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Departments of
* Molecular Virology and Microbiology and
Immunology, Baylor College of Medicine, and
Veterans Affairs Medical Center, Houston, TX 77030
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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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RV infects and replicates in the epithelial cells of the small intestine. RV and rotaviral Ags are shed into the lumen of the intestine during an infection and are detectable by ELISA. It is thought that control of RV infection is lumenal and dependent upon Ab. Ab, in particular intestinal IgA, correlates with protection from RV infection (3). Studies in mice have correlated the rise in fecal IgA levels after 7 days postinfection (dpi) with the decrease in rotaviral Ag shed (4). However, work in animal models has shown that protection from reinfection can also be established in the absence of IgA (5) and is thought to be modulated by IgG. Studies in infant mice, humans, and rabbits have determined that RV-specific IgM levels increase initially early (before 7 dpi) after infection and then decrease, while IgA and IgG levels increase (6, 7, 8). The correlation of Ab with protection implies a significant role for B lymphocytes in the mediation of immune responses to RV infection, but it is not known what exact contribution B lymphocytes and Ab production make to clearance of the infection.
Mice provide a good model for studying the immune response during a primary RV infection (9). Infant mice exhibit diarrhea starting 1 day after infection with homologous murine strains of RV (EC wild type (ECwt) and epizootic diarrhea of infant mice) and resolve infection by 9 days after infection (4). Infection of infant or adult mice with homologous strains of RV protects the mice against reinfection as adults (4). Naive mice >14 days of age, infected with murine RV, shed detectable fecal RV Ag during a primary infection but do not exhibit diarrhea (10). The kinetics of infection in mice differs slightly from what is observed in humans. Unlike infant mice, human infants can get multiple RV infections up until approximately the age of 5 years (11), and there have been many reports of adult RV infections, particularly in the elderly (11). However, aside from these differences, studies of RV infection in mice can still provide valuable information on the induction of immune responses by the virus.
The mechanism of clearance of a RV infection in the mouse has been difficult to define. Recombinase-activating gene-2-/- and SCID mice (deficient in both B and T lymphocytes) are unable to clear RV infection (12, 13), suggesting that either B or T lymphocytes mediate RV clearance. TCR-deficient mice (lacking functional T cells) effectively clear a primary infection and produce RV-specific Ab, suggesting that clearance is T cell independent (14). However, adoptive transfer of CD8+ T cells into SCID mice results in clearance of RV with a short delay compared with normal mice (15), suggesting that clearance can occur in the absence of B cells or Abs and that control of RV infection can occur at the cellular level. Further evidence for a B cell/Ab-independent clearance mechanism is provided by studies that demonstrated that RV infection in B cell-deficient JHD and µMt mice was efficiently cleared (16). Taken together, these studies in knockout (KO) mice have implicated both B and T lymphocyte subsets in rotaviral clearance, but within the context of an intact immune system the lymphocyte subsets critical for clearance have not been identified.
Local Ig responses in the small intestine of RV-infected mice are first detected 7 days after infection (6, 17), suggesting that there may be significant B lymphocyte activation in the gut-associated lymphoid tissues (GALT) before this time. Because RV Ag has been previously detected in the Peyer’s patches (PP) and mesenteric lymph nodes (MLN) of infected mice (17), we reasoned that the presence of virus in these tissues might stimulate early activation of lymphocytes at these sites and initiate the production of RV-specific Ab. We demonstrate in this work, in outbred CD-1 mice infected with a homologous murine RV, ECwt, that the earliest lymphocyte response to RV infection is a large increase in the percentage of activated B cells, and not T cells, in the PP and MLN. In addition, these B lymphocytes secrete RV-specific IgM and not IgA. The presence of activated B cells in RV-infected TCRKO mice demonstrates that the early response to RV infection is T cell independent.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice were housed in microisolation cages and fed ad libitum.
CD-1 female mice 
6 wk of age (Charles River Breeding Laboratories,
Wilmington, MA), C57BL/6J male and female mice, or TCRKO 
and 
male and female mice (The Jackson Laboratory, Bar Harbor, ME) were
inoculated orally with 100 µl of PBS or 100 µl of PBS containing
105 shedding doses (SD50)
of murine ECwt, as described previously
(18). Fecal samples were collected daily 1–7 dpi and
analyzed by ELISA for Ag shedding and RV-specific Ab against simian
SA11 RV as described previously (18).
Qualitative analysis of PP and MLN
Five mice in each group were euthanized 1–7 dpi by administration of 100 µl of rodent combination anesthesia (37.5 mg/ml ketamine, 1.9 mg/ml xylazine, 0.37 mg/ml acepromazine) and cervical dislocation. PP, MLN, and spleens (SP) were removed and imaged using a Nikon Coolpix990 digital camera (Nikon, Tokyo, Japan). Part of histological analysis was performed by the Center for Comparative Medicine (Baylor College of Medicine, Houston, TX). Briefly, tissues were placed in 10% zinc buffered formalin, embedded in paraffin, sectioned, and stained with H&E following standard protocols. Histology images were recorded using an Olympus IX70 microscope (Olympus, Lake Success, NY).
Quantitative analysis of lymphocyte cell number
All small intestinal PP (typically 8–10) and MLN were harvested and placed in complete RPMI medium consisting of RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% FBS (Summit Biotechnology, Fort Collins, CO), 200 mM L-glutamine, 10,000 U/ml penicillin and streptomycin, 50 mM 2-ME, and 5% NCTC-109 medium (BioWhittaker). Single cell suspensions were made by mechanically disrupting tissue by gently pressing through a 70-µm cell strainer (Fisher Scientific, Pittsburgh, PA). RBCs were lysed using ACK lysing buffer (BioWhittaker) and cell suspensions were washed and resuspended in complete RPMI 1640. Aliquots of cell suspensions were stained with trypan blue and viable cells were counted using a hemocytometer (BD Biosciences, San Diego, CA). Significant differences in cell numbers were determined using a two-way ANOVA with two between-group factors (treatment and dpi).
Flow cytometry
Aliquots of cell suspensions from SP, MLN, and PP containing 2 x 106 cells from each tissue were washed in PBS and centrifuged at 400 x g, and the cell pellets were suspended in 100 µl of PBS containing 2% FBS (Summit Biotechnology) and PE-labeled CD4 (0.06 µg/106 cells; BD Biosciences), CD8 (0.06 µg/106 cells; BD Biosciences), or CD19 (0.06 µg/106 cells; BD Biosciences) and FITC-labeled CD69 (0.06 µg/106 cells; BD Biosciences). Samples were incubated on ice for 30 min in the dark. Cells were washed once with 1 ml of 2% FBS in PBS and the resulting pellet was resuspended in 500 µl of 4% paraformaldehyde (pH 7; Fisher Scientific) and stored at 4°C until analysis. Samples were analyzed using a Coulter EPICS XL-MCL (Beckman Coulter, Hialeah, FL) flow cytometer. Significant differences between the percentages of labeled cells from control and infected groups was determined using a two-way ANOVA with two between-group factors (treatment and dpi).
Fragment cultures
Fragment cultures were performed as previously described (19). Briefly, all intestinal PP and MLN were removed and washed three times in HBSS-Ca-Mg (Life Technologies) supplemented with 25 mM HEPES (Sigma-Aldrich, St. Louis, MO) and 50 µg/ml gentamicin (Life Technologies). Tissues were suspended in 48-well plates containing 400 µl of GALT medium (H-Y medium (Sigma-Aldrich) containing 10 mM HEPES, 10% FBS, 4 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin, and 0.25 µg/ml amphotericin B). Samples were incubated for 4–6 days at 37°C with 95% humidity and 5% CO2. Supernatants were removed and specific Ab amounts were determined by ELISA (18). Differences in the amount of RV-specific Ab produced by each tissue were analyzed using a Student t test. A value of p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Quantitation of fecal RV Ag levels of CD-1 mice orally infected
with a homologous strain of murine RV, ECwt
(4), revealed that CD-1 mice shed RV Ag within 24 h
after infection and rapidly resolved infection between 5 and 7 dpi
(Fig. 1
). This is in contrast to mice
deficient in both B and T lymphocytes, which continually shed RV
Ag, suggesting that lymphocytes mediate clearance of RV infection
(12, 13). However, the phenotype and numbers of
lymphocytes activated early during a primary RV infection have not been
identified. Because the virus enters via the gastrointestinal tract, we
examined PP and MLN, two sites of early inductive immune responses
after exposure to enteric pathogens. Additionally, we examined the
response in the SP, classically thought to be involved in systemic
immune responses. Isolation of the three tissues revealed dramatic
increases in the size of PP and MLN, but not SP, of
ECwt-inoculated mice between 2 and 5 dpi compared
with PBS-inoculated mice (4 dpi, shown in Fig. 2, A–C).
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Histological examination of PP and MLN sections from
ECwt-inoculated mice revealed a hyperplasia of
lymphocytes within both tissues without any detectable increase in
germinal center formation (Fig. 2
, D–G). The
distribution and numbers of peanut
agglutin+CD19+ cells (germinal center
B cells) did not differ between infected and uninfected sections of
either tissue at 4 dpi (data not shown). Additionally, tingible
body macrophages indicative of Ag processing were observed in the PP
sections from ECwt-inoculated mice at 2 and 4 dpi
but not in PBS controls (data not shown). Because histological analysis
indicated an increase in the number of lymphocytes, single cell
populations were quantified from the SP, PP, and MLN from both control
and infected groups. Anatomical and histological observations were
confirmed; there were significant increases in total lymphocyte numbers
in the PP and MLN of RV-inoculated mice (Fig. 3) compared with PBS-inoculated mice
(p < 0.05), with no increase in lymphocyte
numbers in the SP (p > 0.05, data not shown).
Therefore, at the time of active RV replication in the intestine there
are significant increases in the total number of lymphocytes in the PP
and MLN, indicating that lymphocytes within these sites are potentially
important for clearance of infection and in the initiation of the
primary Ab response.
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We next sought to determine whether a specific subset
of lymphocytes was activated in response to RV infection in the PP and
MLN of ECwt-infected mice. The percentage of
lymphocytes expressing CD69, an early marker of activation, and
lymphocyte cell surface markers CD4 (Th cells), CD8 (cytotoxic T
cells), and CD19 (B cells) in each tissue was determined using a flow
cytometer. The percentage of CD4+,
CD8+, CD19+,
CD4+CD69+, or
CD8+CD69+ in the PP did not
differ between PBS-inoculated mice and RV-inoculated mice (Fig. 3).
Significant increases in the percentage of
CD19+CD69+ B lymphocytes
were observed in the PP 2–3 dpi and in the MLN 3–6 dpi (Fig. 3;
n = 5). When taken together with the overall increase
in lymphocyte number, the flow cytometry data indicate that, following
a RV infection, there is an overall increase in the total number of
each subset of lymphocytes in the PP and MLN but only a specific and
large increase in the number and percentage of activated B cells.
A small but significant increase in the percentage of CD8+CD69+ T lymphocytes accompanied the increase in the percentage of activated B cells in the MLN at 4 dpi, but there was no increase in the percentage of CD8+CD69+ T lymphocytes at any time point in the PP. No significant increases in the percentage of activated B or T lymphocyte populations was observed at any time point in SP or in the lamina propria from infected mice (data not shown). Similar results showed that the percentage of lymphocytes expressing two other activation markers, CD25 and CD44, also were increased in the CD19+ B cell, but not T cell, population early after primary infection (data not shown). These data indicate that murine RV infection of an immunologically intact mouse results in the activation of a significant percentage of B lymphocytes in the intestine as early as 2 dpi, in the absence of any significant increases in the percentage of CD4+ Th cells. Additionally, a statistically significant increase in the percentage of activated CD8+ T cells was observed at only one time point in the MLN. Taken together, these results suggest that the earliest immunological responses to RV infection is a predominately T cell-independent B cell response in the PP and MLN.
B lymphocytes are activated in RV-infected TCRKO mice
To prove that T cells were not required for the early massive B
cell activation we observed in the PP and MLN early after a primary
infection, PP and MLN from TCRKO mice after 3 dpi were examined
for increases in the percentage of activated B cells after
ECwt infection. As shown in Fig. 4, a significantly higher percentage of B
lymphocytes from the PP and MLN of RV-infected TCRKO mice and wild-type
mice express the activation marker CD69 compared with their respective
control mice. Because TCRKO mice do not contain T cells, the relative
percentage of activated B cells compared with the total B cell
population of the tissue appears larger in TCRKO mice. When adjusted
for the absence of T cells, the percentage of activated B cells
compared with the percentage of total B cells isolated from the tissue
is the same in wild-type mice of the same background compared with
TCRKO mice. Results in the TCRKO mice confirm the lack of involvement
of T cells in the initial B cell immune response to RV infection in the
PP and MLN.
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To confirm RV specificity of the B cell response observed as early
as 2 dpi, we tested fragment cultures from the MLN and PP for
RV-specific Ab. Other groups have shown that, after infection with RV,
a serum IgM response is observed as early as 3 dpi, while serum IgA is
not detectable until 7 dpi (9). However, we and others
have demonstrated that fecal samples from RV-infected mice have no
detectable IgM but, like the serum response, IgA is present by 7 dpi
(Refs. 4 and 9 and data not shown). TCRKO
mice have detectable, albeit lower, levels of fecal IgA detectable by 7
dpi (14). To determine whether the increased percentage of
activated B cells detected in the PP and MLN early after a primary RV
infection in mice might be capable of locally producing either
RV-specific IgM or RV-specific IgA, we used fragment culture
followed by ELISA. PP and MLN fragments from outbred CD-1, C57BL/6J,
and TCRKO mice collected at either 4 or 10 dpi were placed in culture
for 4 days, and media were collected and tested for RV-specific IgM and
IgA Ab. RV-specific IgM, but not IgA, Ab was detected at 4 dpi in PP
and MLN fragment cultures established from
ECwt-infected CD-1 mice but was not detected in
mock-inoculated mice (Fig. 5A). At 10 dpi, both
RV-specific IgM and RV-specific IgA are present in the PP and
MLN fragment cultures (Fig. 5B). Ileal intestinal segments
were used as a control for IgA production. Similar results were
observed at 4 dpi in the C57BL/6J strain (Fig. 5, C and
D). RV-specific IgM was detected in fragment cultures of PP
from ECwt-infected TCRKO mice 4 dpi (Fig. 5C); however, no IgM was detectable in the MLN (Fig. 5D). This can be attributed to our observation that there is
a short delay in onset of Ag shedding in the TCRKO mice compared with
their wild-type C57BL/6J controls (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Further analysis of the lymphocyte populations from the PP and MLN of
ECwt-infected mice revealed a large increase in
the percentage of activated (CD69+,
CD25+, CD44+) B lymphocytes
in the PP and MLN, but not SP, during the first week following RV
infection. There was an absence of concomitant detectable increases in
the percentage of activated CD4+ and
CD8+ T lymphocytes in these tissues during
infection, with the exception of a small but significant increase in
the percentage of activated CD8+ T lymphocytes in
the MLN at one time point (4 dpi) (Fig. 3). Because our analysis is
limited to 1–7 dpi, we do not know when or at what site CD4 or CD8 T
cell responses occur. However, because RV-specific CD8 T cells are
reported by others (15), activation of CD8 T cells must
occur after 7 dpi. Together, the data suggest that there is an initial
early T cell-independent B cell response possibly followed by a later T
cell activation phase, and that the mechanism that likely mediates
clearance of RV infection in the context of a immunocompetent mouse is
a T cell-independent B cell response. It is possible that other
gut-associated immunological sites harbor populations of activated T
cells early in infection. However, this seems unlikely because the PP
and MLN are thought to be the sites of induction of intestinal immune
responses. Preliminary studies of the activation status of lamina
propria lymphocytes (the effector site of the gut immune system) also
failed to reveal any significant differences in activated T or B cell
populations in RV-infected mice compared with uninfected mice (data not
shown), suggesting that early activation of T cells is not occurring in
the lamina propria.
Although flow cytometry is a widely accepted method for quantitating the activation status of lymphocytes, it is possible that the flow cytometry studies that we performed to detect activated lymphocyte populations after a murine RV infection might have missed small, but biologically important, increases in the percentages of activated T cell subsets. Therefore, we tested whether TCRKO mice, which have no T cells present in the PP and MLN, would develop the same increase in the percentage of activated B cells. TCRKO mice clear RV infection and produce RV-specific Ab after infection (14), suggesting that RV has the capability of activating B cells in the absence of T cells. Our detection of significant increases in the percentage of activated B lymphocytes in the MLN and PP from RV-infected TCRKO mice confirms that B cells in the PP and MLN do not require the presence of T cells to become activated in response to RV. However, the results in TCRKO mice do not eliminate the possibility that other immune cells, including dendritic cells or NK cells, are required to initiate B cell activation following a RV infection in the mouse.
We did not detect any significant differences in the percentages of
CD4+, CD8+,
CD19+,
CD4+CD69+, and
CD8+CD69+ present in the PP
and MLN of RV-infected mice. However, the overall number of lymphocytes
is increased in the PP and MLN by hemocytometer counting at many time
points. When the overall number of lymphocytes in each tissue, obtained
by hemocytometer, is multiplied by the percentage of each phenotypic
subset examined in our study, obtained by flow cytometry, the PP and
MLN from RV-infected mice had an increased number of each phenotypic
subset of lymphocytes compared with the respective subset from control
mice. This difference is not attributable to differences in percentages
of each type (Fig. 3) but is due to the overall increases in lymphocyte
numbers obtained by hemocytometer (Fig. 3). This overall increase in
lymphocyte number in the PP and MLN results from one of two mechanisms:
the local proliferation of PP and MLN lymphocytes or a general influx
of naive lymphocytes into the PP and MLN. Currently studies are under
way to test these hypotheses. Previous studies have demonstrated that
trafficking mechanisms using the integrin
47 have been shown to
have an important role in targeting B lymphocytes to the intestine
following RV infection (21, 22). Alternatively, many
chemokines and cytokines are implicated as chemoattractants in B cell
recruitment to specific sites (23) and could be possible
factors that account for the increase in the percentage of activated B
cells in the PP and MLN following RV infection.
We could not directly assess the RV specificity of the activated B
cells because as activated B lymphocytes differentiate into RV-specific
Ab-producing plasma cells they lose expression of both CD69 and the B
cell marker CD19. Therefore, to confirm RV specificity of the B cell
response, we tested fragment cultures for RV-specific Ab. We found that
the B cells present in the PP and MLN at 4 dpi from RV-infected mice
produced RV-specific IgM but not IgA (Fig. 5A). By 10 dpi,
when RV shedding has ceased, RV-specific IgA can be detected (Fig. 5B). We do not observe increases in percentages of activated
T cells, which are classically thought to control Ab isotype class
switching from IgM to IgA, but Macpherson et al. (24) have
recently demonstrated that some Ags can stimulate class switching to
IgA in the intestine in the absence of T cell help. Our data suggest
that RV may fall into this class of Ags. Further investigations into
the immune responses after 7 dpi (at which time Ag is no longer
detectable in fecal samples) will be necessary to conclude that RV does
not induce any T cell activation, but our fragment culture data
describing the local Ab response in two GALTs indicates that production
of IgA in these tissues occurs in the absence of T cell activation. The
RV Ag specificity of the T cell-independent Ab response in CD-1 and
TCRKO mice is not known. It is possible that all RV Ags can stimulate
an Ab response, but Franco and Greenberg (14) hypothesized
that the RV-specific fecal IgA is directed against the VP6
structural component of the virus. To more directly characterize
the Ab produced by RV-specific B cells in the PP and MLN, ELISPOT
studies will be necessary and are under way.
Ab responses to viral proteins are typically thought to be induced through primarily T cell-dependent pathways. However, studies on many viruses in T cell-deficient mouse models clearly indicate that many viruses can act as T cell-independent Ags and induce the production of Abs without T cell help. Polyomavirus infection in T cell-deficient mice resulted in the production of virus-specific IgM and IgG, which was protective against the induction of disease (25). Several recent studies suggest that other viruses, such as vesicular stomatitis virus, lymphocytic choriomeningitis virus, Pinchinde virus, murine CMV, and vaccinia virus, can also induce IgM, followed by the production of isotype-switched IgG and IgA in the absence of T cell help (26). However, the ability of a virus to produce T cell-independent Ab responses in T cell-deficient mice does not necessarily mean that T cell-independent Ab responses are biologically relevant to the immune response to that same virus in a mouse that is immunologically intact. Our studies provide the first direct evidence in vivo that a T cell-independent B cell response likely mediates clearance of a primary RV infection within the context of an intact immunocompetent host.
The data presented in this work strongly suggest that the early Ab response to RV is T cell independent and is the first report of an enteric viral infection causing T cell-independent activation of B lymphocytes in vivo detectable very early after infection (within 48 h). Understanding the mechanisms involved in inducing this immune response will contribute significantly to the development of vaccines against RV and other viruses that infect across mucosal surfaces.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Margaret E. Conner, Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail address: mconner{at}bcm.tmc.edu
3 Abbreviations used in this paper: RV, rotavirus; PP, Peyer’s patch; MLN, mesenteric lymph node; SP, spleen; KO, knockout; GALT, gut-associated lymphoid tissue; SD50, shedding dose 50; ECwt, EC wild type; dpi, day postinoculation/infection.
Received for publication July 9, 2001. Accepted for publication March 21, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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47 independent pathway for CD8+ T cell-mediated intestinal immunity to rotavirus. J. Clin. Invest. 106:1541.[Medline]
47 integrin expression but does not require IgA antibody production. J. Immunol. 166:1894.This article has been cited by other articles:
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)
or 105 SD50 of ECwt in PBS (•).
Fecal samples were collected from individual mice 1–7 dpi and analyzed
for rotaviral Ag shedding. Results are reported as mean OD ± SD
(n = 5).
