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*
Department of Microbiology and Interdisciplinary Immunology Program, University of Iowa, Iowa City, IA 52242;
Department of Animal Science, Iowa State University, Ames, IA 50011;
Institute of Microbiology, Novy Hradek and Prague, Czech Republic; and
National Animal Disease Center, Ames, IA 50011
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is a considerable body of evidence indicating that switch
recombination is mediated through costimulation by helper T cells
involving CD40 and B7-1/B7-2 (3, 4, 5, 6, 7). For example, switch
recombination in mice to IgG1 and IgE requires costimulation through
CD40 and either IL-4 or IL-13 (8), whereas TGF-
mediates the switch to IgA (3). Switch recombination
together with somatic mutation and receptor editing are major molecular
events that occur as part of the germinal center
(GC)3 reaction
(5, 9, 10, 11, 12). Because the GC reaction occurs in response to
stimulation with T-dependent Ags, switch would not be expected to occur
in an environment free of T-dependent environmental Ags. Nevertheless,
there are reports of IgA present as early as the second trimester in
human fetuses (13, 14, 15). IgA-containing cells have been
reported to be present in 180-day fetuses (16), and
transcripts can be detected on day 110 (17). In addition
to IgA, C
2 and C4 transcripts have also been detected in fetal
human liver (18), and switch can even occur at the time of
VDJ rearrangement (19). Because LPS alone can stimulate
switch recombination (20, 21, 22) by an unknown mechanism (N.
Maizels, unpublished observations) the requirements for T
dependence and the GC reaction are not absolute. Although
lymphotoxin-
knockout mice are unable to form GC and to somatically
mutate their variable region genes, they are still capable of producing
IgG Abs to some Ags (23).
These observations raise the question of whether switch recombination is exclusively Ag driven and whether it is coupled to somatic mutation in vivo. Unfortunately, the human fetus is a poor model because active transport of maternal IgG to the fetus progressively increases during the third trimester, and eventually concentrations in cord blood exceed maternal levels just before birth (24, 25, 26). Furthermore, allergen-primed T cells can be found in the human fetus suggesting that allergens also can cross the placenta after they have been encountered by the pregnant mother either alone or as part of transported IgG immune complexes (27, 28, 29, 30). Although such cells could be of maternal origin, analysis of microsatellite DNA suggests that they are indeed of fetal origin (27). Responsive cells can be found even in the first 6 mo of pregnancy (29, 30), suggesting that allergens may cross the placenta alone during this stage of pregnancy and do not require shuttle as IgG immune complexes. Thus, low-level stimulation of the human fetus by T-dependent Ags that cross the placenta alone or as immune complexes may account for any fetal switched isotypes found.
Unlike the human, it has been generally believed that maternal Igs do not enter the circulation of the fetal piglet (24, 25) although there is evidence that switched isotype Igs, i.e., IgG and IgA, are present in fetal piglet serum (31, 32). Some have maintained that these are due to contamination by maternal blood (33, 34) resulting either from placental damage or contamination during collection. Although contamination may explain the IgG and IgA in fetal sera, it cannot explain the occurrence of IgA transcripts in late-term and newborn piglets (35, 36) or the presence of IgA- and IgG-secreting cells (SC) (37). This suggests that isotype switch is spontaneous and independent of environmental Ag in this species or that it results from environmental Ags such as virus, which can cross the six-layer epitheliochorial placenta of the pig by unknown mechanisms (38, 39) just as HIV crosses the placenta in humans (40).
Here we report that IgG and sometimes IgA and IgM can be detected in
fetal serum as early as 38 days in utero (114 days of gestation, DG)
and that IgG concentrations are 
10-fold higher than IgM
concentrations throughout gestation. The concentration of the switched
isotypes markedly increases in the last 15 DG with IgA levels equal to
or exceeding IgM levels and IgG levels nearly 20-fold higher. With rare
exception, the isotype profile of normal fetal piglets and those
infected with the PRRS (porcine respiratory and reproductive syndrome)
virus, are statistically distinct from the profile in maternal serum.
These exceptions notwithstanding, fetal serum Igs do not appear to
result from placental leakage or contamination by maternal blood during
collection. We also show that transcription of IgA and IgG first occurs
midway through gestation, is especially pronounced in the thymus, and
generates a highly polyclonal repertoire of switched isotypes. This
repertoire is not hypermutated like that in fetal piglets that have
encountered environmental Ag though viral infection. Further evidence
that this polyclonal repertoire of switch isotypes develops
intrinsically is demonstrated by its development in a noninductive site
of the mucosal immune system of piglets maintained in germfree
isolators.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Yorkshire x Meishan F1 crosses from Iowa State University (Ames, IA) and Minnesota minipigs x Vietnam Asian crosses from Novy Hradek (Czech Republic) were the sources of all normal fetal samples. Yorkshires from the National Animal Disease Center (Ames, IA) were used for viral inoculation studies and Yorkshires from a local supplier at South Dakota State University (Brookings, SD) were used in all isolator piglet studies. Animals were hand-mated, and all mothers were healthy and normal at the time of euthanization. The fetuses were immediately removed from the gravid uterus. Blood from >130 normal fetuses was collected when fetuses were at or near 40, 50, 60, 70, 90, 95, 105, and 110 days of age. The umbilical cord was carefully washed with water and saline to remove any traces of maternal blood and samples were then collected from the umbilical vein. Blood samples were also collected from 1) 27 mother pigs, including those of the fetuses studied, 2) the jugular vein or retro-orbital sinus of 29 colostrum-deprived newborn piglets, and 3) 36 fetal piglets experimentally infected in utero with an attenuated variant of the PRRS virus. Infection with the PRRS virus should theoretically allow us to test whether VDJs expressed with switched isotypes in animals with known exposure to environmental Ag differed in diversification from those in normal fetuses. Fetal liver was collected from 30-, 40-, and 50-day fetuses. Spleen was collected from all 40-day and older fetuses, and a variety of lymphoid tissues were collected when animals were 70 days and older.
Preparation of cDNA
Total RNA was prepared using TRIzol reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. RNA integrity was verified by staining with ethidium bromide after agarose electrophoresis in formaldehyde-containing gels. First-strand cDNA was prepared as follows: Five micrograms of total RNA and 1 µl of the oligonucleotide mixture (IgG anti-sense CH2 (5 pmol/µl), IgM anti-sense CH2 (5 pmol/µl), IgA anti-sense CH3 (5 pmol/µl), and a random hexamer (10 pmol/µl)) were mixed together, and water was added up to a total volume of 15 µl. The mixture was heated at 85°C for 10 min and immediately cooled on ice. The first-strand cDNA synthesis solution contained 6 µl of 5x first-strand cDNA buffer, 20 U of RNasin, 300 U of Moloney murine leukemia virus reverse transcriptase, 100 µg/ml BSA, and 1 mM DTT and water to 15 µl. The denatured RNA sample was transferred into the first-strand cDNA synthesis mixture and incubated at room temperature for 10 min, then at 37°C for 40 min, and finally at 50°C for 10 min.
Relative Cµ, C, and C transcript expression
First-strand cDNA and the isotype-specific anti-sense
primers together with a FR1-5' end primer (see Table I
) were used to amplify segments of Cµ,
C, and C. -actin segments were amplified from these same
tissues using the -actin primer set described in Table I. All
reactions were performed in a 25-µl volume containing 2.5 µl of PCR
buffer, 5 U of KlenTaq (AM Peptides, St. Louis, MO), 7.5 pmol of
primers, 0.5 µl of cDNA, 2.5 mM dNTP, and 20 µl of
dH2O. Amplification was performed in a MJ-200 DNA
engine under the following conditions: Initial denaturation was for 1
min at 94°C, followed by 30 cycles of denaturing at 94°C for
40 s, and then annealing at 58°C for 30 s followed by an
extension at 72°C for 20 s. All products were then examined by
agarose electrophoresis and Southern blotting to verify their size and
identity. Five microliters of each PCR product (Cµ, C, C, and
-actin) from the same cDNA preparation was applied to wells in a
96-well membrane transfer apparatus (Pierce, Rockford, IL) in 200 µl
of 20x SSC and then blotted onto a nylon membrane as previously
described (41, 42). The transferred DNA was then
cross-linked to the nylon membrane by exposure to UV radiation. After
prehybridization in a buffer containing 5x Denhardts solution, 0.2%
SDS, 3x SSC at 53°C for 1 h, the membrane was transferred to a
tube containing the same hybridization buffer and a
32P-end-labeled probe specific for Cµ, C,
C, or -actin. Hybridization was conducted for 4 h or
overnight (no differences were observed between 4 h and
overnight). The membranes were rinsed twice in medium stringency buffer
(0.125x SSC, 0.1% sodium pyrophosphate, and 0.1% SDS) for 20 min
each. The membranes were then wrapped in Cling Wrap (Glad,
Danbury, CT), and the signals were measured in a Packard Instant
Imager (Palo Alto, CA). The membranes were then stripped, monitored for
residual radioactivity, and hybridized with a different
32P-end-labeled probe. Representative raw data
are shown in Fig. 2 and Table II. In each
case, controls containing only single isotype PCR products were used,
and these served to test for any cross-hybridization (see Fig. 2).
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-actin probe was designated "relative
transcription"; mean data from 4–6 animals at each time point for
each tissue/organ are presented in Fig. 3. Because the measured signal
depends on the sp. act. of the probe used, relative transcription is
only quantitatively valid for comparing a particular isotype among
tissues but not directly for comparing different isotypes. Furthermore,
all assays must be conducted on samples from all animals simultaneously
to avoid bias caused by differences in the sp. act. of the
32P-probes used.
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Sequence analysis of thymic transcripts
Anti-sense Cµ, C, and C primers were used together with
a FR1 sense primer to amplify the rearranged VDJs transcribed with IgM,
IgA, and IgG, respectively, from the thymus of both normal and
PRRS-infected fetuses. The resultant PCR products were then cloned into
pBluescript as previously described (35), and 33
IgM-, 34 IgA-, and 32 IgG-associated VDJ clones were selected at random
for sequencing. Clones were sequenced using the T3 primer and the
four-color automated Applied Biosystems (Foster City, CA)
system. Sequences were analyzed for identification of
VH usage, for mutation frequency in
complementarity-determining region (CDR)1, CDR2, and CDR3, CDR3 length,
DH usage and length, and N and P region additions
in the manner previously described (47, 48). Eight of the
clones were duplicates; Table V summarize
only the data from the 91 unique clones.
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The serum levels of these Igs were quantified by sandwich ELISA.
In the case of IgA, the globulin fraction of an -chain-specific
rabbit anti-IgA was used (B5321) for capture, whereas mAb 1459 was
used for detection (provided by Dr. K. Nielsen, Animal Disease Research
Institute, Nepean, Ontario, Canada). mAb 5C9B12 was used to capture IgM
(provided by Dr. P. Paul, Iowa State University, Ames, IA) while
detection was accomplished using rabbit anti-swine L chain conjugated
to alkaline phosphatase. IgG was captured using the globulin fraction
of a -chain-specific polyclonal antiserum (B784 + 5) and detected
using the same anti-L chain conjugate described above for measuring
IgM. Methods for the preparation of all polyclonal Abs and purified
IgM, IgG, and IgA reference standard sera have been previously
described (36, 49, 50, 51). All sandwich assays were conducted
as previously described (47, 50, 52, 53).
Recovery experiments, designed to test the effect of contaminating 500
µl of several fetal sera with 1 µl of an adult serum, were
performed by quantitation of IgG, IgA, and IgM in the fetal sera before
and after "spiking" and in the adult serum used for spiking. The
relationship of the concentrations of IgG, IgA, and IgM was established
by expressing IgA and IgM levels relative to IgG and assigning the
latter a value of 100. This generates an isotype profile (see Fig. 1B).
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The CDR3 segments of the isotype-specific cDNAs described above were amplified using nested FR3 and anti-sense FR4 primers, which anneal to all porcine VDJ rearrangement (35, 42). These were separated on 6% polyacrylamide gels as described above and elsewhere (48). The resultant spectratypes were scanned, and different tissues of the same individual piglets were compared according to isotype.
Detection of Ig-SC
The number of spontaneous Ig-SCs of all isotypes was determined
among thymocytes, splenocytes, BM leukocytes, and lymphocytes isolated
from cord blood, liver, PBMC, Peyer’s patches, and mesenteric lymph
nodes (MLN) by the ELISPOT assay using isotype-specific mAbs as
previously described (37). The following murine
anti-swine mAbs were used as capture Abs: anti-IgM (LIG-4; Ref.
54) and anti-IgG (23.3 1a) and anti-IgA
(27.9.1; both obtained from van Zaane and Holst; Ref. 55).
Detection of captured Igs was visualized using a mixture of
biotinylated anti-
and -
mAbs 27.7.1 and 27.2.1 and the
streptavidin-HRP system. Data are expressed as 1) the number of Ig-SCs
(according to isotype) per 106 cells (Table III),
and 2) the total number of Ig-SC in each organ examined by multiplying
Ig-SC per million times the total number of leukocytes recovered from
each organ (Table IV).
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The Ig profiles in the serum of fetal piglets of different age (DG) and treatment (e.g., PRRS-infected vs normal fetuses) were compared by Student’s t test to the mean profile established using the sera of 27 birth mothers. We tested the null hypothesis that normalized IgA and IgM levels in the sera of fetuses and newborns was identical with that in the sera of gilts. The same statistical treatment was applied to the comparison of mutation frequencies in transcripts from the thymus of normal vs PRRS-infected fetuses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IgG was detected in the sera of all 38-day and older fetuses,
whereas IgM and IgA was only detected in 2/7 of the 38-day samples and
in only 1/6 of the 57-day samples (data not shown). Thereafter, all
three isotypes were detected in every specimen and increased in
concentration with fetal age (Fig. 1
A). In all very young fetuses
and in animals 70 days and older, IgG was present at concentrations
10-fold higher than IgM levels. Because this concentration
relationship, i.e., isotype profile, is superficially reminiscent of
that in the sera of the gilts (but at a 1000-fold lower concentration),
it seemed probable that the Igs in fetal serum were of maternal origin
and their age-dependent increase was the result of increasing low-level
placental leakage or contamination during the collection process. The
extent to which such contamination of fetal sera with maternal serum
could alter fetal Ig levels was tested by adding 1 µl of maternal
serum to 500 µl of four different fetal serum samples containing very
low levels of Igs, i.e., 0.734 ± 0.38 µg/ml IgG. When the
spiked fetal sera were measured, their Ig levels increased 43-fold. The
adult serum used for spiking contained 16.98 mg/ml IgG, 487 µg/ml
IgA, and 5.08 mg/ml IgM. The mean concentrations of Igs
recovered from four spiked fetal sera were 31.8 ± 1.8 µg
IgG/ml, 916 ± 121 ng/ml IgA, and 9.86 ± 0.47 µg/ml IgM.
The expected values were 33.9 µg/ml IgG, 975 ng/ml IgA, and 10.16
µg/ml IgM. Thus, the spiked fetal sera gave exactly the same profile
as the maternal serum used for spiking (Fig. 1B). Based on
the outcome of this in vitro test, we reasoned that contamination
during collection could be tested by comparing the isotype profiles of
fetal and maternal sera. IgM and IgA concentrations were normalized to
that of IgG and the latter assigned a value of 100. Representative
isotype profiles of fetuses of different age, newborn piglets, and of
their birth mothers are shown in Fig. 1B. Relative IgM
levels were always significantly lower than in maternal sera, and IgA
levels especially increased in late gestation, attaining values 2-fold
higher than IgM in colostrum-deprived newborns. This difference is
significant by Student’s t test (Fig. 1, A and
B). When the isotype profiles of individual birth
mothers were compared with the isotype profiles of their own offspring,
there was no correlation (data not shown) similar to the lack of
correlation seen with nonlitter-segregated animals (Fig. 1B).
Virally infected fetuses have Ig levels and profiles that differ from normal fetuses
Because porcine Parvovirus and the arterivirus
responsible for PRRS have been reported to cross the placenta by an
unknown mechanism, we wondered whether especially the Igs of switch
isotypes in fetal serum represented an immune response to an
environmental Ag. Thus, we infected fetuses in utero at 50 DG and
monitored the immune response using a PRRS-specific ELISA (data not
shown). The fetal response to PRRS virus is latent, perhaps because the
number of target cells for the arterivirus is limiting
(56). An anti-PRRS virus response is only weakly
detectable by day 95, but such fetuses show a >2-fold elevation of
IgG, >10-fold increase in IgA, and >5-fold increase in IgM compared
with normal piglets of nearly the same age (data not shown). At 110
days, when anti-PRRS ELISA titers were robust, IgM levels were
elevated 50-fold and IgG levels 5- to 10-fold compared with age-matched
normal fetuses. This yielded serum Ig levels much greater than seen in
normal age-matched fetuses (data not shown) and an isotype profile that
differed from that in the sera of gilts because of relatively lower
levels of IgA and higher levels of IgG (Fig. 1B).
IgM, IgA, and IgG are transcribed in fetal piglets
Representative raw hybridization results are shown in Fig. 2. As shown, cross-hybridization values
were equal or less than background scanner values except for the IgG
probe in which 1.2% cross-reactivity with IgA was observed. Table II
gives scanner results obtained when equal volumes of isotype or
-actin PCR products were hybridized with 32P
isotype or -actin probes. Representative data were selected from
those with very low transcription (IgA and IgG in 60 DG spleen) to
those with high levels of transcripts (90 DG and 110 DG thymus). Table II provides insight into animal variation and assay reproducibility
including cases of extreme variation. The latter were typically
encountered when values close to background were encountered (IgA and
IgG in 60 DG spleen). Nevertheless, an overall coefficient of variation
(C.V.) of <12% was obtained between replicates. Extreme animal
differences were seldom seen, although animals 2 and 3 differ nearly
10-fold in IgA thymic transcription but only 3-fold in IgM
transcription.
When transcription of the IgM, IgG, and IgA were expressed relative to
-actin for 4–6 animals at each time point, IgM transcription was
observed in all lymphoid tissues available at 50 DG (Fig. 3). IgM relative transcription was
especially pronounced in spleen and BM at and after 60 DG. Although
some individual splenic samples showed trace amounts of transcripts for
switch isotypes (Table II), pronounced transcription was not observed
until 60 DG in thymus (Fig. 3). Noteworthy is that pronounced
transcription of these switched isotypes was confined to the thymus, in
sharp contrast to that seen for IgM (Fig. 3). However, hybridization of
a Cµ probe with thymic cDNA from 90- and 110-day fetuses was
pronounced (Table II, Fig. 2). By 110 days, transcription of both IgG
and IgA was also emerging in the IPP (Fig. 3). In data not shown in
Fig. 3, IgG and IgA transcription could also be detected in the MLN of
late term fetuses, and IgA transcripts could be detected in the parotid
gland of 110-day fetuses (Fig. 4).
Measured as relative transcription, this was less than that seen in the
IPP (data not shown).
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Representative CDR3 spectratypes for IgM, IgA, and IgG are
presented in Fig. 5. Spectratypic
analysis is CDR3 length analysis. Because CDR3 is the result of the
recombination of the VH,
DH, and JH segments by a
mechanism that involve segment trimming, N region additions, and P
nucleotide additions, it is the major source of Ab and T cell
repertoire diversity. CDR3 is most important for both Ab
(57) and T cell specificity (58). Because in
all lymphoid tissues studied except the fetal parotid (Fig. 4) the IgM
spectratype was polyclonal, only the IgM spectratype at 60, 90, and 110
days in BM is shown. Although significant relative transcription of IgG
and IgA appeared at 60 days in fetal thymus (Fig. 3), the CDR3
spectratype of transcripts of both switched isotypes was very
oligoclonal. In 70-day and older fetuses, the prominent IgA transcripts
in thymus were as polyclonal as those for IgM, and the same is true
after 90 days for IgG transcripts (Fig. 5). This observation, together
with data on relative transcription (Fig. 3), suggests that switch
recombination is a major event with broad clonal participation in the
thymus.
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although the IgG spectratype was polyclonal at 90 and 110 days (Fig. 5). In general, the IgG spectratype in spleen, IPP, and BM tended to be
more polyclonal than that for IgA transcripts, although individual
variations were observed (Fig. 6).
Because the fetus is free from environmental Ag, these differences are
either the results of random chance or are genetically determined. We
observed no tendency for CDR3 lengths in any oligoclonal pattern to
favor very short (12–18 nt) or long (45–60 nt) sequences, and in
60-day thymus, both were observed in the IgG spectratype (Fig. 5). In
cases where IgG and IgA transcript spectratypes were oligoclonal,
seldom was the exact same spectratype observed in different fetuses of
the same age (Fig. 6). Thus CDR3 length differences between tissues and
individuals appear random.
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It is generally regarded that B cell migration to noninductive
sites in the mucosal immune system is driven by antigenic stimulation
and amplified if Ag is present at the site (59). Fig. 4
shows that both IgM and IgA can be recovered from the fetal parotid
gland, although few IgA clones are represented. However, piglets
maintained germfree for 6 wk show development of a polyclonal
spectratype for both IgA and IgM, suggesting that this clonal expansion
is either spontaneous or driven by food protein. Because piglets
maintained germfree do not respond to fluorescein-keyhole limpet
hemocyanin or trinitrophenyl-Ficoll administered without
adjuvant if not colonized (J. E. Butler and D. Francis,
unpublished data) and no Abs to dietary proteins can be detected (J. E.
Butler, P. Weber, and D. Francis, unpublished data), expansion
to a polyclonal repertoire in the parotid gland of germfree
piglets does not appear to be driven by environmental Ag.
The fetal thymus may be a major source of IgA and IgG during fetal life
The transcription data presented above indicate that C and C
transcripts are prominently expressed in the thymus. Because both
switched isotypes are also present in serum, we wondered whether the
number of IgA- and IgG-SC in thymus was correlated with serum IgG and
IgA levels. Table IV shows that 70% of all Ig-SC detected by
ELISPOT in the tissues studied were recovered from the thymus. Analyzed
according to isotype, IgA- and IgG-SCs were only detected in the
thymus, whereas IgM-SC were found in both spleen and thymus (Table III). On day 105, no Ig-SCs were observed in fetal liver, blood, BM,
omentum, MLN and Peyer’s patches. However, although serum IgG levels
are >25-fold higher than serum IgA, the number of IgA and IgG-SCs in
thymus are essentially equal.
Switched thymic transcripts in normal fetuses generally resemble those in germfree piglets, not those in piglets or adults exposed to environmental Ags
A total of 44 thymic VDJ transcripts from several normal late term
fetuses, representing those expressed with IgM, IgA, and IgG, and 47
transcripts from two PRRS-infected fetuses were analyzed. These
transcripts were not unique to the thymus because >95% used the same
four porcine "fetal VH genes"
(VHA, VHB,
VHC, and
VHE)4
that characterize the fetal preimmune repertoire, and all but five of
the 91 transcripts used either DHA or
DHB. The results of this analysis are summarized
in Table V. In uninfected fetuses
1) relative DH usage
(DHA:DHB = 0.28 for
all normal transcripts) resembled that seen in germfree piglets (0.27)
rather than that of colonized and conventional animals
(DHA:DHA > 1.7), and
usage of DHB was especially favored in IgG
transcripts; 2) total CDR3 length in IgM and IgA transcripts did not
significantly differ from that reported for >220 fetal VDJs
(48), although because of shorter
DHB segments, more N region additions were
required to achieve the same CDR3 length; 3) the CDR3s of IgG
transcripts were shorter than those of IgA and IgM because of fewer N
region additions; 4) the formula used for VH
usage (VHA + VHC +
VHX)/(VHB +
VHE) in IgM and IgA transcripts yielded values
(0.9) that resembled those in germfree animals (0.5), whereas in the
case of IgG transcripts, this formula yielded notably higher values
(e.g., 3.8) than in germfree piglets; and 5) the overall frequency of
mutations (CDR1, CDR2, and CDR3; column 6, total mutation) was similar
to that in transcripts from germfree piglets and fetal VDJ in general
(48) but much lower than that in colonized piglets and
adult swine.
Although the overall mutation frequency resembled that of
germfree piglets, i.e., <0.03, the mutation frequency was not
randomly distributed among the CDRs. The CDR3 region was virtually
nonmutated in all normal fetal transcripts, whereas the frequency of
mutation is significantly higher in CDR2 of normal IgA transcripts, but
not in IgG transcripts, compared with CDR2 in IgM transcripts (Table V). Furthermore, when the total mutation frequency was compared, VDJs
transcribed with switched isotypes were significantly more mutated than
those associated with IgM in both normal and virus-infected piglets
(Table V). Age-matched PRRS-infected animals displayed
DH ratios and VH usage
profiles indicative of repertoire diversification, and these were
shifted in the direction of those from colonized piglets and
conventional adult swine that received known exposure to environmental
Ag. Somatic mutation in thymic transcripts from PRRS-infected fetal
piglets was concentrated in CDR2 and CDR3, in contrast to normal
fetuses, in which mutations in CDR3 were nearly absent. Specifically,
mutation frequencies were 10-fold higher in CDR3 of PRRS-infected
animals than in normal fetuses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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10-fold higher level of IgG than IgM is superficially reminiscent to
what is seen in the sera of the gilts, the fetal isotype profile is
distinct from that of their mothers (Fig. 1B). Nevertheless,
contamination during the collection process is possible and is
especially problematic when collecting <0.5 ml of blood from the
umbilicus of very young fetuses. The impact of such an event was
demonstrated in fetal samples experimentally contaminated with maternal
serum (1 µl/500 µl). This test resulted in sera with an isotype
profile identical with that of the serum used for contamination and
statistically distinct from the isotype profile of all normal fetuses
(Fig. 1B). Because IgA and IgM were detected in only a
minority of samples before DG70, reliable mean values were not
available for calculating isotype profiles before DG70. This leaves
open to speculation whether any serum Igs before DG70 are of fetal
origin. On one hand, only 100–500 µl can be collected from such
small fetuses, so contamination with 0.1 µl of maternal blood is
conceivable. In contrast, trace levels of IgG transcripts were detected
at DG50 in spleen, leaving unresolved the possibility that serum IgG
detected at DG57 could also be of fetal origin. The argument against
contamination during the collection from older fetuses is supported by
the observation that Ig levels increase, not decrease, with fetal age
(Fig. 1A). If the 0.5-ml sample collected on day 38 was
contaminated with 1 µl of maternal blood, the Ig levels should be
much higher than if 1 µl of maternal blood contaminated the <10-ml
samples collected from 90- and 110-day-old fetuses. Many fetal sera
gave an isotype profile with significantly lower levels of IgM and
higher levels of IgA than are seen in maternal serum (Fig. 1B), and newborns had absolute IgA levels higher than those
for IgM (Fig. 1A). Thus the switched isotypes in fetal serum
must result from a previously undescribed selective transport or from
de novo synthesis. The latter does not fit the paradigm established
using the mouse model but is supported by the transcription and ELISPOT
data reported here, whereas the former does not fit the paradigm
established for any group III mammal.
Although some IgG in fetal sera may result from low level selective
transport, the magnitude of this transport and the methods used to
study it are the subject of another study (61). Here we
have presented unambiguous evidence that the switched isotypes
are both transcribed and secreted by the fetus. Data on transcription
(Figs. 2, 3, 5, and 6) show that IgG, IgA, and IgM are all
transcribed in utero, and even those for switched isotypes are
pronounced on DG60 in thymus, i.e., during the first half of gestation.
Not only is the thymus a prominent site for the transcription of these
switched isotypes (Figs. 2 and 3), but the resulting repertoire is
polyclonal (Fig. 5), and ELISPOT data indicate that both IgA- and
IgG-SCs are present in thymus but not in other tissues that were
studied (Tables III and IV). The less polyclonal and/or oligoclonal
CDR3 spectratype observed with switched isotypes in spleen, IPP, and BM
presumably reflects the paucity of IgG and IgA cells in these organs.
The surprising polyclonality seen in spleen for switched isotypes
despite low relative transcription in this organ, and the high degree
of polyclonality seen in thymus, may indicate that splenic IgG and IgA
cells are immigrants from the thymus.
Although we have observed no hypermutation in rearranged VDJs in DNA
during fetal life in piglets (46, 48) and the overall
mutation frequency in all fetal transcripts was similar to that in
germfree piglets (Table V), VDJs associated with IgA had a
higher total frequency of mutations and mutations in CDR2 when compared
with IgM transcripts from the same animals (Table V). Furthermore,
VH usage in IgG transcripts was heavily skewed
away from VHB usage; this phenomenon is normally
seen in diversified repertoires of colonized and conventional adult
animals (Table V). This trend would be anticipated for thymic
transcripts from fetuses actively producing Abs to an environmental Ag.
This was observed in those from PRRS-infected fetuses, where
VH usage is skewed away from
VHB (a VH81x analog) to
preferential usage of VHA and
VHC. Although somatic mutations in IgG and IgA
transcripts from normal fetuses are rare in comparison to colonized
isolator piglets and adults, they are more frequent than in IgM
transcripts from the same animals. Thus switch recombination appears to
parallel repertoire diversification even when not driven by
environmental Ag. Our in vivo findings in a nongenetically manipulated
mammal are consistent with evidence from CD40-deficient (62, 63), µMT/µMT heavy chain minilocus transgenic
(64), and lymphotoxin- knockout mice (23),
which indicates that switch recombination can proceed in the absence of
antigenic stimulation and/or GC formation. In fact, studies with 3-83
transgenic mice show that the switch from IgM to IgG2a precedes GC
formation (65), whereas it has been shown that some
patients with X-linked hyper IgM syndrome can somatically mutate their
VDJs without GC formation (66).
The ratio of IgG- to IgA-SC in thymus is nearly equal, whereas the
serum IgG/IgA ratio is 10:1. Thus, IgG must 1) be secreted at a
greater rate, 2) preferentially accumulate in the blood vascular
system, 3) be in part derived from maternal blood by selective
transport, or 4) be synthesized elsewhere than the thymus. Secretion
outside the thymus was not detected (Tables III and IV). In mammals
with the exception of primates, most serum IgA is dimeric (67, 68), and its short half-life (6 days) apparently results from
its rapid transport to exocrine body fluids (68). In
contrast, most IgG is not transported extravascularly, so the higher
serum IgG levels we report could result from the longer serum IgG
half-life, i.e., 20 days. Although the thymus may account for most
fetal serum IgA, the exact origin of all fetal IgG remains unknown and
awaits further experimentation. In support of de novo synthesis of all
fetal IgA, late-term fetuses and colostrum-deprived newborns have Ig
profiles with relative IgA levels much higher than their mothers and
exceeding those of IgM (Fig. 1, A and B). This
apparent increase in IgA production just before birth may reflect
increased IgA production in sites such as the IPP in the last few days
before parturition (Fig. 3).
We show elsewhere (61) that some low-level IgG placental
transport occurs, although its impact on protective passive immunity is
likely to be minimal because even in fetuses with the highest IgG
levels, these are >1000-fold lower than in day-old piglets that suckle
their dam (33). However, transfer of some maternal IgG
across the placenta might provide an avenue for viral transfer and
other environmental Ags if they could enter the fetus as part of immune
complexes. The delivery of Ag in this manner could stimulate an immune
response that could subsequently result in switch recombination and
explain the transcription and secretion of the switched isotypes we
report here. If this scenario is correct, it occurs with minimal effect
on repertoire diversification but could explain the repertoire
diversification observed in thymic IgA and IgG transcripts compared
with IgM transcripts in normal fetuses (Table V). Although it can be
maintained that viral Ags and viral infections are not representative
of all environmental Ags, the repertoire diversification observed in
response to infection with PRRS virus parallels those resulting from
bacterial colonization. Thus it is unlikely that the switch
recombination and VDJ diversification we observed in fetal piglets
results from a unique environmental Ag. Thus, switch recombination in
normal swine fetuses must 1) result from stimulation with fetal Ag, 2)
be driven by cytokines released in an Ag-free environment, or 3) be a
stochastic event in unstimulated B cells in this species. Regardless of
mechanism, data from both normal and PRRS-infected fetuses show that
switch recombination is associated with repertoire diversification.
The reason why the epicenter for isotype switch recombination is the
fetal thymus is unexplained. There is no precedent indicating that the
thymus can act as a site of Ag presentation by B cells or as a site for
the B cell GC reaction. However, evidence that B cells and Ig-SC can be
found in the thymus dates back to 1965 (69). More
recently, evidence has been presented indicating that B cell
lymphogenesis occurs in the mouse thymus (43), and we have
shown that the DNA of porcine thymus (but not the cDNA) is the only
lymphoid DNA that shows a CDR3 spectratype consistent with that of an
unselected pro-B cell population (48). It is not
transcripts from this presumed pro-B cell population that are analyzed
in Table V. Rather, we and others have also shown that isotype-switched
plasma cells are present in both the bovine and porcine thymus
(37, 70), with IgA production being especially pronounced
in cattle (70). These IgG and IgA plasma cells are
predominantly found in the medulla, and we have sampled these by
micromanipulation and shown them to contain the type of VDJs described
in Table V (M. Sinkora, unpublished data).
Akashi and colleagues (45) calculated that
3 x 104 B cells leave the mouse thymus,
of which 2/3 were developed intrathymically. Thus, low level
transcription of IgG and IgA in many lymphoid tissues of the fetal
piglet (Fig. 3) could be due to immigrant cells derived from the
thymus. Although significant relative IgG and IgA transcription occurs
first in the 60-day fetal thymus, this does not rule out switch
recombination in other lymphoid tissues. The appearance of both IgG and
IgA transcripts in fetal IPP (Fig. 3) would suggest that if the IPP of
swine functions as a primary lymphoid tissue for repertoire
diversification as reported for sheep (42), this
diversification also involves cells with switched isotypes. We have no
evidence that porcine IPP, although anatomically similar to the
IPP of sheep (71), functions as a primary lymphoid tissue.
Thus, it behaves in a manner distinct from the IPP of sheep and cattle
(70, 71). We have observed no significant somatic mutation
in any lymphoid tissue of fetal or germfree isolator piglets
(46, 47, 48) as has been reported in sheep and cattle
(72, 73). Because the type of switch recombination we
report here has not been observed to occur in fetal hindgut lymphoid
tissues of chickens, sheep, or rabbits, neither phylogeny nor
anatomical homology is a reliable predictor of repertoire
diversification in higher vertebrates. Thus, the in vivo piglet model
continues to offer a rich opportunity to address a variety of topics
concerning repertoire development in a natural and noncontrived
environment.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John E. Butler, Department of Microbiology, University of Iowa, 51 Newton Road, Iowa City, IA 52242-1109. E-mail address: john-butler{at}uiowa.edu
3 Abbreviations used in this paper: GC, germinal center; DG, days of gestation; CDR, complementarity-determining region; PRRS, porcine respiratory and reproductive syndrome; IPP, ileal Peyer’s patch; SC, secreting cell; MLN, mesenteric lymph node; BM, bone marrow; C.V., coefficient of variation.
4 Fetal and newborn piglets develop their preimmune repertoire using four different VH genes that account >90% of all VH usage (46 47 ). In addition, almost all DH usage can be accounted for by DHA and DHA.
Received for publication May 11, 2001. Accepted for publication July 16, 2001.
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cooperate to induce anti-CD40 activated naive human B cells to secrete immunoglobulin A. J. Exp. Med. 175:671.1 genes on the expressed and unexpressed chromosomes of lipopolysaccharide-activated normal murine B-cells. J. Immunol. 143:334.[Abstract]
/ T-cell receptors. Annu. Rev. Immunol. 16:532.
diversification in cattle. J. Immunol. 161:5438.This article has been cited by other articles:
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  J. E. Butler, N. Wertz, P. Weber, and K. M. Lager Porcine Reproductive and Respiratory Syndrome Virus Subverts Repertoire Development by Proliferation of Germline-Encoded B Cells of All Isotypes Bearing Hydrophobic Heavy Chain CDR3 J. Immunol., February 15, 2008; 180(4): 2347 - 2356. [Abstract] [Full Text] [PDF]
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J. E. Butler, C. D. Lemke, P. Weber, M. Sinkora, and K. M. Lager Antibody Repertoire Development in Fetal and Neonatal Piglets: XIX. Undiversified B Cells with Hydrophobic HCDR3s Preferentially Proliferate in the Porcine Reproductive and Respiratory Syndrome J. Immunol., May 15, 2007; 178(10): 6320 - 6331. [Abstract] [Full Text] [PDF]
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J. E. Butler, P. Weber, and N. Wertz Antibody Repertoire Development in Fetal and Neonatal Piglets. XIII. Hybrid VH Genes and the Preimmune Repertoire Revisited J. Immunol., October 15, 2006; 177(8): 5459 - 5470. [Abstract] [Full Text] [PDF]
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J. E. Butler and N. Wertz Antibody Repertoire Development in Fetal and Neonatal Piglets. XVII. IgG Subclass Transcription Revisited with Emphasis on New IgG3 J. Immunol., October 15, 2006; 177(8): 5480 - 5489. [Abstract] [Full Text] [PDF]
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J. E. Butler, D. H. Francis, J. Freeling, P. Weber, and A. M. Krieg Antibody Repertoire Development in Fetal and Neonatal Piglets. IX. Three Pathogen-Associated Molecular Patterns Act Synergistically to Allow Germfree Piglets to Respond to Type 2 Thymus-Independent and Thymus-Dependent Antigens J. Immunol., November 15, 2005; 175(10): 6772 - 6785. [Abstract] [Full Text] [PDF]
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J. E. Butler, N. Wertz, J. Sun, H. Wang, P. Chardon, F. Piumi, and K. Wells Antibody Repertoire Development in Fetal and Neonatal Pigs. VII. Characterization of the Preimmune {kappa} Light Chain Repertoire J. Immunol., December 1, 2004; 173(11): 6794 - 6805. [Abstract] [Full Text] [PDF]
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C. D. Lemke, J. S. Haynes, R. Spaete, D. Adolphson, A. Vorwald, K. Lager, and J. E. Butler Lymphoid Hyperplasia Resulting in Immune Dysregulation Is Caused by Porcine Reproductive and Respiratory Syndrome Virus Infection in Neonatal Pigs J. Immunol., February 1, 2004; 172(3): 1916 - 1925. [Abstract] [Full Text]
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J. E. Butler, P. Weber, M. Sinkora, D. Baker, A. Schoenherr, B. Mayer, and D. Francis Antibody Repertoire Development in Fetal and Neonatal Piglets. VIII. Colonization Is Required for Newborn Piglets to Make Serum Antibodies to T-Dependent and Type 2 T-Independent Antigens J. Immunol., December 15, 2002; 169(12): 6822 - 6830. [Abstract] [Full Text] [PDF]
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