| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
RI+ Cells Through Interaction with the VH3 Region of IgE1
*
Division of Clinical Immunology and Allergy, University of Naples Federico II, and
Istituto Nazionale Tumori Giovanni Pascale, Naples, Italy; and
Unité d’Immunopathologie Humaine, Institut National de la Santé et de la Recherche Médicale U430, Hôpital Broussais, Paris, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-chain of Fc
RI was unaffected by this
treatment. Three human VH3+ monoclonal IgM
concentration-dependently inhibited pFv-induced secretion of IL-4 and
histamine from basophils and of histamine from human lung mast cells.
In contrast, VH6+ monoclonal IgM did not
inhibit the release of IL-4 and histamine induced by pFv. These results
indicate that pFv, which acts as an endogenous superallergen, interacts
with the VH3 domain of IgE to induce the synthesis and
release of IL-4 from human FcRI+
cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
35%), C (HBC; 43%), A, and E
(1, 2). pFv is an acidic sialoprotein (pI, 4.0) with a molecular
mass of 175 kDa that dissociates into 85-kDa monomers during SDS-PAGE.
pFv binds to Ig from various mammalian and nonmammalian species (3); it
binds the variable domain of heavy chains (VH),
irrespective of Ig class, subclass, allotype, and light (L) chain type
(4). Silverman and Bouvet recently demonstrated that the binding of
this molecule involves mainly the human VH3 family (5, 6).
Because, like staphylococcal protein A (6), pFv binds outside the
Ag-binding site of Ab (4), it has been allocated to a new group of B
cell superantigens (5, 7). A wide spectrum of hepatic and extrahepatic tissue injuries, not necessarily directly related to the cytotoxic effects of HBV and HCV infections, are associated with acute and chronic viral hepatitis (8, 9). Skin rashes, urticarial reactions involving skin mast cells, and cutaneous and systemic vasculitis occur in patients with viral hepatitis (8, 9, 10, 11). Human basophils and mast cells, through the release of vasoactive and proinflammatory mediators (histamine, etc.) (12, 13) and the elaboration of various cytokines (IL-4, etc.) (14, 15, 16, 17, 18, 19, 20, 21), play a role in cutaneous and systemic vasculitis (12) and stimulate the collagen synthesis (22, 23) characteristic of the fibrotic response in chronic liver diseases (24).
Several laboratories have demonstrated that human basophils express and
secrete such cytokines as IL-4 (17, 18, 19), IL-13 (19, 20), and macrophage
inflammatory protein (MIP)-1 (21). The striking correlation between
IL-4 mRNA and protein secretion from basophils activated by
IgE-mediated stimuli suggested that basophils are a major source of
IL-4 (18). This important cytokine promotes isotype switching and
induction of IgE synthesis by B cells (25, 26, 27), down-regulates the
production of proinflammatory cytokines by monocytes and macrophages
(28, 29), stimulates the expression of VCAM-1 on endothelial cells
(30), and displays antitumor activity in vivo (31). IL-4 also regulates
the cytokine profile and function of CD4+ T cells (26, 32).
We have previously demonstrated that pFv induces the release of
proinflammatory mediators from human basophils and mast cells isolated
from lung parenchyma and skin tissue, presumably by interacting with
the IgE bound to FcRI (33). This study has been undertaken to
investigate the mechanism of the effects of pFv on the release of
mediators from human basophils and mast cells and to evaluate whether
this protein induces IL-4 synthesis and secretion in human basophils.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following were purchased: 60% HClO4 (Baker
Chemical, Deventer, The Netherlands); PIPES, hyaluronidase,
collagenase, chymopapain, and elastase type I (Sigma, St. Louis, MO);
HBSS and FCS (Life Technologies, Grand Island, NY); deoxyribonuclease I
(Calbiochem, La Jolla, CA); RPMI 1640 with 25 mM HEPES buffer, Eagle’s
MEM (Flow Laboratories, Irvine, Scotland); Dextran 70, Percoll, and
protein A-Sepharose (Pharmacia, Uppsala, Sweden). Rabbit
anti-human-Fc Ab was a generous gift from Drs. Teruko and Kimishige
Ishizaka (La Jolla Institute for Allergy and Immunology, La Jolla, CA).
The mAb against the -chain of FcRI was a generous gift of Dr.
John Hakimi (Roche Research Center, Hoffmann-La Roche, Nutley, NJ).
Preparation of pFv
pFv was purified by size permeation through a set of three 16 x 900-mm columns of S300 Sephacryl (Pharmacia) from the stool extracts of two patients (FAR and TER) affected by HCV hepatitis. Under these conditions, pFv elutes in the 175-kDa fraction as confirmed by HPLC with a calibrated Superose 12 column (Pharmacia) (1). Purified fractions were extensively dialyzed against PBS to remove both sodium deoxycholate and sodium azide and were stored at 4°C under sterile conditions.
Ig-binding test
pFv preparations were assayed for Ig-binding activity by a nonimmune ELISA-like method (1). Polyclonal normal IgG (2.5 µg/ml) or F(ab')2 fragments were coated on Nunc plates. After three washes with 0.1% Tween 20 in PBS, the plates were saturated with a PBS solution containing 1% BSA and 0.05% Tween 20. The diluted extracts were left overnight at 4°C to bind the coated Ig. Ig-binding molecules were then revealed by their nonimmune binding to 0.5 µg/ml of peroxidase-labeled human IgG or F(ab')2 fragments after a 1-h incubation at 37°C in BSA-Tween PBS. This assay thus detected only bivalent or multivalent molecules. Results were considered positive when the OD was >0.1 U at 492 nm and more than twofold the OD of a negative control. For each preparation of pFv, the activity giving 0.5 OD at 492 nm was obtained with the following dilutions: 1:200 (TER pFv) and 1:128 (FAR pFv). The appropriate concentration of pFv as ponderal value was measured by this assay as described elsewhere (6).
Absorption experiments
For pFv absorption, 9 vol of sample were incubated twice with 1 vol of IgG-coated protein A-Sepharose beads under constant mixing at 37°C for 1 h. The supernatant was then checked for loss of pFv activity, extensively dialyzed, and stored at 4°C under sterile conditions. To remove Ig, 9 vol of sample were incubated with 1 vol of anti-F(ab')2 immunosorbent. The latter was obtained by CNBr insolubilization of specific Abs raised in sheep, a species unreactive with pFv (3). In both cases, unabsorbed samples had been incubated with uncoated Sepharose 4B.
Buffers
The PIPES buffer (P) used in these experiments contains 25 mM PIPES, pH 7.37, 110 mM NaCl, 5 mM KCl. PCG buffer contains, in addition to P, 5 mM CaCl2 and 1 g/L dextrose (33); pH was titrated to 7.4 with sodium bicarbonate. PBS contains (g/L): NaCl, 8.0; Na2HPO4·7H2O, 2.89; KH2PO4, 0.2; KCl, 0.2, pH 7.3. TCF buffer contains (g/L): NaCl, 8.0; KCl, 0.2; NaH2PO4, 0.05; NaHCO3, 0.28; D-glucose, 1.0, pH 7.3; TGMD contains, in addition to TCF, pH 7.3, 0.25 g/L MgCl2·6H2O, 10 mg/L DNase, and 1 g/L gelatin (34).
Purification of human monoclonal IgE and IgM
IgE myeloma proteins were purified from the sera of two myeloma patients (PP and ADZ) by repeated gel filtration on Sephadex G-200, followed by elution through a Sepharose CL-4B column. RIA showed no IgG, IgM, or IgA contamination (35). Monoclonal IgM were purified from the sera of patients with Waldenstrom’s macroglobulinemia by gel permeation in a diluted buffer as described (36). Variable regions of these monoclonal IgM were determined using a well-characterized panel of primary sequence-dependent VH and VK family-specific reagents that identify previously described framework regions (6).
Purification of peripheral blood basophils
Basophils were purified from peripheral blood cells of normal subjects, ages 21 to 39 yr (mean age, 32.4 ± 4.1 yr), undergoing hemapheris. "Buffy coat" cell packs from healthy volunteers, provided by the Immunohematology Service at the University of Naples Federico II, were reconstituted in PBS containing 0.5 g/L human serum albumin and 3.42 g/L sodium citrate and loaded onto a countercurrent elutriator (model J2-21, Beckman Instruments, Fullerton, CA). Several fractions were collected, and fractions containing basophils in large numbers (>20 x 106 basophils) and of improved purity (>15%) were further enriched by discontinuous Percoll gradients (37). Yields by this technique ranged from 3 to 10 x 106 basophils with a purity from 74–98%, as assessed by basophil staining with Alcian blue and counting in a Spiers-Levy eosinophil counter (38).
Isolation and purification of human lung mast cells (HLMC)
Macroscopically normal lung tissue obtained from patients undergoing thoracotomy and lung resection, mostly for lung cancer, was dissected free from pleura, bronchi, and blood vessels, minced into 3- to 8-mm fragments and dispersed into single-cell suspension as described (34). Yields with this technique ranged between 3–20 x 106 mast cells, and mast cell with a purity ranged between 1 and 8%. The cells were resuspended and incubated overnight in RPMI 1640 containing 25 mM HEPES, 2 mM L-glutamine, 1% gentamicin, and 10% FCS as previously described (13). Mast cells isolated from lung parenchyma were fractionated in a Beckman elutriator fitted with an elutriation apparatus; elutriation fractions with the highest purity mast cells were pooled and were further purified by flotation in Percoll density gradients as described (34). The fractions rich in mast cells were then counted by Alcian blue staining (38).
Histamine release assay
Basophils (6 x 104 basophils/tube) or mast
cells (3 x 104 mast cells/tube) cells resuspended
in PCG, and 0.1 ml of the cell suspension were placed in 12 x
75-mm polyethylene tubes and warmed to 37°C; 0.1 ml of each prewarmed
stimulus for release was added, and incubation was continued at 37°C
for 45 min (histamine release) or 4 h (IL-4 secretion). At the end
of this step, the reaction was stopped by centrifugation (1000 x
g, 22°C, 2 min), and the cell-free supernatants were
stored at -20°C for subsequent assay of histamine and IL-4 content.
The cell-free supernatants were assayed for histamine with an automated
fluorometric technique (39). Total histamine content was assessed by
lysis induced by incubation of cells with 2% perchloric acid before
centrifugation. To calculate histamine release as a percentage of total
cellular histamine, the "spontaneous" release of histamine from
basophils (0–10% of the total cellular histamine) and mast cells
(5–20% of the total cellular histamine) was subtracted from both
numerator and denominator. All values are based on means of duplicate
or triplicate determinations. Replicates differed from each other in
histamine content by <10%.
IL-4 ELISA
The harvested supernatants were assayed for IL-4 by using the IL-4 Quantikine High sensitivity kit (R&D Systems, Minneapolis, MN). The standard curve for this kit was run in the same RPMI 1640 medium used for the release experiments (18).
Isolation of cellular mRNA
RNA was isolated by harvesting the basophils from culture wells and centrifuging for 30 s at 10,000 x g. After removing the supernatants, the cell pellet was extracted with RNAzol B (Tel-test, Friendswood, TX), which is a modified guanidinium thiocyanate single-step procedure. In this procedure, RNA is extracted from an aqueous phase after the addition of CHCL3. The isopropanol precipitated RNA was washed once with 70% ethanol, dried, resuspended in 25 µl of diethylpyrocarbonate-treated water, and stored at -80°C.
RT-PCR and quantitative PCR
An aliquot of total cellular mRNA was reversed transcribed to
cDNA and PCR expanded using the GeneAmp RNA PCR Core Kit (Perkin-Elmer,
Nieuwerkerk, The Netherlands) according to the manufacturer’s
protocol. In this protocol, RT was performed on 2 µl of the RNA
extract (10–20% of the total RNA extracted). The RT mix (5 mM
MgCl2) was incubated for 20 min in a Perkin-Elmer Cetus
thermocycler followed by 2 min at 95°C to inactivate the reverse
transcriptase. Buffers, dNTPs (final concentration, 0.4 mM each),
Amplitaq polymerase (1 U/50 µl reaction), and paired primers (0.5
µM each) were added to RT tubes (bringing MgCl2 to 2 mM)
and the PCR reaction cycled according to the following protocol:
denaturation at 95°C for 15 s, annealing at 60°C for 15 s
and at 72°C for 30 s. IL-4 was cycled 56 times before a 15-min
incubation at 72°C for a final extension. The primers for IL-4 and
IFN-
were performed and synthesized by a commercial source (Life
Technologies Italia, Milan, Italy) based on the known cDNA sequences of
the following cytokines: IL-4, 5' primer (5'-ATG GGT CTC ACC TCC CAA
CTG CT) and 3' primer (5'-GTT TTC CAA CGT ACT CTG GTT GGC); IFN-, 5'
primer (5'-ATG AAA TAT ACA AGT TAT ATC TTG GCT TT) and 3' primer
(5'-GAT GCT CTT CGA CCT CGA AAC AGC AT).
An aliquot (10 µl) of the reaction product was visualized on 2% agarose in buffer containing 0.5 µg/ml of ethidium bromide. As a negative control, an aliquot of each RNA sample was subjected to PCR amplification without the reverse transcription step.
In some experiments, an aliquot of total cellular mRNA was reverse-transcribed to cDNA and PCR expanded for quantitative PCR using a Cytopress detection kit (BioSource, Camarillo, CA). This technique is a competitive PCR in which a known copy number of an exogenously synthesized DNA, known as the internal calibration standard (ICS), is mixed with the sample cDNA prior to amplification. The ICS was constructed to contain PCR primer binding sites identical to the IL-4 cDNA and a unique capture binding site that allows the resulting ICS amplicon to be distinguished from the IL-4 amplicon. The Cytopress kit contains IL-4 primers, one of which is biotinylated, to be included in the PCR mix. During amplification, biotin-labeled primer is incorporated into both ICS and IL-4 amplicons. After PCR, the amplicons are denaturated and hybridized to either ICS or IL-4 sequence-specific capture oligonucleotides. Capture oligonucleotides are prebound to microtiter wells. The captured sequences are detected and quantified by the addition of an enzyme-streptavidin conjugate followed by substrate. The signal generated in the reaction is proportional to the amount of amplicon present. Since the ICS is amplified at an efficiency identical to the IL-4 cDNA, it can serve as a standard for IL-4 cDNA quantitation. The number of copies of IL-4 in each PCR reaction is calculated from the ratio of the total OD for the IL-4-specific well to the total OD for the ICS well and the input copy number of the ICS. The following formula is used to calculate the starting copies of IL-4 cDNA in the PCR reaction: [(total IL-4/total ICS OD) x (2 x ICS input copy number)] = starting copy number of IL-4 cDNA. Factor 2 is used to correct for dsDNA ICS. The copy number has been adjusted for any dilution done on cDNA before amplification according manufacturer’s protocol.
Statistical analysis
The results are the mean ± SEM. The data subjected to linear regression were calculated by the least squares method (y = a + bx) in which a was the y-axis intercept and b the slope of the line. The correlations were calculated by Student’s t test (40).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In a first series of experiments, we compared the
histamine-releasing activity of increasing concentrations of pFv
purified from patients FAR and TER on histamine release from human
basophils. Low concentrations of pFv (0.1–1 ng/ml)
concentration-dependently released histamine from basophils purified
(64–93%) from seven donors. pFv was at least 100 times more potent
than anti-IgE (Fig. 1
). pFv had no
effect on basophil viability (data not shown). The same preparations of
pFv (FAR and TER) absorbed with protein A-Sepharose coated with human
polyclonal IgG, which were negative for Ig binding, did not induce
histamine release. This result indicates that pFv was responsible for
mediator secretion from basophils by binding to Ig.
|
In a second series of experiments, we evaluated the effects of
increasing concentrations of pFv (FAR and TER) on histamine and IL-4
release from human basophils. Fig. 2
shows that pFv (0.1–1 ng/ml) concentration-dependently induced the
release of histamine and IL-4 from purified (60–96%) basophils. The
same preparations of pFv (FAR and TER) absorbed with protein
A-Sepharose coated with normal IgG did not induce histamine or IL-4
release in any subject studied.
|
In eight experiments, using basophils enriched to 95–98% purity,
cells were challenged with a wide range of concentrations of pFv
(0.3–1 ng/ml) obtained from patients FAR and TER (Fig. 3). pFv concentration-dependently induced
both IL-4 and histamine release from basophils. A significant
correlation was found between the percent histamine release and IL-4
secretion induced by pFv (rs = 0.70;
p < 0.001). These results are in agreement with the
hypothesis that pFv induces IL-4 secretion from basophils.
|
Fig. 4 compares the kinetics of
histamine and IL-4 release from basophils challenged with an optimal
concentration (0.5 ng/ml) of pFv. Histamine release induced by maximal
stimulation was complete within 5 min, whereas IL-4 release was
complete in 2–3 h. The average t1/2 for
histamine release was 3.5 ± 1.5 min, whereas the average
t1/2 for IL-4 release was 79.5 ± 8.5 min
(p < 0.001).
|
The studies described above support the hypothesis that human
basophils secrete IL-4 after stimulation with pFv. Further evidence in
favor of this hypothesis comes from a series of measurements of the
IL-4 mRNA present in resting and in pFv-stimulated basophils. In these
experiments, highly purified basophils (97–99%) were cultured with
recombinant human IL-3 (rhIL-3; 10 ng/ml) for 16 h, washed,
and challenged with anti-IgE (0.3 µg/ml) or pFv (0.5 ng/ml) in
the presence of rhIL-3 (10 ng/ml). We measured IL-4 mRNA expression in
basophils by RT-PCR. In three experiments, one of which is illustrated
in Fig. 5, we found that IL-4 mRNA is
constitutively expressed in human basophils (Fig. 5A);
panel A also shows that pFv induced an apparent increase in
IL-4 mRNA similar to the previously reported stimulation with
anti-IgE (18). In these experiments in which IL-4 mRNA was detected
by RT-PCR, we were unable to find any IFN- mRNA expression in
control basophils or in pFv- and anti-IgE-stimulated basophils
(Fig. 5B). Therefore, IL-4 mRNA was extracted from purified
preparations of basophils uncontaminated by IFN--synthesizing
leukocytes (e.g., lymphocytes and monocytes).
|
shows a representative result of three experiments in which we examined
the effects of anti-IgE and pFv on the secretion of histamine, on
the extracellular protein levels of IL-4, and on the specific IL-4 mRNA
in unstimulated purified (97%) basophils and cells activated by
anti-IgE (0.3 µg/ml) or pFv (0.5 ng/ml). These experiments
confirmed the presence of constitutive expression of IL-4 mRNA and
demonstrated that pFv and anti-IgE increased specific IL-4 mRNA
copies (Fig. 6A). The results shown in panels A
and B demonstrate that pFv and anti-IgE stimulated the
release of IL-4 in parallel to the secretion of histamine from
basophils.
|
Two immunophilin-binding drugs, CsA and tacrolimus
(FK-506), are potent inhibitors of the IgE-dependent release of
proinflammatory mediators from human basophils and mast cells (34, 41, 42). We compared the effects of preincubation of low concentrations of
CsA (8–800 nM) and tacrolimus (1–100 nM) on the release of histamine
and on the secretion of IL-4 from purified basophils activated by pFv.
CsA concentration-dependently inhibited pFv-induced release of
histamine and of IL-4 from basophils at concentrations as low as 240 nM
(Fig. 7A). The inhibition of
IL-4 release ranged from 10% at 8 nM to 90–95% at 800 nM, with a
median inhibitory concentration (IC50) of 12.1 ± 2.1
nM, similar to that calculated from the corresponding inhibition of
histamine release (15.1 ± 5.7 nM). The effects of tacrolimus on
the of release histamine and on the secretion of IL-4 from human
basophils were similar to those observed with CsA (Fig. 7B).
However, tacrolimus was 10 times more potent than CsA; i.e., for
IL-4 release, it had an IC50 of 1.8 ± 0.6 nM, similar
to that calculated from the corresponding inhibition of histamine
release (1.5 ± 1.1 nM).
|
In eight experiments, basophils of a purity from 76–96% were challenged with a range of concentrations of pFv from patients FAR and TER (0.1–1 ng/ml) and anti-IgE (0.03–1 µg/ml). A significant correlation was found between the maximum IL-4 release induced by anti-IgE and that induced by pFv (rs = 0.58; p < 0.01), in agreement with the hypothesis that pFv might induce IL-4 release from basophils by interacting with IgE (data not shown).
Effect of IgE stripping on pFv-induced IL-4 release from human basophils
Brief exposure to low pH removes most of the IgE bound on
basophils, thus markedly reducing the activating properties of
IgE-mediated stimuli (35, 43). Fig. 8
illustrates the representative results of three experiments showing
that brief exposure to lactic acid completely blocks the effect of pFv
and of anti-IgE on IL-4 secretion from basophils. In contrast, the
response to a mAb cross-linking the -chain of FcRI (anti-
FcRI) (33, 44) was not affected by this treatment. As a control,
the same preparation of absorbed pFv, which is negative for Ig binding,
did not induce IL-4 release.
|
To evaluate the mechanism whereby pFv activates basophils, the
protein was preincubated with monoclonal IgM of different
VH families (7). In a group of three experiments, one of
which is illustrated in Fig. 9A, preincubation of basophils
with three preparations of monoclonal IgM (M3, M11, and LAN),
which possess a VH3 domain (7), concentration-dependently
inhibited the histamine releasing activity of pFv. In contrast, a
monoclonal IgM (M14), which possesses a VH6 domain, had a
marginal effect. Similarly, the three preparations of monoclonal
VH3+ IgM concentration-dependently inhibited
IL-4 secretion from basophils, whereas IgM M14 had no effect (Fig. 9B). These results confirm that the binding to
VH3 domain inhibits the binding of pFv to IgE bound to
FcRI on basophils.
|
Lung involvement has been documented in patients with type II
cryoglobulinemia (45), which is associated with HCV infection (46, 47).
pFv from donors FAR and TER concentration-dependently induced histamine
release from HLMC. In contrast, the absorbed preparations of pFv from
FAR and TER, which no longer bind Ig, failed to induce histamine
secretion from HLMC (data not shown). Fig. 10 shows the mean ± SEM of three
experiments in which HLMC were challenged with pFv from patient TER.
Preincubation with the three VH3+ monoclonal
IgM concentration-dependently inhibited the release of histamine from
HLMC. In contrast, the VH6+ IgM M14 had no
effect on pFv-induced activation of HLMC.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI+ cells of such an important cytokine as IL-4 that
possesses several immunoregulatory functions.
The IL-4-releasing activity of purified pFv preparations is due to pFv
itself and not to contamination by natural anti-Id, anti-L
chain, or anti- Abs reacting with IgE. Indeed, this activity was
fully abolished by removal of pFv with insoluble Ig, and it remained
unchanged after Ig absorption with an immunosorbent
anti-F(ab')2. The mechanism of activation of human
inflammatory cells possessing FcRI appears to be mediated by
interaction of pFv with the VH3 region of membrane-bound
IgE. This hypothesis is supported by the observations that: 1) pFv
binds with high affinity all Ig isotypes (4, 33); 2) there is a highly
significant correlation between the maximal mediator release induced by
anti-IgE and that induced by pFv; and 3) basophils from which IgE
had been dissociated by brief exposure to lactic acid no longer
released IL-4 in response to pFv and anti-IgE. These findings agree
with the notion that pFv has six distinct functional Fab binding sites
(48) that enable binding by at least 50% of human
F(ab')2 fragments (5). Therefore, pFv, which has been shown
to funcation as an endogenous B cell superantigen (5, 6, 7), can also
function as an endogenous superallergen, i.e., it reproduces the
releasing activity of specific allergens and of anti-IgE on human
FcRI+ cells (49, 50).
This study provides insight into the mechanism of interactions between
pFv and IgE bound on the basophil/mast cell membrane. Three different
monoclonal IgM with a VH3 domain inhibited the release of
histamine and of IL-4 induced by pFv from basophils, whereas an IgM
with a VH6 domain had no effect. This confirms that the
releasing activity of pFv depends on the binding to an Ig
structure not located in the CH constant regions but in the
VH3 domain, a fragment common to all Ig classes and
subclasses. Since pFv does not bind free or combined L chains (4), the
Ig structure in question must be the VH domain of IgE, as
demonstrated by direct evidence using Ig fragments (4). Structural
variations between VH families explain why the
VH6+ IgM M14 failed to inhibit the releasing
effect exerted by pFv on FcRI+ cells, whereas the three
VH3+ IgM exerted a concentration-dependent
inhibition.
Our results provide the first evidence that an endogenous
superallergen, pFv, largely released during viral infections, can
induce the synthesis and secretion of such an important cytokine as
IL-4 from human basophils. Interestingly, the activity of pFv is
extremely potent, which raises the possibility that it exerts important
effects in vivo. The activity of IL-4 has been linked to several
immunoregulatory functions including the synthesis of IgE by B
lymphocytes (25, 26, 27), adherence and selective eosinophil and basophil
endothelial transmigration (30), antitumor activity in vivo (31), and
development of T lymphocytes expressing a Th2 phenotype (25, 26, 27). Given
the biologic importance of IL-4, our findings could have biologic
relevance. pFv, found in normal liver (1), occurs as a free molecule in
the gut lumen of patients with hepatitis A, B, C, and E (2). It might
be envisioned that pFv released in the blood of patients with acute and
chronic infection with HCV and HBV contributes, through the release of
IL-4 from FcRI+, to the increased serum IgE levels found
under these conditions (51, 52, 53).
Our data represent the first indication that viruses can induce in vivo
the release of an endogenous superallergen (pFv) that acts on human
FcRI+ cells to release cytokines. This novel finding
might be of interest not only to students of allergic diseases
in which basophils, mast cells, and their mediators play a key
pathogenetic role (14, 16, 34). There is increasing evidence that human
basophils (17, 18, 19, 20, 21) and mast cells (14, 15, 16, 54, 55) synthesize a growing
list of cytokines and chemokines, thus playing a more complex
immunoregulatory role than previously anticipated (56). Therefore, the
mechanism of basophils/mast cell activation by pFv represents a new
model for a pathogenetic link between viral infections,
FcRI+ cell activation, and cytokine release.
Our previous in vitro (34, 41) and in vivo (42) studies
demonstrated that immunophilin-binding drugs (i.e., CsA and
tacrolimus) exert anti-inflammatory effects by inhibiting the
IgE-dependent release of proinflammatory mediators from human
FcRI+ cells. Here, we demonstrate that CsA and
tacrolimus also concentration-dependently inhibit the pFv-induced
release of IL-4 from human basophils. Based on these findings, it would
appear that some of the effects of this class of
anti-inflammatory/immunosuppresive drugs exerted on
basophil-dependent inflammatory reactions (57, 58) reflect actions of
these drugs also on the secretion of IL-4 from FcRI+
cells.
Besides an increase of the secretory "immune exclusion" (48), the
pathophysiologic role of pFv is largely unknown. One may speculate that
pFv released in the digestive tract provides a stimulus for IL-4
secretion from FcRI+ cells, which triggers B cells to
produce IgE. IgE bound to mast cells might play a role in the
elimination of helminthic infections (59, 60), thus providing an
explanation of a new ancestral role of such an endogenous
superallergen as pFv in defense mechanisms. Indeed, the possibility of
a parallel anti-infectious role of a pFv-related superantigen bound
to natural Abs has been raised (2, 61).
The concept that superallergens from diverse biologic origins can induce IL-4 secretion from human basophils is both without precedent and of potential interest. Staphylococcus aureus protein A (62) and the HIV-1 retrovirus envelope protein (gp120) have a VH3 family-directed specificity (63, 64) that may contribute to the immunodeficient state present in AIDS patients, presumably through release of IL-4 (65, 66). It might, therefore, be important to investigate whether gp120 can modulate IL-4 secretion from human basophils.
In conclusion, we demonstrate that pFv can activate human
FcRI+ cells to release cytokines, probably through
interaction with the VH3 region of IgE. This finding
supports the hypothesis that the in vivo release of pFv, previously
recognized as a B cell superantigen (5, 7) and here identified as a
superallergen, might be pathophysiologically relevant.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Gianni Marone, Division of Clinical Immunology and Allergy, University of Naples Federico II, Via S. Pansini 5, 80131 Napoli, Italy. E-mail address:
3 Abbreviations used in this paper: pFv, Fv fragment-binding protein; anti-FcRI, anti- chain mAb of high affinity receptor for IgE; Anti-IgE, rabbit IgG anti-Fc fragment of human IgE; CsA, cyclosporin A; HBV, hepatitis B virus; HCV, hepatitis C virus; HLMC, human lung mast cells; ICS, internal calibration standard; L chain, light chain; rhIL-3, recombinant human IL-3; P, PIPES buffer containing 25 mM PIPES, pH 7.37, 110 mM NaCl, and 5 mM KCl; PCG, PIPES buffer containing 2.0 mM CaCl2 and 1 g/L D-glucose; TCF, Ca2+- and Mg2+-free Tyrode’s buffer; TGMD, TCF buffer containing 0.25 g/L MgCl2·6H2O, 10 mg/L DNase, and 1 g/L gelatine.
Received for publication April 14, 1998. Accepted for publication July 9, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
/cachectin. Nature 346:274.[Medline]
, which induces endothelial leukocyte adhesion molecule 1. Proc. Natl. Acad. Sci. USA 88:4220. by human basophils. J. Immunol. 157:1207.[Abstract]
production is cytokine-dependent. Proc. Natl. Acad. Sci. USA 94:3189. and and prostaglandin E2. Proc. Natl. Acad. Sci. USA 85:6880., interleukin 1, and prostaglandin E2. Proc. Natl. Acad. Sci. USA 86:3803.RI): analysis of functional domains of the -subunit with monoclonal antibodies. J. Biol. Chem. 266:11245.E (IgE) in conditions other than classical allergy. J. Allergy 45:30.[Medline]
by human mast cells: increased anti-IgE-dependent secretion after IgE-dependent enhancement of mast cell IgE-binding ability. Lab. Invest. 77:185.[Medline]
RI expression in the rat intestinal mucosa and tongue is enhanced during Nippostrongylus brasiliensis infection and can be up-regulated by in vivo administration of IgE. J. Immunol. 158:3805.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  S. LOFFREDO, F. AYALA, G. MARONE, G. DELFINO, S. STRANGES, and G. MARONE Immunopathogenesis of Psoriasis and Pharmacological Perspectives J Rheumatol Suppl, August 1, 2009; 83(0): 9 - 11. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Gessner, K. Mohrs, and M. Mohrs Mast Cells, Basophils, and Eosinophils Acquire Constitutive IL-4 and IL-13 Transcripts during Lineage Differentiation That Are Sufficient for Rapid Cytokine Production J. Immunol., January 15, 2005; 174(2): 1063 - 1072. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Schramm, F. H. Falcone, A. Gronow, K. Haisch, U. Mamat, M. J. Doenhoff, G. Oliveira, J. Galle, C. A. Dahinden, and H. Haas Molecular Characterization of an Interleukin-4-inducing Factor from Schistosoma mansoni Eggs J. Biol. Chem., May 9, 2003; 278(20): 18384 - 18392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Genovese, G. Borgia, L. Bjorck, A. Petraroli, A. de Paulis, M. Piazza, and G. Marone Immunoglobulin Superantigen Protein L Induces IL-4 and IL-13 Secretion from Human Fc{varepsilon}RI+ Cells Through Interaction with the {kappa} Light Chains of IgE J. Immunol., February 15, 2003; 170(4): 1854 - 1861. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. de Paulis, G. Florio, N. Prevete, M. Triggiani, I. Fiorentino, A. Genovese, and G. Marone HIV-1 Envelope gp41 Peptides Promote Migration of Human Fc{epsilon}RI+ Cells and Inhibit IL-13 Synthesis Through Interaction with Formyl Peptide Receptors J. Immunol., October 15, 2002; 169(8): 4559 - 4567. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Arock, E. Schneider, M. Boissan, V. Tricottet, and M. Dy Differentiation of human basophils: an overview of recent advances and pending questions J. Leukoc. Biol., April 1, 2002; 71(4): 557 - 564. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. H. Falcone, H. Haas, and B. F. Gibbs The human basophil: a new appreciation of its role in immune responses Blood, December 15, 2000; 96(13): 4028 - 4038. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Genovese, J.-P. Bouvet, G. Florio, B. Lamparter-Schummert, L. Bjorck, and G. Marone Bacterial Immunoglobulin Superantigen Proteins A and L Activate Human Heart Mast Cells by Interacting with Immunoglobulin E Infect. Immun., October 1, 2000; 68(10): 5517 - 5524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Patella, G. Florio, A. Petraroli, and G. Marone HIV-1 gp120 Induces IL-4 and IL-13 Release from Human Fc{epsilon}RI+ Cells Through Interaction with the VH3 Region of IgE J. Immunol., January 15, 2000; 164(2): 589 - 595. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-P. Bouvet and V. A. Fischetti Diversity of Antibody-Mediated Immunity at the Mucosal Barrier Infect. Immun., June 1, 1999; 67(6): 2687 - 2691. [Full Text] [PDF]
|
|
|
|
 |
|
 G. Marone, C. S. Ong, T. Ruzicka, S. Reitamo, and T. Bieber Tacrolimus Ointment for Atopic Dermatitis N. Engl. J. Med., December 10, 1998; 339(24): 1788 - 1789. [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
