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IMMUNOBIOLOGY
From The Johns Hopkins Asthma and Allergy Center,
Baltimore, MD.
The human recombinant histamine-releasing factor (HrHRF) was
previously shown to induce histamine release from human basophils from
a subset of donors. The ability of HrHRF to directly induce histamine
release from only certain basophils was thought to involve interaction
between HrHRF and a particular kind of IgE, termed IgE+, on
the surface of these cells. Recent studies disproved the hypothesis
that the IgE molecule or its high-affinity receptor, Fc Histamine-releasing factors (HRFs) comprise a group
of molecules that induce basophil degranulation. Molecules with this
activity include interleukins,1,2
chemokines,3-5 and the previously subcloned IgE-dependent
HRF.6 This human recombinant HRF (HrHRF) (also known as
p237 or translationally controlled tumor
protein8,9) was initially described as a complete
secretagogue for secretion of histamine6 and
interleukin (IL) 410 from basophils from a subset
of allergic donors. Mediator release was thought to be the consequence
of physical interaction between HRF and a certain type of IgE,
termed IgE+, on the surface of the responding
basophils.11 However, several lines of evidence suggest
that the interaction with IgE may not be required for cell activation
mediated by HrHRF. First, HrHRF enhanced anti-IgE-mediated
histamine release, as well as IL-4 and IL-13 protein production, in
donors with IgE Although one study found that eosinophils from a subset of
hypereosinophilic patients have Fc Eosinophils are known to contribute to the pathophysiologic mechanisms
of allergic diseases by secreting proinflammatory granule proteins,
such as major basic protein and eosinophil cationic protein, that
induce damage of bronchial epithelial cells.19,20 Increased numbers of these cells are found in bronchoalveolar lavage
(BAL) fluids and bronchial biopsy specimens from allergic asthmatic
patients during the late phase of an allergic reaction (LPR).21 There is evidence that these cells are capable of
synthesizing, storing, and in some cases, releasing cytokines that
contribute to cell recruitment and activation. Transcription or
translation of the following cytokines has been reported to occur in
eosinophils: IL-1 In this study, we demonstrated that HrHRF directly caused the release
of IL-8 or enhanced TNF- We used 10 × piperazine diethanesulfonic acid (PIPES) buffer
that contained 250 mmol/L PIPES (Sigma, St Louis, MO), 110 mmol/L sodium chloride (NaCl), and 50 mmol/L potassium chloride (KCl), adjusted to pH 7.4. PIPES-albumin-glucose (PAG) buffer was made with
10% 10 × PIPES and contained 0.003% human serum albumin
(Calbiochem-Novexbiochem Corp, La Jolla, CA) and 0.1%
D-glucose. PAG-calcium-magnesium (PAGCM) buffer was made by
adding 1 mmol/L calcium chloride (CaCl2) and magnesium
chloride (MgCl2) to PAG buffer. Percoll (Pharmacia, Piscataway, NJ) was made isotonic by mixing 1 part 10 × PIPES with 9 parts Percoll. This solution was then adjusted to a density of 1.09 g/mL by adding approximately 38 mL of 1 × PIPES to 100 mL of
isotonic Percoll and verifying the results with a
densitometer.33
For preparation of cell lysates, 1 mL of lysis buffer was used; this
consisted of 25 mmol/L HEPES, 5 mmol/L KCl, 119.4 mmol/L NaCl2, 1 mmol/L MgCl2, 0.5 mmol/L
CaCl2 (pH 8), 1 mmol/L sodium orthovanadate, 1% Nonidet
P-40, and 20 µL protease inhibitor mixture (PharMingen, San Diego,
CA). GM-CSF, TNF- HrHRF production
Endotoxin levels in purified HrHRF were determined by a limulus
amebocyte lysate assay (BioWhittaker, Walkersville, MD), according to
the manufacturer's specifications. Additionally, endotoxin was removed
from certain aliquots of HrHRF by using a column containing polymyxin B
immobilized on agarose (Detoxi-Gel; Pierce, Rockford, IL).
Purification of eosinophils
Cell cultures Human eosinophils.
Purified eosinophils were resuspended in medium M199 (Gibco, Grand
Island, NY) containing 20% fetal-calf serum (FCS; Sigma) with or
without GM-CSF (10 ng/mL) and incubated for 30 minutes at 37°C in 5%
carbon dioxide (CO2). Aliquots of
5 × 105 cells were transferred to 48-well
plates containing TNF- AML14-3D10 cells.
The AML14-3D10 subline, a gift of Dr Cassandra Paul (Wright State
University, Dayton, OH), is a human eosinophilic leukemic cell line
that consistently shows eosinophilic granules in 95% of its
cells.34 Cells were cultured in RPMI 1640 (Gibco BRL, Gaithersburg, MD), 8% FCS, 2 mmol/L L-glutamine, 1 mmol/L
sodium pyruvate, 50 µg/mL gentamicin (all from Biowhittaker), and
5 × 10 IL-8 determination Supernatants from stimulated eosinophils were assayed with an IL-8-specific ELISA (Biosource, Camarillo, CA), according to the manufacturer's specifications. The threshold for cytokine detection was 10 pg/mL. In some cases, the values obtained were compared with values determined by using another commercial kit (R&D Systems). No significant differences were observed between the 2 assays.Chemotaxis experiments Chemotaxis experiments were performed by using the modified Boyden chamber technique described previously.35 Briefly, 25 µL of PAGCM buffer or various concentrations of the stimuli in the same buffer were placed in the lower chamber in triplicate. A 5-µm pore-sized polycarbonate membrane (Nucleopore Corp, Pleasanton, CA) separated the upper and lower chamber. Eosinophils (105) resuspended in PAGCM buffer were placed in each well of the upper chamber on top of the membrane. The chamber was then incubated for 30 minutes at 37°C in 5% CO2 and air, after which the chamber was disassembled. The membrane was removed, washed in PAG buffer to remove the nonmigrating eosinophils from the upper surface, scraped, and stained with Wright stain. Eosinophils from 10 high-power fields of triplicate wells were identified and counted.Ca++ mobilization assay The Ca++ mobilization assay we used was a modification of a procedure described for human basophils by MacGlashan et al.36 Purified eosinophils were loaded with 1 µmol/L Fura 2-AM (Molecular Probes, Eugene, OR) for 30 minutes at 37°C in RPMI-1640 (Gibco BRL) containing 2% FCS and 0.32 mmol/L EDTA. In some experiments, eosinophils were incubated for 2 hours at 37°C in 5% CO2 with 1 ng/mL of PT or its -oligomer and Fura 2-AM
was added during the last 30 minutes of the incubation. The cells
(5 × 105) were washed once with PAG buffer and
resuspended in 200 µL of PAG for loading in the microscope
observation chamber. A 15-µL cell suspension was placed on a
siliconized (Sigma Cote; Sigma) coverslip that comprised the base of
the observation chamber. After 5 to 10 minutes of settling time, the
cell drop was overlaid with 1 mL of PAGCM buffer at 37°C. The
temperature, which was measured by a probe placed next to the settled
cells, was brought to a stable 36.5°C and the stimulus (dissolved in
1 mL of prewarmed buffer) was added. Intracellular changes in
Ca++ were monitored with a Zeiss Axiovert microscope with
epifluorescence capacity as described previously.37
Flow cytometry to determine surface IgE expression Cells were incubated for 30 minutes at 4°C in phosphate-buffered saline containing 0.2% bovine serum albumin (PBS-BSA) and 3.6 mg/mL human IgG with saturating concentrations of receptor-specific antibody or an equivalent concentration of an irrelevant isotype-matched control antibody. Surface IgE was detected by using a fluorescein isothiocyanate-conjugated polyclonal goat antihuman IgE (Kirkegaard and Perry, Gaithersburg, MD). The Fc RI  chain was investigated by using an IgG1 mouse antihuman
monoclonal antibody (mAb), 22E7 (provided by Dr J. Kochan, Hoffman-La
Roche Inc, Nutley, NJ). FcRII (CD23) was assessed by using mAb 9P.25
(Immunotech Inc, Westbrook, ME). Anti-FcRI mAb 22E7 is known to
detect the high-affinity IgE receptor on human
basophils,38 and anti-FcRII mAb 9P.25 detects the
low-affinity IgE receptor on human B cells.39 An IgG1 isotype control mAb was purchased from Sigma. Cells
were washed with PBS-BSA, incubated with 1:150 dilutions of
R-phycoerythrin-conjugated F(ab')2 goat antimouse IgG
antibody (Biosource) for 30 minutes at 4°C in the dark, washed,
resuspended in PBS with 0.2% BSA, and assayed immediately with a flow
cytometer (EPICS Profile II; Coulter, Hialeah, FL). Results
were expressed as mean fluorescence intensity.
Cell lysates and Western blots to determine the presence of the
Fc RI chain, at a dilution of 1:300.
After a 15-minute washing, the secondary antibody, sheep antimouse
horseradish peroxidase (Amersham Life Sciences, Piscataway, NJ), was
diluted 1:20 000 in PBS-T and incubated for 1 hour. After the final
washing step (6 times for 6 minutes each in PBS-T), the blot was
developed by using SuperSignal chemiluminescent substrate (Pierce) and
exposed to Hyperfilm ECL (Amersham Life Sciences).
Statistical methods Results are expressed as the mean ± SEM. Where indicated, cells incubated under different conditions were compared by using the Student t test. In the Ca++ mobilization experiments, the Student t test was used to compare time points before and after the addition of the stimulus. Specifically, the t test was used to compare the area under the curve for a relevant time period by using the initial phase of the calcium response, which is important in the PT response.
IL-8 production by HrHRF in eosinophils requires priming with GM-CSF To determine the optimal conditions for cytokine production by eosinophils stimulated with HrHRF, freshly isolated eosinophils were incubated with HrHRF, TNF-, and IL-5 alone and with a combination of
these 3 cytokines. None of these cytokines alone induced significant IL-8 secretion. Only the combination of TNF- and IL-5 induced significant cytokine production, which was only 400 pg/mL greater than
the value for the medium control (data not shown). Although significant
IL-8 production by peripheral blood eosinophils stimulated with TNF-
alone has been reported,23 other authors showed that priming of eosinophils with GM-CSF is required for optimal IL-8 secretion.28 Therefore, we tested whether preincubation of
eosinophils with GM-CSF would alter IL-8 secretion. Incubation of
eosinophils from 4 separate donors with GM-CSF for 30 minutes before
stimulation greatly enhanced the responsiveness of these cells to
TNF- but not to IL-5 (Figure 1). In
the presence of GM-CSF, HrHRF induced some IL-8 production above the
value for the medium control; however, HrHRF enhanced IL-8 secretion
induced by TNF- or IL-5. The magnitude of the enhancement
with HrHRF and TNF- was greater than that with HrHRF and IL-5.
Additionally, this enhancement of IL-8 production was comparable to
that induced by the combination of TNF- and IL-5 (Figure
1).
Although the HrHRF preparations produced in baculovirus used for these experiments contained low but variable amounts of endotoxin, we do not believe that contaminating endotoxin accounted for our results. Although there is one report that 10 ng/mL of lipopolysaccharide (LPS) induced IL-8 secretion from human eosinophils,40 we used a polymyxin B column to remove endotoxin and our HrHRF preparation contained 10 000-fold less endotoxin than the amount reported as necessary to induce IL-8. Thus, HrHRF that contained 8.5 ng/mL of endotoxin on limulus amebocyte lysate assay was placed on a Detoxi-Gel column containing polymyxin B immobilized on agarose and an 8500-fold reduction in endotoxin was achieved. This resulted in a barely detectable level of 1 pg/mL of endotoxin in the assay with eosinophils. Additionally, preparations that contained almost undetectable amounts of endotoxin (1 pg/mL) behaved similarly to those with higher levels of endotoxin (350 ng/mL). Moreover, when preparations of HrHRF containing 350 ng/mL of endotoxin were used, LPS at a concentration of 350 ng/mL did not reproduce the results (n = 4, data not shown). Although HrHRF alone did not significantly increase IL-8 production in
the 4 donor samples used to produce the results shown in Figure 1,
there was a trend toward an increase. Therefore, we examined samples
from additional donors. In 12 experiments using eosinophil samples from
7 different donors, HrHRF induced significant production of IL-8 by
eosinophils primed with GM-CSF, even in the absence of TNF-
IL-8 production by eosinophils requires protein synthesis It is known that some cytokines are both stored in a preformed state in granules and newly synthesized. For example, TNF- is
released from mast cells after cross-linking FcRI as a result of
both release from preformed cytoplasmic granule stores and de novo
synthesis.41 We questioned whether de novo protein
synthesis is required for cytokine-induced IL-8 production from
eosinophils. Stimulated eosinophils were incubated in the presence or
absence of 1 µmol/L cycloheximide. As shown in Figure
4, cycloheximide inhibited IL-8
production by all the stimuli. However, in 2 of 3 experiments, the
combination of HrHRF and TNF- induced small but measurable amounts
of IL-8 from cycloheximide-treated cells. Because cycloheximide did not
decrease cell viability, the decrease in HrHRF-stimulated IL-8
production was probably due to decreased protein synthesis.
Continued presence of HrHRF in cultures is necessary for enhanced IL-8 production We questioned whether incubation of eosinophils with HrHRF for short periods would provide the necessary signal to enhance IL-8 secretion induced by TNF-. Therefore, eosinophils treated with
GM-CSF were incubated for 1 hour with medium or HrHRF, washed, and
stimulated with additional medium or TNF- without HrHRF. The culture
continued for 24 hours. When HrHRF was present during only the first
hour of culture, no enhancing effect on TNF--induced IL-8
production was observed (1963 pg/mL versus 1778 pg/mL). In contrast,
when HrHRF was present throughout the culture period, 3913 pg/mL of
IL-8 was generated. Decreased IL-8 production in cells primed with
HrHRF was not a consequence of cell washing because comparable levels
of IL-8 were produced in cultures that had been washed and then
replenished with the appropriate cytokines (3848 pg/mL versus 3913 pg/mL).
IL-8 production by stimulated eosinophils is
inhibited by a modulator of G i subunit.13 In
contrast to these results with basophils, PT (1 ng/mL) moderately
reduced IL-8 secretion from GM-CSF-primed eosinophils that was induced
by HrHRF, TNF-, or the combination of HrHRF and TNF- (Figure
5). This reduction was a consistent
finding. To provide a control, cells were also stimulated in the
presence of the PT -oligomer that does not affect Gi
function. In the experiments shown in Figure 5, the PT -oligomer did
not affect IL-8 secretion induced by the various stimuli. These
experiments used a suboptimal concentration of TNF- (10 ng/mL),
which was also shown to have synergy with HrHRF for IL-8
production.
HrHRF induces Ca++ mobilization in eosinophils from some donors that is inhibited by PT Because human eosinophils are known to possess the receptor for GM-CSF,42 we investigated the effect of HrHRF on the Ca++ response in the absence of GM-CSF. Unlike the previous experiments, the initial Ca++ experiments did not include use of GM-CSF to prime the eosinophils. Figure 6 shows the average Ca++ response of eosinophils (4 different donors) preincubated with HrHRF in the presence of PT or the PT-oligomer. HrHRF induced an elevation
in Ca++ in eosinophils preincubated with the PT
-oligomer (P < .05), whereas in cells pretreated with
PT, the Ca++ response induced by either HrHRF or
platelet-activating factor (PAF), the positive control, was completely
ablated. Under these experimental conditions, Ca++
mobilization occurred in 4 of 6 samples from different donors tested.
However, in samples from a total of 9 donors examined over months,
Ca++ responses to HrHRF occurred in only 25% (9 of 36 experiments). The lack of responsiveness of some cell preparations was
not due to a general inability of the cells to mobilize
Ca++ because eosinophils always responded to stimulation
with PAF, regardless of the effect of HrHRF. In general, the
HrHRF-induced Ca++ response was less vigorous than that
induced by PAF.
Because HrHRF induced a Ca++ response only 25% of the
time, we subsequently preincubated the cells with GM-CSF. Preincubation of cells with GM-CSF, TNF- HrHRF is chemotactic for human eosinophils in vitro Teshima et al43 reported that recombinant p26 HRF injected into the peritoneum of ovalbumin-sensitized mice caused eosinophil recruitment within 4 hours. Because of this report, we performed eosinophil chemotaxis studies. Purified eosinophils were stimulated with HrHRF or PAF, and chemotaxis was assessed by using Boyden microchambers. In 3 experiments, the number of eosinophils migrating in the medium-control condition was 24 ± 4. The number of eosinophils migrating in response to HrHRF (0.4 µmol/L) was 54 ± 14, or 225% of the medium-control value; to HrHRF (0.24 µmol/L), 58 ± 6, or 243%; and to HrHRF (0.12 µmol/L), 18 ± 7, which was not above the medium-control value. In comparison, the results with the 2 positive controls were that 473 ± 65 eosinophils (1971%) migrated in response to PAF (10 7mol/L) and 157 ± 105 eosinophils (653%) migrated
in response to RANTES (100 ng/mL).
rHRF stimulates IL-8 production in the human eosinophilic cell line AML14-3D10 To demonstrate the effect of HrHRF on an eosinophilic cell line, AML14-3D10 cells were stimulated in the presence or absence of GM-CSF. Results were similar to those achieved with human eosinophils in the presence of GM-CSF: HrHRF stimulated IL-8 production to a level 2224 pg/mL above that with medium alone. Unlike eosinophils, AML14-3D10 cells did not need priming with GM-CSF for this to occur. This is not surprising, since these cells produce and use GM-CSF in an autocrine fashion.44 Even in the absence of GM-CSF, HrHRF produced a level of IL-8 in the AML14-3D10 cell line that was 1217 pg/mL above that with the medium control (n = 3).The effect of HrHRF on eosinophils and AML14-3D10 cells is not dependent on IgE It was previously reported that human eosinophils do not express FcRI but do have an intracellular pool of it.17 We
reproduced these data: our flow cytometry studies detected no
extracellular expression of IgE, FcRI, or FcRII on either
human eosinophils or AML14-3D10 cells (data not shown). We next
investigated whether the AML14-3D10 cells, like human eosinophils, had
an intracellular pool of FcRI. Western blot analysis of
AML14-3D10 cell lysates incubated with a mAb specific for FcRI
showed no band, whereas the matched, positive-control basophil lysates
had a distinct band of approximately 50 kd (Figure
7). Therefore, unlike human eosinophils,
AML14-3D10 cells have no detectable FcRI. Thus, HrHRF activates
eosinophils and AML14-3D10 cells through a mechanism that does not
depend on IgE or the FcRI receptor.
The principal findings of this study are that HrHRF can activate a
cell type other than the basophil and that it acts like a
cytokine. Our experiments demonstrated that HrHRF causes IL-8 production from eosinophils and AML14-3D10 cells primed with GM-CSF and
from AML14-3D10 cells in the absence of GM-CSF. Additionally, HrHRF is
chemotactic for eosinophils in vitro in the absence of GM-CSF, although
it is not known whether the concentrations required for chemotaxis have
in vivo relevance. HrHRF also induced Ca++ mobilization in
a subset of allergic donors in the absence of GM-CSF coculture. Because
there was no difference in viability or cell number in cultures
incubated with GM-CSF, we believe that GM-CSF may provide an activation
signal allowing cells to respond to HrHRF, TNF- The necessity of GM-CSF for IL-8 secretion from eosinophils stimulated
by other stimuli, such as RANTES or PAF, was reported previously.28 In contrast, Nakajima et al23
showed that preincubation with GM-CSF was not required for IL-8
secretion from eosinophils when 25 ng/mL TNF- In addressing the specificity of HrHRF activation of eosinophils, we
also considered the following. Human eosinophils express Mac-1
(CD11b),45 a molecule capable of binding various ligands, including LPS.46 Our finding of no difference in IL-8
production between experiments using preparations containing barely
detectable levels of endotoxin and experiments using preparations with
high endotoxin levels rules out endotoxin as the stimulus for the
results observed. Additionally, although both
neutrophils47 and monocytes48 were previously
shown to secrete IL-8 on stimulation with TNF- It might be argued that IL-8 production by HRF that requires GM-CSF and
TNF- HrHRF induced increases in intracytoplasmic Ca++ that were completely ablated by preincubation of eosinophils with PT. However, the Ca++ responses induced by HrHRF were observed in only 25% of donors. The reason for this low response rate is unclear. It may have been a consequence of variability among different eosinophil preparations. It is unlikely that it was due to differences in expression of a putative HRF receptor because HrHRF always had an effect on cytokine production that was independent of Ca++ mobilization. It is intriguing that both secretion and Ca++ mobilization
in eosinophils induced by HrHRF were inhibited by PT, whereas the HrHRF-induced histamine release from basophils from IgE+
donors was not inhibited by PT (data not shown). One possible explanation for these findings is that there may be differential HrHRF
signaling in these 2 cell types. There is precedent for this. The CC
chemokine, RANTES, showed 2 types of Ca++ signaling in
T-cell lines.59 The initial transient peak was sensitive
to PT, whereas the second, delayed Ca++ peak was inhibited
by protein tyrosine kinase inhibitors. These different Ca++
responses are associated with different functional responses. Although
one could propose that this difference is due to the presence of 2 different HrHRF receptors, as is the case with chemokine receptors,59 another likely explanation is the use of
different guanine nucleotide binding proteins (G proteins) in HrHRF
signaling in these 2 cell types. Studies of cardiac phenomena and of
bradykinins have found that different types of cells express different
amounts of G proteins.60-62 Additionally, the CC chemokine
MIP-1 Another difference between HrHRF activation of basophils and HrHRF
activation of eosinophils was observed in the HrHRF priming experiments. Priming of basophils with HrHRF for 15 minutes and subsequent washing of the cells did not diminish histamine release (data not shown). This suggests a high-affinity interaction between HrHRF and the basophil. On the other hand, preincubation of eosinophils with HrHRF for 1 hour and washing abolished the enhancement of TNF- Eosinophils from selected patients with
hypereosinophilia15 and eosinophils obtained after antigen
challenge16 were reported to express Fc
We thank Ms Nancy Dietz for secretarial support.
Submitted September 1, 1999; accepted May 18, 2000.
Supported by National Institutes of Health (NIH) grant RO1AI 20253 (D.W.M.) and NIH grant RO1 AI 32651 (S.M.M.) and partly supported by Bristol-Myers Squibb Pharmaceutical Research Institute.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Susan M. MacDonald, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD; e-mail: smacdona{at}mail.jhmi.edu.
1. MacDonald SM, Schleimer RP, Kagey-Sobotka A, Gillis S, Lichtenstein LM. Recombinant IL-3 induces histamine release from human basophils. J Immunol. 1988;142:3527[Abstract]. 2. White MV, Yoshimura T, Hook W, Kaliner MA, Leonard EJ. Neutrophil attractant/activation protein-1 (NAP-1) causes human basophil histamine release. Immunol Lett. 1989;22:151[Medline] [Order article via Infotrieve]. 3. Kuna P, Reddigari SR, Schall TJ, Rucinski D, Sadick M, Kaplan AP. Characterization of the human basophil response to cytokines, growth factors, and histamine releasing factors of the intercrine/chemokine family. J Immunol. 1993;150:1932[Abstract]. 4. Alam R, Forsythe P, Stafford S, et al. Monocyte chemotactic protein-2, monocyte chemotactic protein-3, and fibroblast-induced cytokine: three new chemokines induce chemotaxis and activation of basophils. J Immunol. 1994;153:3155[Abstract]. 5. Bischoff SC, Krieger M, Brunner T, et al. RANTES and related chemokines activate human basophil granulocytes through different G protein-coupled receptors. Eur J Immunol. 1993;23:761[Medline] [Order article via Infotrieve]. 6. MacDonald SM, Rafnar T, Langdon J, Lichtenstein LM. Molecular identification of an IgEdependent histamine-releasing factor. Science. 1995;269:5688.
7.
Burckhard G, Gaestel M, Böhm H, Bielka H.
cDNA sequence coding for a translationally controlled human tumor protein.
Nucleic Acids Res.
1989;17:8367 8. Böhm H, Benndorf R, Gaestel M, et al. The growth-related protein p23 of the Ehrlich ascites tumor: translational control, cloning and primary structure. Biochem Int. 1989;19:277[Medline] [Order article via Infotrieve]. 9. Sanchez J-C, Schaller D, Ravier F, et al. Translationally controlled tumor protein: a protein identified in several nontumoral cells including erythrocytes. Electrophoresis. 1997;18:150[Medline] [Order article via Infotrieve].
10.
Schroeder JT, Lichtenstein LM, MacDonald SM.
An immunoglobulin E-dependent recombinant histamine-releasing factor induces interleukin-4 secretion from human basophils.
J Exp Med.
1996;183:1265 11. MacDonald SM, Lichtenstein LM, Proud D, et al. Studies of IgE-dependent histamine releasing factors: heterogeneity of IgE. J Immunol. 1987;139:506[Abstract]. 12. Schroeder JT, Lichtenstein LM, MacDonald SM. Recombinant histamine-releasing factor enhances IgE-dependent IL-4 and IL-13 s |
Top
Abstract
Introduction
chains of Fc
20°C until analysis by
enzyme-linked immunosorbent assay (ELISA). In some experiments,
cycloheximide (1 µmol/L) (Sigma) was added at the beginning of the
culture. Cycloheximide did not affect cell viability as determined by
staining with erythrosin B (Sigma).
G(s), G(o), and G(z)