ARTICLE |
Correspondence to: Henri P. Salmon, PII, INRA, Centre de Recherches de Tours, 37380 Nouzilly, France. E-mail: salmon@tours.inra.fr
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Summary |
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The mammary gland (MG) develops new vasculature and is colonized by lymphocytes, primarily T-cells, during pregnancy. In contrast, during lactation it is colonized primarily by IgA-containing B-cells (c-IgA cells). To explain this difference, we analyzed the spatiotemporal relationships between lymphocytes that expressed peripheral or mucosal homing receptors (HR) and the location of their vascular counterreceptors using quantitative immunohistochemical techniques. We observed that the density of ß7+/CD3+ T-cells varied with the amount of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1)-stained area. Both increased during pregnancy to peak at delivery, decreased rapidly in early lactation to a steady level in mid- and late lactation, and returned to resting values after weaning. Although 60% of these ß7+/CD3+ T-cells scattered in the epithelium co-expressed Eß7, whereas the remaining 40% in association with blood vessels were
4ß7, these results are consistent with a role of MAdCAM-1 in the localization of
4ß7+ T-cells. In contrast to T-cells, ß7+/c-IgA+ B plasmablasts (~30% of total c-IgA cells) were located at the alveolar confluence, and their numbers increased in mid- and late lactation when MAdCAM-1 density plateaued. However, both T-and B-cells decreased after weaning. These results show an association between MAdCAM-1 expression level and recruitment of T-cells that does not hold for c-IgA B cells. Furthermore, the recruitment and accumulation of
4ß7+ c-IgA cells are reminiscent of locally produced chemoattractants. (J Histochem Cytochem 47:15811592, 1999)
Key Words: homing receptors, vascular addressins, mammary gland, CD3+ cells, c-IgA+ cells, PNAd, MAdCAM-1, immunohistochemistry, image analysis
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Introduction |
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The mammary gland (MG) is an exocrine gland located under the skin which produces secretions contributing to the protection of the suckling against a variety of infectious diseases by the so-called enteromammary immunological link (
The role of these T-cells has not yet been defined. They may be involved in epithelial cell growth during pregnancy, or they may be involved in protection against infections (
The recruitment of activated (blast)/memory lymphocytes into mucosal (i.e., gut) and peripheral (i.e., skin) tissues is believed to occur in the same manner as in lymphoid organs. This process involves the spatial and temporal interactions of homing receptors (HR) with corresponding tissue-specific vascular addressins (4ß7+ cell localization (
4ß7 CD45RB T-cell recruitment (
Given the cutaneous origin of the MG (4ß1 with vascular cell adhesion molecule-1 (VCAM-1) or cutaneous lymphocyte-associated antigen with E-selectin (
To understand the homing programs for T- and B-lymphocytes trafficking into the MG during its development, we followed the localization and kinetics of lymphocyte subsets in relation to their HR and corresponding vascular addressins, using quantitative immunohistochemistry. We found that differences in the extent of trafficking of 4ß7 lymphocytes in the MG are related to the level of MAdCAM-1 expression for T-cells but not for c-IgA cells.
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Materials and Methods |
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Mice
Nulliparous, specific pathogen-free female Balb/c mice obtained from our breeding colony were fed a breeding diet (UAR Factory; Epinay sur Orge, France). They were used at 68 weeks of age at Days 4, 12, or 19 of pregnancy (P4, P12, and P19, respectively; Day 1 of pregnancy determined by vaginal plaque detection), at Days 1, 4, 12, or 18 of lactation (L1, L4, L12, and L18 respectively), and at Day 4 after weaning (25 days postpartum). For the lactating mice, the newborns were left with the mother and allowed to suckle until sacrifice and tissue recovery.
Tissue Samples and Sections
Mice were anesthetized and injected IV with Lukonyl blue 7080 (BASF) to visualize the blood vessels, then sacrificed by cervical dislocation 10 min later (
The first abdominoinguinal MG (fourth pair) with their attached lymph node (superficial inguinal LN), as well as small intestine with Peyer's patches and mesenteric lymph node, was removed and snap-frozen in liquid nitrogen. Serial sections of MG were cut at 7 µm. These cryostat sections were dried, fixed in cold acetone for 20 min (
Antibodies
Except where indicated, all Abs used were purified rat monoclonal antibody (MAbs). Mature T-cells were detected with anti-mouse CD3 (KT3, IgG2a; Serotec; Pantin, France), c-IgA cells using R5-140, IgG1 (Pharmingen; Le Pont de Claix, France) or goat anti-mouse IgA-conjugated alkaline phosphatase (Caltag; Le PerrayYvelines, France), cells expressing ß7 integrin using hybridoma culture supernatant (SN) M293, IgG2a (generously given by P.J. Kilshaw), cells expressing 4 integrin using PS/2, IgG2a (Serotec), cells expressing
E (
M290) integrin using M290, IgG2a (Pharmingen), cells expressing L-selectin using MEL-14, IgG2a (Cedarlane; Le PerrayYvelines, France), pro-B through the mature B-cells using anti-mouse CD19 (1D3, IgG2a; Pharmingen), pro-B through mature and activated T- and B-cells using anti-mouse CD45R/B220 (RA3-6B2, IgG2a; Caltag).
The presence of MAdCAM-1 was detected using mouse endothelial cell antigen 367 (MECA 367), IgG2a (Pharmingen), panendothelial cell antigen using MECA 32, IgG2a (Pharmingen), PNAd using hybridoma culture SN MECA 79, IgM (generously supplied by C. Kieda), VCAM-1 using M/K-2, IgG1 (Caltag), and E-selectin using 10E9.6, IgG2a (Pharmingen).
Anti-mouse MHC I (ER-HR 52, IgG2a, Bale Biochimie Bachem; Voisins-le-Bretonneux, France) was used as a positive control. Purified IgG2a, (R35-95, isotype standard; Pharmingen), purified IgM (IR202, myeloma; UCR Bruxelles), and a goat anti-rat IgG2a-conjugated alkaline phosphatase (Bethyl; Montpuçon, France) were used as isotype-matched negative control Abs.
Immunohistochemical Staining
Frozen sections were thawed, encircled using a pap pen (Immunotech; Marseille, France), and were first covered with PBS1% BSA for 30 min to remove the nonspecific background and then with avidin in excess for 10 min to block endogenous biotin activity (avidinbiotin kit; Dako, Glostrup, Denmark). After washing in PBS, sections were incubated in 0.03% hydrogen peroxide for 5 min at room temperature (RT) to remove nonspecific peroxidase activity (Peroxidase Blocking Reagent; Dako) and then stained using a three-stage biotinstreptavidinperoxidase technique.
After incubation with the primary MAb at optimal concentrations for 1 hr at RT, sections were washed three times and incubated for 1 hr with secondary antibody, biotin-conjugated rabbit anti-rat IgG/IgM (Dako). After further washes, the sections were incubated for 45 min with preformed peroxidase-labeled streptavidin complex (Dako). The horseradish peroxidase bound to the sections was revealed with amino-9-ethylcarbazol substratechromogen (Dako) and sections were counterstained with hematoxylin solution (Gill No.1; Sigma, St Louis, MO) and mounted in aqueous medium (Faramount; Dako). In the case of c-IgA/ß7 double immuno-staining, c-IgA was first detected using goat anti-IgA-conjugated alkaline phosphatase (0.5 µg/ml). The phosphatase reaction was developed with naphthol AMSX-P (Sigma) and Fast Blue BB Salt (Sigma) in the presence of levamisole (Sigma) as previously described (
Quantitative Evaluation of Data
Counts were made on stained cells present in adjacent fields in each section using a x25 objective and a x10 eyepiece. The cells counted were those that showed clearly defined membrane and/or cytoplasmic staining (c-IgA). Distinction between plasmablasts (or B-blasts) and plasma cells was made using criteria of size, nucleus location, and nucleus/cytoplasm ratio. The c-IgA cells with greater quantities of cytoplasm than nucleus size (cytoplasm/nucleus ratio >1) were categorized as mature plasma cells and large cells with cytoplasm/nucleus ratio 1 as plasmablasts.
All lymphocytes and plasma cells were counted at x250 magnification (field diameter 0.576 mm) in counts of at least 100 cells in 30140 microscopic fields, representing the whole area of three randomly selected sections (
Analysis of the stained area per tissue area unit covered by stained endothelial cells was quantified by a computer-assisted image analyzer (VISILOG; NOESIS, Velizy, France). A stable light source and a fixed threshold to elimate background (assessed by isotypic control stained slides) and light intensity were used throughout the measurements. To compare the extent of MECA 32 staining in relation to that of MECA 367, two adjacent sections were mounted per slide and three slides per MG were selected at random. Evaluation of the amount of stained area was made on adjacent fields in each section as above. Briefly, the red-stained area of light microscopic images was captured through a green filter and results in pixels were converted into µm2. The stained areas were determined in 30140 microscopic fields (covering the entire histological section) from three randomly selected sections at x200 magnification (field surface 0.13 mm2). Because the SEM of stained areas for each section was less than 10%, the stained areas were calculated from two to three mice as the means ± SEM of MECA 367- or MECA 32-stained area (µm2) per mm2 of MG tissue, to improve the accuracy of the results.
All the results were obtained from microscopists blind to groups and to Abs during analysis.
Photomicrographs
Photomicrographs of representative fields were taken on Kodak Ektrachrome tungsten film (64 T).
Statistical Analysis
Assumptions for the Gaussian distribution of values were made by the KolmogorovSmirnov test. In cases of nonGaussian distribution, non-parametric tests were used instead. Paired Student's t-test was used to compare the number of labeled cells within the same time period and results were considered significantly different at p<0.05. The mean number of positive cells per area unit between time periods was compared by variance analysis and statistical differences were checked by the Bonferroni test. The 2 test was used to compare the proportion of ß7+/IgA+-labeled cells between the lactation stages. Variation in the number of labeled cells or in stained area at a given developmental stage was analyzed by linear regression using GraphPad Prism 2.01 software (GraphPad Software; San Diego, CA). The rates of variation between HR and vascular addressins were compared by F-test on the slopes of regression lines. Correlation between two HR was performed by Spearman test, whatever the stage.
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Results |
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General Architecture of the Developing MG
The MG of virgin mice is mainly composed of fatty tissue and a highly organized system of ducts with terminal end buds that are the major sites of growth and lateral buds (Figure 1A). Vascularization is mainly located in the fatty tissue, with some capillary vessels around the epithelial cells.
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Development and differentiation of the gland take place during pregnancy and lactation. The lateral buds differentiate and subdivide progressively during pregnancy, giving rise to small alveolar buds (P4, Figure 1B). The mammary alveoli start to form larger alveolar lobules (P12, Figure 1C). A rapid increase in the number and size of alveoli and a decrease in fatty tissue occur during the second half of pregnancy, resulting in the development of fully differentiated secretory lobules (P19, Figure 1D). The lobule, the secretory unit of the MG, consists of a cluster of alveoli around the single small duct. The alveolar wall is a single layer of epithelial cells (Figure 1D and Figure 1E). The morphological changes are minimal during lactation, and the glandular epithelium alveoli continue to predominate over fat cells (Figure 1E). The MG reduces after weaning owing to loss of alveolar tissue, and the fatty tissue content correspondingly increases (Figure 1F).
Natural Recruitment of Lymphocytes in Mouse MG from the Virgin Stage and During Pregnancy, Lactation, and Involution
Kinetics and Localization of T- and B-Lymphocytes.
Immunohistochemical analysis of MG for various cell surface antigens showed inverse patterns of T- and c-IgA+ B-lymphocytes during pregnancy and lactation, respectively (Figure 2). The number of CD3+ T-cells per microscopic field increased linearly during pregnancy, from six in virgin mice to a peak value of 17 CD3+ cells at Day 19 of pregnancy (P19) (p<0.01). It then decreased rapidly during lactation to a value not significantly different (p>0.05) from that of virgin mice at Day 12 of lactation (L12), and increased again significantly (p<0.05) in involuting MG.
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The numbers of CD19+ and B220+ B-cells did not vary during pregnancy and lactation, amounting to 0.2 and 2.5 cells per field, respectively (Figure 2). As expected, in control tissues there were high numbers of these cells in the B-follicles of Peyer's patches, mesenteric and MG lymph nodes, but a low number in the gut lamina propria (not shown).
The number of c-IgA+ cells increased slightly in late pregnancy and early lactation, i.e., from 0.6 at P12 to 4 at L4 (slope significant, p<0.01), then increased abruptly during lactation to a peak value of 29 cells at L18 (p<0.001). In mid (L12) and late (L18) lactation, c-IgA+ cells were approximately three and six times more abundant than T-cells, respectively. Four days after removal of the suckling pups, the number of c-IgA+ cells decreased abruptly, from 29 to 12 (p<0.001).
Studies of tissue localization (Figure 3A) showed that the majority of CD3+ T-cells were scattered in the epithelium of ducts (virgin), buds (pregnancy), and alveoli (lactation), with some in the connective tissue between the epithelium and the walls of underlying blood vessels but never in fatty connective tissue. The c-IgA+ plasmablasts were observed crossing blood vessel walls at the confluence of several alveoli and were never found in the epithelial layer, in contrast to CD3+ cells. The plasma cells were located in perialveolar connective tissue (Figure 3D) near the confluence of several alveoli. They were dispersed in early lactation, then arranged in single file along capillaries in mid- and late lactation, but never in clusters at any stage. Using morphological criteria (Figure 3E), the c-IgA+ B-cells comprised ~30% plasmablasts and ~70% mature plasma cells.
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Even in perialveolar connective tissue, the CD3+ and c-IgA+ cells were not found in close association when observed by c-IgA/CD3 double immunostaining (not shown).
It therefore appears that the MG tissue is mainly colonized at specific locations by T-cells during pregnancy and by mature B-cells, B220-/CD19-/c-IgA+ plasmablasts/plasma cells, during mid- and late lactation.
Expression of Homing Receptors of T- and B-Lymphocytes.
Colonization of MG may result from differential expression of peripheral or mucosal HR on T- and B-lymphocytes. Among the peripheral HRs, no L-selectin+ lymphocytes were detected in the MG at any stage of its development, whereas they were present in secondary lymphoid tissues (not shown). In contrast, the 4 and ß7 integrin subunits of the mucosal HR were expressed on a variable fraction of MG lymphocytes, depending on the developmental stage of the MG (Figure 4), a feature that was confirmed by morphological analysis.
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During pregnancy, the number of ß7+ cells and CD3+ cells per field increased at the same rate (slopes not significantly different; p>0.05) and had a similar tissue localization (Figure 3A and Figure 3B), suggesting a co-expression of ß7 and CD3. However, the number of ß7+ cells was lower than that of CD3+ cells. Because very few B-cells were present during pregnancy (Figure 2), the constant difference (CD3+ - ß7+) of ~5 cells per field corresponds to CD3+/ß7- cells, which probably represent the same cells as those already present in MG before pregnancy. During lactation, the number of ß7+ cells remained fairly constant (slope not significant), in contrast to the number of CD3+ and c-IgA+ cells. It could be seen (Figure 4) that during mid- (L12) and late (L18) lactation, the numbers of ß7+ cells were intermediate between those of CD3+ and c-IgA+ cells, i.e., ~13 cells per field. Because ß7+ cells outnumbered CD3+ cells during this period, ß7 integrin could be present on cells other than CD3+ cells, such as c-IgA+ cells. To confirm this hypothesis, we performed double ß7 and IgA immunostaining. We observed proportions of ß7+/c-IgA+ labeled cells at L4, L12, and L18 which were not significantly different (2; p>0.05), averaging 30 ± 3% (n = 3) (Table 1). Moreover, morphological examination showed that most of the ß7+/c-IgA+-labeled cells (Figure 3G compared to 3F) appeared to be plasmablasts. We can therefore conclude that the ß7 integrin is co-distributed on CD3+ and on c-IgA+ cell plasmablasts during lactation.
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In contrast with the situation seen during lactation, Figure 4 shows that the number of ß7+ cells significantly decreased (p<0.05) in involuting MG compared to the number of CD3+ cells.
We next researched which -chain type (
4 or
E) was associated with ß7 in the HR
ß heterodimer. During pregnancy, the numbers of
4+ cells (Figure 4) were highly correlated with numbers of ß7+ cells (r = 0.83; p<0.01). However, the rate of increase in
4+ cells during pregnancy was lower than that of ß7+ cells (respective slopes 0.43 ± 0.06 and 0.73 ± 0.05; p<0.01). The differences between ß7 and
4 values corresponded to the number of
Eß7+ lymphocytes. Most of the
Eß7+ cells (85%) were situated in the epithelium (Figure 3C) and represented approximately 60% of the total number of ß7+/CD3+ T-cells. During lactation, the number of recruited
4+ cells increased significantly (slope significant; p<0.05) to peak in late lactation (L18), equalling the number of ß7+ cells. These
4+ cells were located in the subepithelial tissue near blood vessels at the confluence of several alveoli, as were c-IgA+ plasmablasts. It can therefore be concluded that ß7 is associated with
E on intraepithelial CD3+ cells and with
4 on both subepithelial CD3+ cells and c-IgA+ lymphoblasts.
Vascular Addressins in MG and Lymphoid Organs from the Virgin Stage and During Pregnancy, Lactation, and Involution
Because the transendothelial migration of cells from the blood to the tissues is critically influenced by the presence of vascular addressins, we examined MG sections to look for the presence of peripheral lymph node addressin (PNAd), MAdCAM-1, and VCAM-1, the counterreceptors of L-selectin, 4ß7, and
4ß1, respectively.
Morphometric analysis of areas stained with mouse endothelial cell antigen 32 (MECA 32), an MAb that recognizes an antigen expressed by most endothelial cells (
Approximately 4% of the endothelium area stained by MECA 32 was also stained by MECA 367, an anti-MAdCAM-1 MAb, in virgin mice (Figure 5). This basal level of MAdCAM-1 expression was occasionally present (one microscopic field of four) on endothelial cells of venules adjacent to epithelial ducts but was never found on fat pad blood vessels (Figure 6A).
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During pregnancy there was a linear increase in the MAdCAM-1-stained area (slope = 2612 ± 214) with a maximum at P19, reaching the same value as MECA 32 (p>0.05) (Figure 5). This increase in stained area included not only a higher proportion of MAdCAM-1+ endothelial cells in close contact with epithelial cells, from ~50% at P4 (Figure 6B) to almost every cell at P12, but also an increase in extent of endothelial cell staining, both luminal and cytoplasmic (Figure 6C). A decrease in stained area (Figure 6D) could be expected at P19 due to the stretching of the blood capillaries. However, it was compensated for by the decrease of fatty tissue whose vessels never expressed MAdCAM-1. Therefore, at the end of pregnancy all blood vessels were associated with alveoli, so that almost all endothelial cells in the whole MG section expressed MAdCAM-1. In conclusion, the increase in MAdCAM-1 staining during pregnancy resulted mainly from an increased proportion of positive cells on increased vascularization.
In early lactation (Figure 5), there was a decrease of 80% in the MAdCAM-1-stained area, which was sharper than that of MECA 32 (8%) (respective slopes -2547 ± 197 and -268 ± 186; p<0.0001). In mid- (L12) and late lactation (L18), we observed a steady level (p>0.05) of 20% of maximal MAdCAM-1 expression that was still six times higher than the virgin value (p<0.01). Concomitantly with MECA 32, MAdCAM-1 expression further decreased in involuting MG to return to the virgin basal level (p>0.05). Figure 6E6H show that the decrease in the extent of MAdCAM-1 staining during lactation is a result of a lower number of positive vessels, the loss of staining occurring from capillaries around the alveoli (Figure 6E and Figure 6F) to the medium-sized blood vessels (Figure 6G) to persist slightly on some capillaries near the remnants of some alveoli (one microscopic field of five) for 4 days after weaning (Figure 6H).
Interestingly, blood vessels of MG as well as those of the gut lamina propria were consistently free of MECA 79 staining, an anti-PNAd MAb (not shown). Furthermore, in inguinal lymph node (LN) contained in MG, no MAdCAM-1+ endothelial cells were observed in spite of the increase in PNAd concomitant with the increase in LN high endothelial venule (HEV) size. It has been recognized that 4ß7 binds to VCAM-1 (
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In conclusion, among the vascular adhesion molecules studied, only the mucosal addressin MAdCAM-1 was present in MG, with maximal expression in late pregnancy, followed by a rapid decrease during early lactation to a steady level in mid- and late lactation, and the involution was characterized by a decrease both in the length of MAdCAM-1-positive blood vessels and in the number of blood vessels.
Comparative Evolution of the Lymphocytes Bearing HR and MAdCAM-1 Expression in MG during Developmental Stages
Extravasation of lymphocytes from the blood into tissues occurs as a process involving the spatial and temporal interactions of HR with vascular addressins. We compared the kinetics of the amount of MAdCAM-1-stained area with those of the numbers of ß7+/CD3+ and ß7+/c-IgA+ cells, whose values were adjusted to the same unit area (Figure 5).
The rate of increase in numbers of ß7+/CD3+ cells during pregnancy was similar to that of MAdCAM-1 expression (respective slopes of 2082 ± 127 and 2653 ± 174). This relationship was again observed when both values decreased during lactation (common slope of -1726). The synchronization during both stages indicated that ß7+/CD3+ cells did not accumulate in the MG, and we could therefore calculate a ratio between ß7+/CD3+ cells and MAdCAM-1 area corresponding to a fairly constant ratio of one ß7+/CD3+ cell per 1000 µm2 of MAdCAM-1 staining.
Conversely, the number of ß7+/c-IgA+ cells increased (slope = 1248 ± 129) during lactation, in contrast to MAdCAM-1 expression, which decreased (slope = -1893 ± 244) during the same period. We thus observed a ratio of ß7+/c-IgA+ cells per 1000 µm2 of MAdCAM-1 staining, which increased from 0.2 at L4 to a maximum of 3 at L18. After weaning, both ß7+/c-IgA+ cells and MAdCAM-1 density decreased at the same rate (common slope of -1942).
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Discussion |
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Our results extend the inverse pattern of CD3+ T- and c-IgA+ B-lymphocytes seen in the rat (
We extend to pregnancy the report on the absence of MECA 79 staining on lactating MG blood vessels (
Morphological criteria, the absence of L-selectin, and the low proportion of CD19 and B220 markers on B-cells indicate that the MG recruits plasmablasts/plasma cells (4ß7, as are plasma cells in the human gut (
Our findings of similar spatial and temporal relationships between the amount of MAdCAM-1-stained area (i.e., the number of stained endothelial cells per unit area) and the number of ß7+/CD3+ cells are consistent with the proposal that MAdCAM-1 is responsible for T-cell recruitment in the MG. This is substantiated by the decrease in number of 4ß7+ cells that we observed during involution. Moreover, the absence of VCAM-1 expression on capillaries of the MG is in favor of a unique interaction of
4ß7+ cells with MAdCAM-1. ß1 integrin staining was not performed, and therefore modulation of association of
4 with ß1 cannot be determined, but if this were the case, it is unlikely that
4ß1 would be involved in lymphocyte extravasation into MG because VCAM-1 (counterreceptor of
4ß1) is not expressed on MG capillaries at any developmental stage. Our observation of a concomitant decrease in T-cells and quantity of MAdCAM-1 in the same MG area during lactation supports the hypothesis of dynamic equilibrium of cells coming and going rather than an accumulation during both pregnancy and lactation. We therefore calculated that ß7+/CD3+ T-cells are extracted from the blood at a constant low ratio of one ß7+/CD3+ cell per 1000 µm2 of MAdCAM-1 area, in accordance with the results of lymphocyte traffic per unit of HEV area (
Eß7+ lymphocytes into the lumen of alveoli, combined with the fact that some
4ß7+ cells (activated) may recirculate in the blood (
There are several hypotheses to explain the increased recruitment of c-IgA+ cells specifically during lactation. At first sight, the inverse pattern of T- and c-IgA+ blast kinetics suggests competition to homing in the MG. However this explanation is unlikely, first because our results showed that T- and c-IgA+-cells had different locations in the MG, and second because other authors found that no competition could be demonstrated between [125I]-iododeoxyuridine-labeled T- and B-cell homing in the gut (
In conclusion, our results suggest that a common mechanism is set in place to recruit both T- and c-IgA B-cells via the MAdCAM-1/4ß7 interaction, but locally produced B-cell chemoattractants may cause the differences in recruitment.
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Acknowledgments |
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Supported by grants from the Institut National de la Recherche Agronomique and Conseil Régional du Centre.
We thank Drs D. Marc and P. Velge for review of the manuscript, Dr C. Taragnat for technical assistance in quantitative image analysis, Dr C. Kieda (CNRS; Orleans, France), and Dr P. J. Kilshaw (AFRC Babraham Institute; Cambridge, UK) for generously donating MAbs, and H. Leroux for animal care.
Received for publication March 10, 1999; accepted July 20, 1999.
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