski. 1997. Platelet-activating factor stimulates interleukin-6 production by human endothelial cells and synergizes with tumor necrosis factor for enhanced production of granulocyte-macrophage colony stimulating factor. Inflammation 21:145.[Medline]
44. Ricci, M., A. Matucci, O. Rossi. 1997. IL-4 as a key factor influencing the development of allergen-specific Th2-like cells in atopic individuals. J. Invest. Allergol. Clin. Immunol. 7:144.[Medline]
45. Ricci, M., A. Matucci, O. Rossi. 1997. Source of IL-4 able to induce the development of TH2-like cells. Clin. Exp. Allergy 27:488.[Medline]
46. Arpin, C., J. Déchanet, C. Van Kooten. 1995. Generation of memory B cells and plasma cells in vitro. Science 268:720.[Abstract/Free Full Text]
47. Tew, J. G., J. Wu, D. Qin, S. Helm, G. F. Burton, A. K. Szakal. 1997. Follicular dendritic cells and presentation of antigen and costimulation to B cells. Immunol. Rev. 156:39.[Medline]
48. Imai, Y., M. Yamakawa. 1997. Morphology, function and pathology of follicular dendritic cells. Pathol. Int. 46:807.
49. Kroncke, R., H. Loppnow, H. D. Flad, J. Gerdes. 1996. Human follicular dendritic cells and vascular cells produce interleukin-7: a potential role for interleukin-7 in the germinal center reaction. Eur. J. Immunol. 26:2541.[Medline]
50. McManus, L. M., D. S. Woodard, S. I. Deavers, R. N. Pinckard. 1993. PAF molecular heterogeneity: pathobiological implications. Lab. Invest. 69:639.[Medline]

This article has been cited by other articles:

				Y. Matsumura, S. N. Byrne, D. X. Nghiem, Y. Miyahara, and S. E. Ullrich A Role for Inflammatory Mediators in the Induction of Immunoregulatory B Cells J. Immunol., October 1, 2006; 177(7): 4810 - 4817. [Abstract] [Full Text] [PDF]

				D. P. Harris, S. Goodrich, K. Mohrs, M. Mohrs, and F. E. Lund Cutting Edge: The Development of IL-4-Producing B Cells (B Effector 2 Cells) Is Controlled by IL-4, IL-4 Receptor {alpha}, and Th2 Cells J. Immunol., December 1, 2005; 175(11): 7103 - 7107. [Abstract] [Full Text] [PDF]

				S. Al-Darmaki, K. Knightshead, Y. Ishihara, A. Best, H. A. Schenkein, J. G. Tew, and S. E. Barbour Delineation of the Role of Platelet-Activating Factor in the Immunoglobulin G2 Antibody Response Clin. Vaccine Immunol., July 1, 2004; 11(4): 720 - 728. [Abstract] [Full Text]

				B. Johansson-Lindbom, S. Ingvarsson, and C. A. K. Borrebaeck Germinal Centers Regulate Human Th2 Development J. Immunol., August 15, 2003; 171(4): 1657 - 1666. [Abstract] [Full Text] [PDF]

				M. Donnard, L. Guglielmi, P. Turlure, C. Piguet, M.J. Couraud, D. Bordessoule, and Y. Denizot Membrane and Intracellular Platelet-Activating Factor Receptor Expression in Leukemic Blasts of Patients with Acute Myeloid and Lymphoid Leukemia Stem Cells, September 1, 2002; 20(5): 394 - 401. [Abstract] [Full Text] [PDF]

				Q. Zhuang, Y. Bastien, and B. D. Mazer Activation Via Multiple Signaling Pathways Induces Down-Regulation of Platelet-Activating Factor Receptors on Human B Lymphocytes J. Immunol., September 1, 2000; 165(5): 2423 - 2431. [Abstract] [Full Text] [PDF]

				Y. Ishihara, J.-B. Zhang, S. M. Quinn, H. A. Schenkein, A. M. Best, S. E. Barbour, and J. G. Tew Regulation of Immunoglobulin G2 Production by Prostaglandin E2 and Platelet-Activating Factor Infect. Immun., March 1, 2000; 68(3): 1563 - 1568. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire