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Departments of
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Medicine,
Pediatrics,
Pathology, and
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Molecular and Human Genetics,
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Howard Hughes Medical Institute, and
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Department of Microbiology and Immunology, Baylor College of Medicine, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4 mRNA and protein. In conclusion,
mice with targeted deletion of the IgA switch and constant regions are
completely deficient in IgA and exhibit altered expression of other Ig
isotypes, notably IgM, IgG2b, IgG3, and IgE, but otherwise have normal
lymphocyte development, proliferative responses, and cytokine
production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Secretory IgA in mucosal secretions can bind Ags, thereby limiting their absorption, inhibiting bacterial attachment to mucosal surfaces, and neutralizing a variety of viruses that might otherwise gain access to the body through mucosal surfaces. Because IgA does not efficiently activate complement, a potentially host-damaging inflammatory response does not occur. IgA in serum is able to bind and neutralize Ags, such as those present on micro-organisms, and may help to neutralize autoantigens or clear small amounts of food Ags that enter the body. Neutralization of autoantigens or foreign Ags may prevent inappropriate immune responses to these Ags. Support for this latter hypothesis is provided by the finding of an increased incidence of autoimmune diseases in subjects with IgA deficiency 3, 4 .
IgA deficiency is the most common humoral immunodeficiency, occurring with an incidence of 1:500 to 1:1000 5 , and it can be associated with IgG subclass deficiencies 6 . When IgA and IgG subclass deficiencies occur concomitantly, there is significant morbidity, manifested primarily as recurrent sinopulmonary infections. The availability of a small animal model of IgA deficiency could greatly facilitate an understanding of the pathophysiology of IgA deficiency, including any increased susceptibility to infections and autoimmune diseases. Such a model would also facilitate study of the in vivo function(s) of IgA. We report on the development and characterization of a murine model of selective IgA deficiency made by targeted deletion of the IgA switch and constant regions in embryonic stem (ES)4 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A plasmid (pm5'I
) containing a 3.5-kb
XbaI/AvrII DNA fragment immediately 5' of the
I exon was previously subcloned from the E-6 phage 7 into
pKS- (Stratagene, La Jolla, CA) 8 , and a plasmid
(pm3'C(RI-XbaI)) containing a 2.9 kb
EcoRI/XbaI DNA fragment from the 3' end of the
IgA constant region was subcloned from E-6 into pKS-. A
targeting vector was constructed with the 3.5-kb
XbaI/AvrII DNA fragment as the upstream
homologous fragment and the 2.9-kb EcoRI/XbaI DNA
fragment as the downstream homologous region (Fig. 1
). For positive selection a neomycin
resistance cassette containing a PGK promoter and bovine growth hormone
polyadenylation signal 9 was inserted between these two homologous
regions in the same transcriptional orientation as the endogenous
locus, while for negative selection a herpes simplex thymidine kinase
(HSV-tk) gene 10 was inserted 5' of the upstream homologous
region.
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Collection of serum and nasal, pulmonary, and intestinal secretions
Blood was collected from the axillary plexus of anesthetized mice and allowed to clot at room temperature, and serum was obtained following centrifugation and stored at -70°C until assayed. Nasal secretions were collected by instillation of 0.5 ml of PBS through a pipette tip inserted into the exposed trachea and directed toward the nasal cavity. Fluid was collected as it exited the nose. Pulmonary secretions were collected by inserting a pipette tip into the exposed trachea, ligating the trachea proximal to this site, and lavaging the lungs with 0.5 ml of PBS. Nasal and pulmonary secretions were tested for the presence of contaminating blood using Chemstrips (Boehringer Mannheim, Indianapolis, IN).
Intestinal secretions were collected from the small intestine using a modification of a previously described method 12 . Briefly, small intestines (from pylorus to cecum) were isolated and carefully filled for 10 min at room temperature with 2 ml of PBS containing a protease inhibitor solution consisting of 0.1 mg/ml soybean trypsin inhibitor (Sigma, St. Louis, MO), 50 mM EDTA, and 1 mM PMSF (Boehringer Mannheim). The intestinal content was then carefully transferred to a test tube, vigorously vortexed, and centrifuged for 10 min at 650 x g at 4°C. The supernatant was transferred to a microfuge tube, and PMSF was added to a final concentration of 1 mM. The solution was mixed by vortexing and was centrifuged at 13,000 x g for 20 min at 4°C. The resulting supernatant was mixed with PMSF and sodium azide to final concentrations of 1 mM and 0.01%, respectively, and was incubated for 15 min at 4°C. Thereafter, 50 ml of FCS was added per 1 ml of secretion, and the solution was centrifuged for an additional 20 min at 13,000 x g at 4°C. Supernatants were removed, and secretions were stored at -70°C until analysis.
Isolation of spleen and Peyer’s patch lymphocytes and in vitro culture of B cells
Spleen and Peyer’s patch non-T cells were obtained as previously described 13 . Briefly, a single cell suspension of spleen or Peyer’s patch cells was prepared, and RBCs were removed by hypotonic lysis. T cells were depleted by incubation with a mixture of anti-Thy 1.2 (New England Nuclear, Boston, MA; final concentration, 1/1000), anti-Lyt 2.2, and anti-L3T4 mAb (Accurate Chemical Co., Westbury, NY; final concentration for both, 1/1000) in cytotoxicity medium (RPMI 1640 with 0.2% BSA) followed by complement lysis (Low Tox-M rabbit complement, Accurate Chemical Co.).
Production of IgA in vitro was assessed as previously described 8 . Briefly, B cells (0.5–2 x 106/ml) were cultured in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% FCS (BioWhittaker), 15 mM HEPES (Life Technologies, Grand Island, NY), 5% NCTC 109 medium (BioWhittaker), 5 x 10-5 M 2-ME (Sigma, St. Louis, MO), 2 mM glutamine (Life Technologies), and 100 U/ml penicillin/100 µg/ml streptomycin (Life Technologies) and incubated at 37°C and 6% CO2 in a humidified atmosphere in 96-well flat-bottom plates or 25- or 75-cm2 flasks (Corning, Corning, NY). LPS was added at the initiation of culture, while cytokines were added on day 1 of culture. Escherichia coli LPS (Sigma, 0127:B8) was used at a final concentration of 10 µg/ml, rIL-2 at 100 U/ml (PharMingen, San Diego, CA), rIL-5 at 100 U/ml 14 , and TGF-ß (R&D Systems, Minneapolis, MN) at 1 ng/ml.
Measurement of Ig isotypes and Abs to hemagglutinin
Isotype-specific sandwich ELISAs were performed as previously described 8 using an mAb 7, 8, 9, 10, 11, 12, 13, 14 previously shown to be specific for mouse IgA 15 . Serum and secretory Ig levels are presented as the mean ± SD from analysis of three to six mice per group.
In vitro Ig production was measured in culture supernatants after 7 days of incubation. Cultures for in vitro Ig secretion by both spleen and Peyer’s patch B cells, for each condition analyzed were performed in triplicate. Two separate experiments were performed.
To determine the ability of IgA-deficient mice to develop serum and secretory Ab responses following immunization, mice were immunized with killed whole influenza virus (B-Yamagata) and cholera toxin (75 µg of hemagglutinin/10 µg of cholera toxin orally and 25 µg of hemagglutinin/1 µg of cholera toxin intranasally) on days 0, 11, and 20. Mice were sacrificed on day 33, and serum, nasal secretions and intestinal secretions were collected. Abs against B-Yamagata were measured by standard ELISA assays using plates coated with whole virus (2 µg/ml) in carbonate buffer. Abs of defined isotypes were detected using alkaline phosphatase-conjugated isotype-specific Abs (Southern Biotechnology Associates, Birmingham, AL). End-point titers were calculated based on the highest dilution giving an OD reading >2 SD above the same dilution from an unimmunized control animal.
Flow cytometric analysis of spleen and Peyer’s patch lymphocytes
Flow cytometric analysis was performed on Peyer’s patch and spleen lymphocytes as previously described 8 using a Coulter Profile II (Coulter, Hialeah, FL). Cells were stained with phycoerythrin-labeled goat anti-mouse IgM (Southern Biotechnology Associates) and fluorescein-labeled anti-mouse IgA 7, 8, 9, 10, 11, 12, 13, 14 . Forward and side scatter gates were set to include live lymphocytes, and 10,000 events were collected. Results shown are from one of two experiments that gave similar results.
Histopathology and immunohistochemistry
Portions of thymus, spleen, mesenteric lymph nodes, jejunum, and terminal ileum were placed in formalin. In addition, portions of jejunum and distal ileum were snap-frozen in Cryoform (International Equipment Co., Needham, MA) and liquid nitrogen. For histopathology, sections were routinely stained with hematoxylin and eosin, then analyzed by light microscopy. For immunohistochemistry, cryostat sections, cut 6 µm thick, were blocked with hydrogen peroxide, sodium azide, and BSA. They were then covered with FITC-labeled rat monoclonal anti-mouse IgA (Accurate Chemical Co.) at a 1/50 dilution in PBS at 0–4°C overnight. After rinsing in PBS, a horseradish peroxidase-conjugated secondary rabbit Ab against FITC (Dako, Carpinteria, CA) at a dilution of 1/50 was applied for 2 h at 21°C. After rinsing, color was developed with diaminobenzidine-nickel chloride as the substrate, and tissues were counterstained with Nuclear Fast Red.
RNA amplification by PCR for IFN- and IL-4
Total cellular RNA was extracted from fresh mouse lymphocytes
(2–10 x 106) or those stimulated or unstimulated
with influenza virus for 3 or 7 days or with PHA (2 µg/ml) for 3 days
using the RNeasy Mini Kit (Qiagen, Chatsworth, CA). First-strand cDNA
preparation and PCR amplification were performed according to the
established protocol (Perkin-Elmer/Cetus, Emeryville, CA) 16 .
Briefly, cDNA synthesis by RT was performed using an RT kit from
Perkin-Elmer/Cetus. The RT mixture was prepared by adding 4 µl of 25
mM MgCl2, 2 µl of 10x PCR buffer II, 8 µl of 40 mM
dNTP, 0.2 µl of RNase inhibitor, 1 µl of Moloney leukemia virus
reverse transcriptase, 1 µl of random hexamers, and 1 µl of sample
RNA (200 µg/ml) and was brought to 20 µl with RNase-free water. The
mixture was overlayed with 50–100 µl of mineral oil (Sigma) and was
incubated at room temperature for 10–20 min and in a Robocycler (PCR
machine, Stratagene, La Jolla, CA) at 42°C for 15 min, at 99°C for
5 min, and at 6°C for 5 min. A PCR master mix for 40 PCR reactions
(each 25 µl) was prepared by adding 100 µl of 10x PCR buffer, 80
µl of 40 mM dNTP, 2 µl of downstream and upstream primers (0.4
µM/ml of murine IFN- and IL-4 amplimer sets (Clontech, Palo Alto,
CA)), 8 µl of AmpliTaq polymerase, and 684 µl of distilled water
(PCR Kits, Perkin-Elmer/Cetus). A 22-µl aliquot of master mix was
added to each of 0.5-ml microcentrifuge tube (10 tubes/4 groups
for each primer set) followed by 2 µl of RT product. One microliter
of 10-fold serially diluted IFN- and IL-4 mimics template (Clontech,
Palo Alto, CA) was added to each tube in each group and was overlayed
with 50 µl of mineral oil. The PCR reaction was performed in a
thermal cycler (Robocycler, Stratagene) at 94°C for 2 min (one cycle)
followed by 94°C for 1 min and 65°C for 1 min (30 cycles) and an
extension at 65°C for 10 min (one cycle). The PCR products were
analyzed using 1% agarose gel electrophoresis. The concentration of
each cytokine mRNA was determined from overlapping bands of the mimics.
ELISA for IFN- and IL-4
Splenic lymphocytes were cultured at a concentration of
106 cell/ml in RPMI 1640 supplemented with 2 mM
L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin
(JRH Biosciences, Lenexa, KS), 5 x 10-5 M 2-ME, 10
mM HEPES buffer, and 10% heat-inactivated FBS with medium alone or PHA
(2.5 µg/ml) in duplicate in a final volume of 2.0 ml in 12-well
plates. Supernatants from day 3 mitogen cultures were harvested and
tested for the presence of IFN- and IL-4 using a modification of the
sandwich ELISA method 17 . Briefly, 96-well vinyl plastic plate
(Nunc-Immuno Plate MaxiSorp, Nunc, Naperville, IL) wells were coated
with 100 µl of solution (Pierce Coating Buffer, Pierce,
Rockford, IL) containing 0.5–2 µg/ml of capture monoclonal rat
anti-murine IFN- or IL-4 Ab (PharMingen, San Diego, CA). The
plates were incubated overnight at 4°C and washed four times with
Tris-NaCl buffer containing 0.05% Tween-20 (Tris-NaCl-Tween) using an
automatic Titertek Microplate Washer (Titertek, ICN Biomedicals, Costa
Mesa, CA). After blocking for 1 h with SuperBlock buffer in
Tris-buffered saline (Pierce) and washing four times, serial
dilutions of recombinant murine IFN- or IL-4 standard and undiluted
test samples were added to the wells in quadruplicate, and the plates
were incubated at 37°C for 1 h. After four washes, 100 µl of
biotinylated rat anti-murine IFN- or IL-4 Ab (0.0–2 µg/ml in
SuperBlock) was added and incubated at 37°C for 1 h. The plates
were washed, and 100 µl of streptavidin alkaline phosphatase (Pierce)
was added to each well, incubated at 37°C for 1 h, and then
washed four times. Substrate (1–2 tablets of 
-nitrophenol
phosphate/10 ml of diethanolamine buffer, pH 9.8) was then added, and
color was allowed to develop for 10–120 min. Absorbance in each well
was read at a wavelength of 405 nm using a Molecular Devices automatic
microplate reader (Menlo Park, CA). The data were collected using
SOFTmax data reduction software (Molecular Devices). The murine
recombinant IFN- and IL-4 were used to generate standard curves. The
amount of cytokine in the test samples was extrapolated from the
standard curves and was expressed as picograms per milliliter of
cytokine.
Lymphoproliferative response assay
Splenic and mesenteric lymph node lymphocytes were cultured at a concentration of 105 cell/microtiter well in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (JRH Biosciences), 5 x 10-5 M 2-ME, 10 mM HEPES buffer, and 10% heat-inactivated FBS with medium alone, LPS (10 µg/ml), PHA (2.5 µg/ml), or Staphylococcus aureus lysate (SPL; Delmont Laboratories, Swarthmore, PA) in quadruplicate in a final volume of 0.2 ml. After 46 h, cells were pulsed with 0.5 µCi of [3H]TdR (6.7 mCi/ml; ICN Radiochemicals, Irvine, CA) for 18–24 h and harvested on glass-fiber filter mats using a semiautomatic harvester (Skatron, Sterling, VA). Radioactivity ([3H]TdR) incorporation was determined using a beta scintillation counter. Differences between means of counts per minute for mitogens and controls were determined by Student’s t test for each mitogen. Stimulation indexes for each mitogen and Ag (optimal concentration) obtained by dividing its mean counts per minute by the mean control counts per minute were compared among groups by ANOVA 18 .
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A replacement-type targeting vector was generated as shown in Fig. 1
A. This targeting vector contains a 5' homologous DNA
region located immediately upstream of the I exon transcription
start site and a 3' homologous DNA region containing the 3' half of the
IgA constant region downstream of the EcoRI site. Inserted
between the two homologous regions of DNA is a neomycin resistance
gene, while an HSV-tk gene is located upstream of the 5' homologous
region. Homologous recombination by this targeting vector in ES cells
leads to deletion of the I exon, the entire IgA switch region, and
the 5' half of the C region. This approach was taken to ensure that
class switch to IgA would be prevented and that any aberrant DNA
rearrangement at that locus would nonetheless lead to a nonfunctional
IgA molecule.
This targeting vector was transfected by electroporation into the
I.2 ES cell clone 8 . As shown in Fig. 1A, this ES cell
clone had a previous targeting event at the IgA locus on one allele,
leading to deletion of the I exon with replacement by a human HPRT
minigene. The I.2 ES cell clone had previously been used to generate
mice with targeted deletion of the I exon. This ES cell clone was
used in the current studies because it allowed triple selection of
correctly targeted ES cell clones, i.e., loss of the HPRT minigene by
growth in 6-thioguanine (TG), gain of the neomycin resistance gene by
growth in G418, and loss of the HSV-tk gene by growth in gancyclovir
(Ganc). Of 1 x 107 transfected I.2 ES cells, six
triple-resistant (Tgr, G418r,
Gancr) clones were obtained. DNA from these clones were
analyzed by Southern blotting (Fig. 1B), and five of the six
clones are shown. Digestion with KpnI and XhoI
results in a 8.8-kb DNA fragment recognized by a C
probe located
upstream of the targeted region at the wild-type locus and a 10.7-kb
DNA fragment from the I.2 locus previously targeted by the HPRT
minigene. Subsequent targeting of this latter locus by the targeting
vector leads to a 9.6-kb DNA fragment recognized by the C probe. Of
the six ES cell clones obtained, four had correct targeting at the IgA
locus. Additional restriction digestion and Southern blotting were
performed to confirm proper targeting (data not shown).
Two of the correctly targeted ES cell clones were injected into C57BL/6
blastocysts from which chimeric mice were obtained. Two chimeric male
mice with >50% chimerism were bred to C57BL/6 females with successful
germline transmission. Tail DNA from F2 offspring was
digested with KpnI and XhoI and analyzed by
Southern blotting with the C probe (Fig. 1C). Mice on the
129Sv background have the Igha allotype, whereas
C57BL/6 mice have the Ighb haplotype. As a consequence, a
KpnI polymorphism is present at the C locus leading to an
8.8-kb DNA fragment in 129Sv mice and a 6.0-kb DNA fragment in C57BL/6
mice. Thus, three different sized DNA fragments recognized by the C
probe were seen in the F2 offspring-8.8 kb wild-type 129Sv
allele, the 6.0-kb wild-type C57BL/6 allele, and the 9.6-kb 129Sv
targeted allele. Shown in Fig. 1C are two mice that are
homozygous for the 9.6-kb 129Sv targeted allele.
Mice with targeted deletion of the IgA locus are completely deficient in IgA
To assess the effects of targeted deletion at the IgA locus on
expression of IgA in vivo, the following analyses were performed. IgA
levels were measured in the serum and nasal, pulmonary, and
gastrointestinal secretions (Fig. 2A). No detectable IgA (<80
ng/ml) was found in any of these locations in mice with targeted
deletion at the IgA locus, in contrast to wild-type mice, which had
readily detectable and substantial levels of IgA at all these sites.
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). In contrast, 10–15% of lymphocytes
in the Peyer’s patches of wild-type mice expressed surface IgA.
Similarly, no surface IgA-expressing B cells were found in the spleen
of targeted mice, in contrast to wild-type mice (data not shown).
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). B cells from wild-type mice secreted
small amounts of IgA when cultured in medium or LPS. As expected,
production of IgA by wild-type B cells was increased substantially in
the presence of LPS and TGF-ß. In contrast, B cells from both spleen
and Peyer’s patches produced no detectable IgA (<80 ng/ml) even when
cultured with LPS and TGF-ß.
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The levels of other Ig isotypes were measured in serum and nasal,
pulmonary, and gastrointestinal secretions. In serum, IgM and IgG
levels were modestly elevated in targeted animals, being approximately
twice the level seen in wild-type mice (IgM, 382 ± 35 vs 179
± 27 µg/ml (p < 0.001); IgG, 11,629 ±
1,908 vs 6,264 ± 842 µg/ml (p < 0.02);
Fig. 2, B and C). In the case of nasal and
pulmonary secretions, no significant differences were seen in IgM or
IgG levels in targeted animals in comparison to wild-type mice. In
contrast, in intestinal secretions IgM levels were approximately 25
times higher in targeted animals (5.1 ± 4.7 µg/ml) than those
in wild-type mice (0.2 ± 0.1 µg/ml). In addition, intestinal
IgG levels were roughly 3 times higher in targeted animals (29 ±
4 µg/ml) than those in wild-type mice (8.2 ± 1.7 µg/ml;
p < 0.005).
In addition, we measured IgG subclass levels in serum and secretions.
Fig. 5 shows the results for serum IgG
subclasses. A twofold increase in serum IgG1 was seen in targeted mice
(p < 0.01), while no difference was seen in
serum IgG2a levels. In the case of IgG2b and IgG3, there was roughly a
fivefold increase in IgG2b (p < 0.01) and a
sevenfold decrease in IgG3 (p < 0.001) in the
serum of targeted mice (p < 0.005). In
gastrointestinal secretions, IgG1 was increased roughly fivefold
(p < 0.0005), IgG2a was increased twofold
(p < 0.01), IgG2b was unchanged, and IgG3 was
decreased fivefold (data not shown). In nasal secretions, no change was
seen in IgG1, IgG2a, or IgG2b; however, there was a 10-fold decrease in
IgG3. Lastly, in pulmonary secretions no change in IgG1 or IgG2a was
seen; however, there was a twofold increase in IgG2b and a three- to
fourfold decrease in IgG3 (data not shown). It should be noted that
when compared with wild-type littermate control mice,
IgA-/- knockout mice exhibited significantly decreased
IgG3 levels; however, their IgG3 levels were normal compared with those
in parental C57BL/6 and 129 mice (data not shown).
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3 ng/ml) compared with those in wild-type mice
(64 ± 23 ng/ml). Likewise, pulmonary secretions from targeted
mice had substantially lower levels of IgE (2.6 ng/ml) than wild-type
mice (64 ± 39 ng/ml). In the case of nasal and intestinal
secretion, no detectable IgE was found in either targeted or wild-type
mice. Targeted deletion of IgA does not affect development of lymphoid tissues or T and B lymphocytes
IgA-deficient mice developed normally with no apparent increased susceptibility to infection in conventional animal facilities. The size of litters from IgA-deficient mice was comparable to that in wild-type mice, and no increased perinatal mortality was evident. Routine histologic examination of thymus, spleen, mesenteric lymph nodes, jejunum, and terminal ileum showed no differences between IgA-deficient mice and age-matched wild-type controls. Phenotypic analysis of T and B cells in various lymphoid tissues of these mice revealed a normal ratio of T and B cell subsets, such as CD4 T cells, CD8 T cells, and IgM B cells, etc. (data not shown).
Histology and immunohistochemistry of Peyer’s patches and small intestine in IgA-deficient mice
Peyer’s patches were well developed in IgA-deficient mice, with
prominent germinal centers (Fig. 6).
Nonetheless, no IgA-containing plasma cells were detected by
immunohistochemistry in small intestine lamina propria of IgA-deficient
mice, in contrast to frequent IgA plasma cells in the lamina propria of
wild-type mice (Fig. 6).
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Splenic and lymph node lymphocytes from IgA+/+ and
IgA-/- mice were stimulated with PHA, a T cell mitogen;
LPS, a B cell mitogen; PWM, a T cell-dependent B cell mitogen; or SPL,
an ubiquitous Ag that stimulates both T and B cells. Both mice
exhibited similar lymphoproliferative responses to all mitogens with
identical stimulation index values, except for PHA, which led to
significant decrease in proliferation in the IgA-/- mice
(Table I). Lymph node lymphocytes
exhibited a similar pattern of responses as splenic lymphocytes (data
not shown).
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IFN- and IL-4 production and mRNA expression
To assess the ability to produce and express two pivotal
immunoregulatory cytokines for stimulation of Th1-type (IFN-) and
Th2-type (IL-4) responses, spleen cells were stimulated with or without
PHA for 4 days. Cell culture supernatants were harvested and tested for
IFN- and IL-4 by ELISA. Unstimulated splenocytes from both
IgA-/- and IgA+/+ mice produced undetectable
levels of IFN- (Fig. 7A).
However, PHA-stimulated cells elaborated significant amounts of
IFN-. IgA-/- mice produced slightly lower
levels of IFN- than IgA+/+ mice
(p = 0.13). In contrast, unstimulated cells
from both mice produced marked levels of IL-4. While
IgA-/- mice produced statistically
significantly higher amounts of IL-4 than IgA+/+
mice (p < 0.05), the difference may not be
biologically significant. PHA stimulation resulted in increased
production of IL-4; however, both IgA-/- and
IgA+/+ mice produced similar amounts (Fig. 7B).
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and IL-4 production induced by PHA
stimulation and its correlation with increased transcription of IFN-
and IL-4 mRNA, quantitative RT-PCR analysis specific for IFN- and
IL-4 was performed on RNA samples obtained from PHA-stimulated and
unstimulated spleen cells. The intensity of the band for PCR product
for IFN-- specific mRNA in both IgA+/+ and
IgA-/- mice increased 10-fold following
stimulation with PHA (5 x 10-19 mM/µg of RNA)
compared with unstimulated spleen cells (5 x 10-20
mM/µg of RNA; Fig. 8, A and
C). In contrast, IL-4 mRNA was detected at levels
approximately 150 times higher than those of IFN- mRNA in
unstimulated cells (5 x 10–18.5 mM/µg RNA) from both
IgA+/+ and IgA-/- mice.
Following stimulation with PHA, IL-4 mRNA expression increased
approximately 5-fold (5 x 10-18 mM/µg RNA) in
IgA-/- mice but was unchanged in most
IgA+/+ mice (Fig. 8, B and
D).
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and IL-4
secretion parallels mRNA expression. However, the data suggest that
while IFN- is secreted only upon PHA stimulation, IL-4 is
constitutively secreted in unstimulated cells, with increased secretion
following PHA stimulation, especially among
IgA-/- mice. Furthermore, both
IgA+/+ and IgA-/- mice
exhibited generally similar patterns of IFN- and IL-4 secretion and
mRNA expression. Effect of IgA deficiency on systemic and mucosal Ab responses to influenza hemagglutinin
The ability of IgA-deficient mice to develop serum and secretory
Ab responses following immunization was assessed by immunizing mice
mucosally with killed whole influenza virus (B-Yamagata) and cholera
toxin. Abs against B-Yamagata in serum, nasal secretions, and
intestinal secretions were subsequently measured by ELISA. As shown in
Fig. 9, no detectable IgA Abs against
influenza virus were detected in serum or nasal or intestinal
secretions from IgA-deficient mice, whereas wild-type mice gave readily
detectable levels. In contrast, IgM Abs in IgA-deficient mice were
4-fold higher in serum, nasal, and intestinal secretions
(p < 0.01, p < 0.05, and
p < 0.08, respectively) compared with those in
wild-type mice. However, no significant differences were noted in IgG
Abs between IgA deficient and wild-type mice. These studies indicate
that IgA-deficient mice produce no detectable IgA Abs following mucosal
vaccination; however, they produce elevated IgM Abs compared with
wild-type mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While these animals express no IgA, they do express all other isotypes, including IgM, IgG subclasses, and IgE. However, the levels of expression of some other isotypes in serum and secretions are altered in the IgA-deficient mice. Most notably, the level of IgM in intestinal secretions was increased substantially in IgA-deficient mice, although in serum and other mucosal secretions little or no increase was noted. In addition, mucosal immunization of IgA-deficient mice with influenza antigens and cholera toxin led to higher levels of IgM Abs in serum and nasal and intestinal secretions compared with those in wild-type mice. Recent studies have also shown increased IgM Abs in gastric secretions of IgA-deficient mice immunized with Helicobacter felis Ags (our unpublished observations). Significant alterations in IgG subclasses were also seen, with the most notable changes in IgG2b and IgG3. In serum, IgG2b levels were increased roughly fivefold in IgA-deficient mice, with a concomitant decrease in IgG3 of roughly the same magnitude. Alterations of IgG subclasses were also seen in mucosal secretions, with an increase IgG2b and a decrease in IgG3 being the most common. Lastly, IgE levels were substantially lower in IgA-deficient mice.
The reason for altered expression of other Ig isotypes in IgA-deficient mice remains to be elucidated. People with IgA deficiency often have increased levels of IgM in mucosal secretions. This is presumably in compensation for the absence of IgA, and a similar reason might pertain in the IgA-deficient mice. It seems likely that the absence of the IgA switch region in B cells from these mice precludes a class switch to IgA. Consequently, this may lead to forced differentiation of B cells into plasma cells, producing IgM under conditions that would normally favor generation of IgA plasma cells. In regard to the increased expression of IgG2b, a similar argument can be made, since previous studies have shown that TGF-ß induces murine B cells to switch to IgG2b in addition to IgA 19 . The reason for the apparent decreased expression of IgG3 and IgE in IgA-/- mice compared with that in control IgA+/+ littermates remains unclear.
IgA-/- mice exhibited good lymphoproliferative
responses to PHA, a T cell mitogen; LPS, a B cell mitogen; PWM, a T
cell-dependent B cell mitogen; and SPL, a ubiquitous Ag that stimulates
both T and B cells. Even though IgA-/- mice had
statistically significant reduced responses to PHA and anti-CD3 mAb
compared with IgA+/+ mice, the magnitude of the
differences is small, and as such may not be biologically significant.
In addition, IgA-/- mice retain the capacity to
produce and express two pivotal immunoregulatory cytokines for
stimulation of Th1-type (IFN-) and Th2-type (IL-4) responses. Thus,
both IgA+/+ and IgA-/-
mice exhibited somewhat similar patterns of IFN- and IL-4 secretion
and mRNA expression. These data indicate that IgA gene deletion did not
adversely impact on normal T and B cell functions.
IgA was first identified in 1953 when Grabar and Williams characterized ß2A-globulin in serum. Serum IgA was further characterized by Bazin and Heremans in 1959, and in 1963, Chodirker and Tomasi identified secretory IgA. Given that mucosal surfaces represent major areas of contact with the environment, and that IgA is the predominant Ig in secretions and IgA is the major isotype produced by the body 1 , it is reasonable to assume that IgA plays an important role in the mucosal immune response. In this regard, previous studies have suggested that IgA plays a role in protection against pathogens entering through mucosal surfaces as well as in neutralization of toxins in both serum and secretions (for review, see Refs. 1, 20, and 21). Thus, it is surprising that people with IgA deficiency often have no overt susceptibility to infections or other illnesses. Nonetheless, some IgA-deficient patients have an increased susceptibility to respiratory and gastrointestinal tract infections as well as autoimmune diseases. However, despite roughly 40 yr of study, much remains to be answered with regard to the in vivo function of IgA.
IgA-deficient mice will offer a valuable model for studying the function of IgA. For example, previous studies have shown that IgA mAbs against influenza hemagglutinin can protect mice against infection with influenza 22 . Whether IgA Abs are the predominant or key mediator of immunity against influenza, however, has not been addressed. The development of IgA-deficient mice will permit studies to directly address this question. Further, an IgA mAb has also been shown to protect mice against challenge with H. felis 23 , but again the relative importance of IgA Abs for protection against infection in vivo remains to be determined. Likewise, IgA mAbs against rotavirus Ags have been shown to protect mice against rotavirus infection 24 , but the relative importance of these Abs in protection needs to be evaluated.
In addition to its role in mucosal immune responses, IgA immune complexes appear to be involved in the pathogenesis of IgA nephropathy 25 , and previous mouse models of IgA nephropathy have substantiated this 26, 27 . Lastly, IgA has been shown to modify effector functions of eosinophils through binding to IgA Fc receptors 28 . Whether IgA participates in allergic immune responses or immune responses to parasites via its effects on eosinophil function can also be addressed using IgA-deficient mice. Thus, these mice represent an important model for further defining the role of IgA in vivo.
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Footnotes |
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2 Current address: Immunology, Clinical Research, Centocor, 200 Great Valley Parkway, Malvern, PA 19355.
3 Address correspondence and reprint requests to Dr. Innocent N. Mbawuike, Department of Microbiology and Immunology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030. E-mail address:
4 Abbreviations used in this paper: ES, embryonic stem; HSV-tk, herpes simplex thymidine kinase; SPL, Staphylococcus aureus lysate; HPRT, hypoxanthine phosphoribosyltransferase.
Received for publication July 8, 1998. Accepted for publication December 3, 1998.
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