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The Journal of Immunology, 2002, 169: 5649-5659.
Copyright © 2002 by The American Association of Immunologists

Absence of L-Selectin Delays Mucosal B Cell Responses in Nonintestinal Effector Tissues1

Keri L. Csencsits and David W. Pascual2

Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies suggest that lymphocyte trafficking to head and neck lymph nodes, also referred to as cranial-, oral-, nasal-associated lymphoid tissue (CONALT), is L-selectin (L-Sel) dependent, despite coexpression of {alpha}4{beta}7, resulting in their marked reduction in L-Sel-deficient (L-Sel-/-) mice. Consequently, early phase (16 days) Ab responses to cholera toxin (CT) are diminished. The following studies reveal that lack of mucosal effector responses is not caused by loss of inductive immune responses in the L-Sel-/- CONALT. Indeed, there was an increased accumulation of total IgA, but not Ag-specific IgA Ab-forming cells (AFC) in L-Sel-/- CONALT. This increased accumulation was not evident in L-Sel+/+ CONALT. Identification of lymphocyte-homing receptors on L-Sel-/- and L-Sel+/+ CONALT lymphocytes revealed no significant differences in expression of {alpha}4{beta}7, which might contribute to lymphocyte homing in the absence of L-Sel. Studies of CONALT responses during the late phase (6 wk post-intranasal immunization) revealed the number of lymphocytes recovered from L-Sel-/- CONALT was less than L-Sel+/+ CONALT; however, L-Sel-/- CT-specific and total AFC did not vary from 16-day responses, suggesting a defect in CT-specific B cell export. No significant differences in {alpha}4{beta}7 expression between L-Sel-/- and L-Sel+/+ CONALT were noted. Yet, these increases in CONALT AFC correlated with restoration of immunity in L-Sel-/- nasal passages and reproductive tracts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Immunization of mucosal tissues imparts immunity in regional, as well as distal mucosal tissues. Given this distinct advantage, recent vaccine strategies have focused on intranasal (i.n.)3 immunizations as an effective means for stimulating reproductive tract (RT) immunity (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). This indirect method of vaccination, in addition to ease of administration, circumvents concerns regarding epithelial cell turnover and hormonal influences within the RT (13). Furthermore, nasal immunization is more efficient than oral immunization at stimulating effector immunity in the RT (1, 14, 15, 16); however, the factors that mediate selective lymphocyte trafficking from the nasal inductive sites to distal mucosal effector sites have yet to be completely elucidated.

Although widely considered most important for homing to peripheral lymph nodes (PLN) (17, 18, 19), L-selectin (L-Sel)-peripheral node addressin (PNAd) interactions may be required for the effective induction of mucosal immunity following i.n. immunization (20). Our work has shown that all high endothelial venules (HEV) of the nasal-associated lymphoid tissue (NALT), as well as the draining head and neck lymph nodes (LN) referred to as cranial-, oral-, and nasal-associated lymphoid tissue (CONALT), express PNAd, and most naive lymphocyte binding to these tissues is mediated primarily through L-Sel PNAd interactions (21, 22). Moreover, PNAd has been shown to be expressed by the HEV of human tonsil (23), and lymphocyte homing to the nasal passages (NP) and lungs in the human and sheep is mediated by L-Sel-PNAd interactions rather than by mucosal addressin cell adhesion molecule-1 (MAdCAM-1)-{alpha}4{beta}7 interactions (24, 25, 26). Thus, lymphocyte homing to the nasal inductive sites differs greatly from homing in the intestinal inductive site, the Peyer’s patch (PP), in which lymphocyte binding is mediated primarily via MAdCAM-1 interactions with {alpha}4{beta}7 integrin and L-Sel (27, 28, 29, 30, 31, 32).

More important, however, is the role of L-Sel in the trafficking of lymphocytes to the effector mucosal sites. Studies of humans have revealed that L-Sel, as well as {alpha}4{beta}7, are expressed by B lymphocytes stimulated subsequent to oral immunization (33, 34, 35), suggesting a role for L-Sel in the production of immunity in the intestinal effector site. In mice, it is well established that L-Sel is important for the development of PLN immunity. Consequently, L-Sel-deficient (L-Sel-/-) (36, 37) mice display reduced size and cellularity of the PLN, while the cellularity of inductive PP is unaffected (36). Recent work in our laboratory has shown that loss of L-Sel severely compromises the number of effector Ab-forming cells (AFC) in NP and RT, but not in the small intestinal lamina propria (iLP), at 16 days post-i.n. immunization with cholera toxin (CT) (20).

Therefore, L-Sel clearly plays an important role in the recruitment of B lymphocytes to the nonintestinal mucosal effector sites following i.n. immunization. However, the mechanism of the loss of immunity remains unclear. Is the loss of AFC in the nonintestinal effector sites due to a loss in inductive site immunity or a failure of activated B cells to traffic to the mucosal effector sites? The following study addresses these questions and determines that loss of nonintestinal effector immune responses at 16 days postimmunization is not a failure of the induction of immunity, but rather that a loss of L-Sel results in a delay in immune response at mucosal effector sites. Furthermore, we have identified an unexpected ramification of the loss of L-Sel in an increased number of AFC found in L-Sel-/- CONALT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Specific pathogen-free C57BL/6N female mice were purchased from the National Cancer Institute at 5–6 wk of age and maintained in the Animal Resources Center at Montana State University (Bozeman, MT). Breeding pairs of L-Sel-/- mice on a B6 background (36, 37) were purchased from The Jackson Laboratory (Bar Harbor, ME), and colonies were established and maintained in the Animal Resources Center at Montana State University. All mice were kept under pathogen-free conditions in horizontal laminar flow cabinets and were fed sterile food and water ad libitum. The mice were free of bacterial and viral pathogens, as determined by Ab screening and by histopathologic analysis of major organs and tissues. The mice used in these experiments were between 5 and 8 wk of age. Mice were immunized without anesthesia via i.n. drip on day 0 with 5 µg CT (List Biological Laboratories, Campbell, CA) in 10 µl sterile PBS, and boosted on days 7 and 14 postprimary immunization with 2.5 µg CT.

Collection of serum, fecal, and vaginal samples

Blood was collected via saphenous vein. Fresh fecal pellets were collected from individual mice and solubilized in 50 µg/ml of soybean trypsin inhibitor (Sigma-Aldrich, St. Louis, MO) in sterile PBS (10x v/w) by continual vortexing for 30 min at 4°C. After microcentrifugation, supernatants were frozen until assayed. Vaginal secretions were collected by gently pipetting 75 µl of sterile PBS in and out of the vaginal vault of the individual mice, and samples were subsequently subjected to microcentrifugation for 30 min at 4°C. Supernatants were collected and frozen until assayed.

Anti-CT-B ELISA

Falcon Microtest III flexible assay microtiter plates (BD Biosciences, Oxnard, CA) were coated with 50 µl/well of 5 µg/ml B subunit of CT (CT-B; List Biological Laboratories) in sterile PBS and incubated overnight at room temperature. The plates were blocked with 200 µl/well PBS + 1.0% BSA for 1 h at 37°C. Plates were washed three times with PBS and twice with PBS-Tween 20; mucosal samples and serum dilutions in ELISA buffer (PBS + 0.5% BSA + 0.05% Tween 20) were added at 50 µl/well; and plates were incubated at 4°C overnight. Plates were washed, and 50 µl/well of detecting HRP conjugates of goat anti-mouse IgG ({gamma}-chain specific) or goat anti-mouse IgA ({alpha}-chain specific) (1.0 µg/ml; Southern Biotechnology Associates, Birmingham, AL) were added and the plates were incubated at 37°C for 1.5 h. HRP was visualized by the addition of 50 µl/well of 2,2'-azino-bis(3-ethylbenzthiazoline 6-sulfonic acid) diammonium substrate (Moss, Pasadena, CA). OD was determined by reading the plates at 415 nm, using a microtiter plate reader (model EL312; Biotek Instruments, Winooski, VT), and endpoint titers were expressed as the reciprocal of the last sample dilution, giving an absorbance >0.1 over the value of negative control wells (in the absence of biological fluid) after a 1-h incubation. Similar dilutions of mucosal samples and serum from nonimmune mice showed no Ab titer to CT-B.

Tissue isolation and collection

Parotid gland LN (PRLN), submaxillary gland LN (SMLN) or superficial cervical LN (CLN), and CLN (deep CLN) were isolated from C57BL/6N or L-Sel-/- mice. Each set of lymphoid tissue, pooled from five mice, was washed in RPMI 1640 medium. NALT tissues were collected by removing the soft palates, as previously described (21, 38, 39). For cell isolation, the soft palates were placed in a 200 U/ml collagenase type IV solution (Sigma-Aldrich) (40) in RPMI 1640 medium containing 0.08 U/ml DNase (Promega, Madison, WI) in a scintillation vial with a 2-cm magnetic stir bar. The palate was vigorously agitated on a magnetic stir plate for 45 min at 37°C; the resulting cell supernatant was removed and filtered through Nitex (Fairview Fabrics, Hercules, CA); and cells were then washed and resuspended in a complete medium (CM; RPMI 1640 + 10% FBS (HyClone, Logan, UT) + 10 mM HEPES buffer + 10 mM nonessential amino acids + 10 mM sodium pyruvate + 10 U/ml penicillin/streptomycin) or FACS buffer (Dulbecco’s PBS + 2% FBS).

For flow cytometry and ELISPOT analysis, CLN, SMLN, and PRLN were removed and subjected to Dounce homogenization. The resulting cell suspensions were filtered through Nitex fabric, washed with RPMI 1640 medium, and centrifuged at 1500 rpm for 5 min. Cell pellets were resuspended in a FACS buffer or CM.

Isolation of effector site lymphocytes

NP were removed from the head by scraping the turbinates from the nasal cavity in a modification of previous protocol (20, 41). Nasal tissue was digested with 200 U/ml collagenase type IV solution containing 0.08 U/ml DNase in a glass 50-ml flask containing a magnetic stir bar. Following rapid agitation during digestion at 37°C for 30 min, released NP lymphocytes were removed, and fresh collagenase solution was added back to the flask. This procedure was repeated until digestion of the tissue was complete. Isolated cells were washed in CM and resuspended in a 40% Percoll solution (Pharmacia, Uppsala, Sweden), and then they were layered over a 60% Percoll solution and subjected to a gradient centrifugation. Lymphocytes were removed from the interface layer, washed, and resuspended in CM.

For iLP lymphocyte isolation, a modification of previous protocol was performed (20, 42). Intestines were extracted from the mouse, and the PP were carefully removed. Fecal material and mucus were flushed from the intestine using RPMI 1640 medium pushed through a 22-gavage needle. Intestines were then slid onto the needle and flayed open, minced into ~1-mm pieces, and shaken vigorously in CM to remove remaining mucus and fecal material, and the waste was filtered through a mesh screen. Intestinal tissues were then placed in the RMPI 1640 medium containing 5% FBS (HyClone) and 2 mM DTT (Sigma-Aldrich) in a 50-ml Teflon flask containing a magnetic stir bar, and gently agitated on a stir plate at room temperature for 5–10 min. This process resulted in the removal of the intestinal epithelial cell fraction. Supernatant was discarded, and DTT was rinsed from the intestinal pieces with the RPMI 1640 medium. Intestinal tissues were returned to the Teflon flask, then 50 U/ml collagenase type IV solution containing 0.08 U/ml DNase, as previously described, was added, and the suspension was agitated at 37°C. After 10 min, the supernatant, containing iLP cells, was removed and washed, and fresh collagenase was added to the remaining intestine. The process was repeated two more times, and lymphocytes were isolated by Percoll gradient centrifugation, as described above.

In a modification of previous protocol (20, 43), RT, which includes vagina, cervix, and uteri, were removed from mice, and the mucus and epithelium were flushed, using RPMI 1640 medium. RT were then flayed open on a blunted 23-gauge needle, and minced into 1- to 2-mm pieces. These pieces were added to a 200 U/ml collagenase type IV solution in a glass 50-ml flask containing a magnetic stir bar, and cells were released from the RT tissue by vigorous agitation on a magnetic stir plate at 37°C for 1 h. Supernatant resulting from this process was removed, cells were washed in CM, and lymphocytes were isolated via Percoll gradient centrifugation, as described above.

Tissues from five L-Sel.-/- or L-Sel+/+ L mice were grouped in each experiment. One to three million viable lymphocytes/tissue/mouse from NP and iLP and ~50,000 viable lymphocytes/mouse from RT were recovered.

CT-B-specific and total Ab ELISPOT

Mixed cellulose ester membrane-bottom microtiter plates (MultiScreen-HA; Millipore, Bedford, MA) were coated with 5 µg/ml CT-B (List Biological Laboratories) in sterile PBS overnight at room temperature. For total IgA or IgG AFC, wells were coated with 5 µg/ml of goat anti-mouse IgA or IgG (H chain-specific) Abs (Southern Biotechnology Associates) in sterile PBS. The plates were blocked at 37°C for 2 h with CM. A total of 100 µl of cells from each tissue at varying concentrations (2 x 106 to 1.25 x 105 lymphocytes/ml) were added to the wells, and the plates were incubated at 37°C overnight. Cells were removed, and the plates were washed three times with PBS + 0.1% Tween 20 and twice with PBS. For detection of a mouse Ab, 100 µl of 1 µg/ml goat anti-mouse IgG and IgA-HRP conjugates (Southern Biotechnology Associates) were added to the wells, and the plates were incubated overnight at 4°C. After washing, as described above, the wells were developed with 100 µl of 3-amino-9-ethylcarbazole (Moss), and the reaction was allowed to continue until spots developed (~30 min). The reaction was stopped with H2O, the plates were allowed to dry overnight, and AFC were enumerated, using a Stereozoom 5 dissecting microscope (Leica, Buffalo, NY).

Comparison of L-Sel, {alpha}4{beta}7, and {alpha}E{beta}7 expression on lymphocytes

Lymphocytes were stained with FITC-M290 anti-CD103 ({alpha}E integrin; M290; BD PharMingen, San Diego, CA), PE-DATK 32 rat anti-mouse {alpha}4{beta}7 (BD PharMingen), CyChrome rat anti-mouse B220 (RA3-6B2; BD PharMingen), and APC-MEL 14 rat anti-mouse L-Sel (BD PharMingen) for 30 min. FL1, FL2, FL3, and FL4 parameters were set with CaliBrite beads (BD PharMingen) and FACsComp software (CellQuest), and compensations were set. Four-color analysis was performed, using a FACSCalibur (BD Biosciences). Ten thousand events/sample were collected.

Statistical analysis

Results were analyzed using a paired Student’s t test, and p values <=0.05 were considered statistically different. Significant p values were indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Loss of effector B cell responses in i.n. immunized L-Sel-/- mice is not due to loss of NALT and PP Ab production

Our previous work (20) showed that NP and RT IgA and IgG AFC were severely compromised in L-Sel-/- mice, following i.n. CT immunization. To further elucidate the mechanism for this loss of Ab production, we investigated the immune responses by the mucosal inductive tissues in CT-immunized L-Sel-/- and L-Sel+/+ mice. Sixteen days postprimary CT immunization, there were no differences in NALT and PP IgA and IgG responses between L-Sel+/+ and L-Sel-/- mice (Fig. 1Go, A and B). Likewise, there were no significant differences in the presence of total IgG and IgA AFC by L-Sel-/- or L-Sel+/+ NALT and PP.



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  		FIGURE 1. Inductive immune responses are found in L-Sel-/- NALT, PP, and CONALT 16 days postprimary i.n CT immunization. CT-B-specific and total L-Sel+/+ and L-Sel-/- NALT IgA (A) and IgG (B) and CONALT IgA (C) and IgG (D)AFC per 106 lymphocytes were determined by B cell ELISPOT. Results are the mean of four experiments ± SEM. *, p < 0.02; **, p < 0.001; ***, p < 0.01.

 
Total IgA responses are increased in L-Sel-/- CONALT after i.n. immunization

To determine the number of Ag-specific B lymphocytes found in the LN that drain the upper respiratory tract, we next investigated the AFC found in CONALT, at 16 days postprimary i.n. CT immunization. Enumeration of the number of lymphocytes obtained from L-Sel-/- CONALT indicated that these LN were much smaller than L-Sel+/+ CONALT. In fact, the number of lymphocytes/LN/mouse in L-Sel-/- mice averaged 10–25% of the lymphocytes/LN/mouse obtained from L-Sel+/+ mice (Table IGo), supporting the contention that L-Sel deficiency reduces the number of lymphocytes found in the CONALT. Such impairment may account for the reduction in the immune response observed in the NP and RT. Surprisingly, the reduction in the total number of lymphocytes present in L-Sel-/- CONALT did not correspond to a reduction in the frequency of Ag-specific and total AFC responses. When compared per the number of AFC/106 lymphocytes, we observed no significant decrease in the number of CT-B-specific AFC produced in L-Sel-/- CONALT vs L-Sel+/+ CONALT (Fig. 1Go, C and D), with the exception of the PRLN IgA anti-CT-B response. In fact, there were significant increases in the number of total IgA AFC/106 lymphocytes in the L-Sel-/- CONALT. L-Sel-/- SMLN produced an average of 350% more AFC than L-Sel+/+ SMLN (p < 0.01), while L-Sel-/- CLN produced 240% more IgA AFC than did L-Sel+/+ CLN (p < 0.01). The most striking difference between L-Sel-/- mice and L-Sel+/+ mice was observed in the PRLN. In L-Sel+/+ mice, this LN produced very little IgA response, following i.n. immunization (~42 AFC/106 lymphocytes), while, in L-Sel-/- mice, this number was increased by nearly 13-fold (536 AFC/106 lymphocytes; p < 0.001). In addition, CT-B-specific response was significantly increased in L-Sel-/- PRLN (p < 0.02).


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  		Table I. Total CT-B-specific AFC responses in the CONALT at 16 days post-i.n. CT immunizationa

 
It remained perplexing that comparing the frequencies of IgA and IgG CT-B-specific AFC responses between L-Sel-/- and L-Sel+/+ CONALT showed minimal to no differences, while the total IgA responses were significantly enhanced. Instead of analyzing the data by this comparison, the total IgA CT-B-specific AFC responses per LN were examined (Table IGo). In the SMLN and CLN, the IgA CT-B-specific AFC response/LN was significantly reduced by 9.7- and 17.6-fold, respectively. Thus, this takes into account the reduced cellularity of the LN. No significant differences were seen for the PRLN. Likewise, significant reductions in IgG CT-B-specific AFC/LN were seen in the SMLN and CLN by 41- and 27-fold. Again, no significant reductions in the PRLN were observed.

Because CONALT HEV expressed varying levels of MAdCAM-1 (22), it is possible that expression of {alpha}4{beta}7 and subsequent binding to MAdCAM-1 might compensate for the loss of L-Sel in these LN. Therefore, we next studied the expression of {alpha}4{beta}7 and L-Sel on L-Sel+/+ and L-Sel-/- NALT and CONALT. L-Sel staining was observed on 7–14% of the lymphocytes isolated from L-Sel+/+ CONALT (Fig. 2GoA), suggesting a possible role for L-Sel in lymphocyte trafficking to these tissues during an immune response. Yet, the loss of L-Sel did not affect the production of AFC in L-Sel-/- CONALT. Interestingly, our results also showed that B lymphocytes isolated from L-Sel-/- CONALT did not display increased expression of {alpha}4{beta}7 or {alpha}E{beta}7 when compared with B lymphocytes isolated from L-Sel+/+ CONALT (Fig. 2GoB). Thus, the increase in AFC in L-Sel-/- CONALT might be indicative of a delay in lymphocyte trafficking from an inductive site to an effector site, rather than selective trafficking of an {alpha}4{beta}7+ subset.



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  		FIGURE 2. {alpha}4{beta}7 expression is not increased on L-Sel-/- CONALT 16 days post-CT i.n. immunization. Lymphocytes were stained with anti-L-Sel mAb MEL 14, anti-{alpha}A{beta}7 mAb DATK 32, anti-{alpha}E mAb M290, and anti-B220 mAb. L-Sel- and {alpha}4{beta}7 (A)- and {alpha}4{beta}7- and {alpha}E{beta}7 (B)-staining profiles are shown for B220+ lymphocytes. Results are representative of three experiments.

 
Immune responses are restored in L-Sel-/- NP and RT by 6 wk postprimary immunization

Kinetic studies of fecal, vaginal, and serum Ab responses revealed that CT-B-specific IgG and IgA responses in L-Sel-/- effector sites may be delayed. In both L-Sel+/+ and L-Sel-/- mice, serum IgA and IgG Ab responses peaked by day 21 postprimary immunization, and then they declined (Fig. 3Go, A and B), although L-Sel-/- serum IgA responses lagged behind L-Sel+/+ responses until day 27. In RT, L-Sel-/- CT-B-specific IgA responses were significantly lower than L-Sel+/+ responses at day 14, but had surpassed L-Sel+/+ responses by 26 days postimmunization and remained robust 6 wk postimmunization (Fig. 3GoC). Vaginal IgG responses were also compromised in L-Sel-/- mice, as evidenced by the delay, followed by a more rapid decline of immune IgG Abs (Fig. 3GoD). Fecal IgA responses by both strains remained similar until day 26 postimmunization, when the L-Sel-/- response was substantially reduced by day 30 (Fig. 3GoE).



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  		FIGURE 3. Vaginal Ab responses in L-Sel-/- mice are delayed. Serum IgA (A) and IgG (B), vaginal IgA (C) and IgG (D), and fecal IgA (E) CT-B-specific ELISA results are shown 0–42 days post-i.n. CT immunization. Data depict the mean of eight mice/group ± SEM.

 
ELISPOT experiments revealed that by 6 wk postprimary immunization, immune responses in the L-Sel-/- NP had been restored (Table IIGo and Fig. 4Go). CT-B-specific (p = 0.023) and total IgA (p = 0.008) AFC responses per 1 x 106 lymphocytes in the NP were enhanced by 6 wk when compared with similar AFC responses at 16 days (Table IIGo; Fig. 4Go, A and C). Although NP responses in L-Sel+/+ had declined by 6 wk when compared with responses at 16 days, the reverse effect held true for CT-B-specific IgA SFC frequencies in the L-Sel-/- NP (Table IIGo). In fact, L-Sel-/- NP CT-B-specific IgA AFC were significantly higher than those in their L-Sel+/+ counterpart were at this time point. In addition, Ag-specific and total IgG responses in L-Sel-/- NP did not differ significantly from those observed in L-Sel+/+ NP by 6 wk postimmunization (Table IIGo; Fig. 4Go, B and D). Although total IgG and IgA responses in L-Sel-/- RT were reduced at 16 days postimmunization (Fig. 4Go, A and B), by 6 wk postimmunization, AFC frequencies in L-Sel-/- did not significantly differ from those observed in L-Sel+/+ RT. More importantly, CT-B-IgA- and IgG-specific responses were nonexistent in L-Sel-/- mice at 16 days postimmunization, but increased by 6 wk postimmunization, while L-Sel+/+ Ag-specific responses declined over the same time period (Table IIGo). As was the case for CT-B-specific IgA and IgG AFC at 16 days postimmunization, we observed no significant difference between responses in L-Sel+/+ and L-Sel-/- iLP at 6 wk postimmunization (Table IIGo). Furthermore, although total, not CT-B specific, IgA (p = 0.002) and IgG (p = 0.029) L-Sel-/- AFC iLP responses at 16 days were reduced (Fig. 4Go, A and B); there was no significant difference in total frequency of AFC between L-Sel+/+ and L-Sel-/- iLP (Fig. 4Go, C and D) by 6 wk postimmunization. Together, these data indicate that a lack of L-Sel does not permanently affect the effector cell response subsequent to i.n. immunization with CT.


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  		Table II. Effector CT-B-specific AFC responses 16 and 42 days post-i.n. immunizationa

 


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  		FIGURE 4. Total AFC/106 lymphocyte responses are diminished in 16 days, but not by 42 days post-i.n. immunization with CT in L-Sel-/- NP and RT. Total IgA (A, C) and IgG (B, D) in L-Sel+/+ and L-Sel-/- NP, RT, and iLP were determined by ELISPOT at 16 (A, B) and 42 (C, D) days post-i.n. CT immunization. Results are the mean of three experiments ± SEM. *, p <= 0.008; **, p = 0.029; ***, p < 0.05.

 
L-Sellow and {alpha}4{beta}7low lymphocytes are present in effector sites at 6 wk postprimary immunization

Our recent work identified two populations of effector B lymphocytes at 16 days: the L-Sellow/{alpha}4{beta}7low population provided CT-B-specific AFC in NP and RT, and the L-Sellow/{alpha}E{beta}7+ population provided CT-B-specific AFC in iLP (20). To determine whether homing receptor profiles on these effector lymphocyte populations changed by 6 wk immunization, we performed flow cytometry analysis on L-Sel+/+ and L-Sel-/- NP, RT, and iLP. We found that the majority of B220+ lymphocytes in RT and NP were L-Sellow/{alpha}4{beta}7low, which was the same homing receptor phenotype expressed by effector lymphocytes at day 16 (Fig. 5GoA). However, in L-Sel+/+ NP, ~18% of B lymphocytes appeared L-Selhigh. In addition, we found two populations of {alpha}4{beta}7low B220+ lymphocytes in the effector tissues with mean fluorescence intensities (MFI) of 10 and 85, respectively. In the iLP, the majority of B lymphocytes expressed {alpha}4{beta}7 at an MFI of 85. However, in the NP, ~50% of B lymphocytes expressed a lower level of {alpha}4{beta}7. In the RT, this population was increased to 68% of B lymphocytes. Similar populations were observed in L-Sel-/- mice, as well (Fig. 5GoB). These results indicated that although CT-specific and total immune responses are restored in L-Sel-/- mice 6 wk after primary immunization, this change is not due to increased expression of the mucosal homing receptor {alpha}4{beta}7.



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  		FIGURE 5. {alpha}4{beta}7+, but not {alpha}4{beta}7high expression is observed in L-Sel+/+ and L-Sel-/- NP, iLP, and RT 6 wk post-i.n. CT immunization. Lymphocytes were treated with anti-L-Sel mAb MEL 14, anti-{alpha}4{beta}7 mAb DATK 32, anti-{alpha}E mAb M290, and anti-B220 mAb. Staining profiles of L-Sel vs {alpha}4{beta}7 are shown for L-Sel+/+ (A) and L-Sel-/- B lymphocytes (B). {alpha}4{beta}7 vs {alpha}E staining profiles are shown for L-Sel+/+ (C) and L-Sel-/- B lymphocytes (D). No significant changes in staining profiles are observed between L-Sel+/+ and L-Sel-/- effector tissues. Results are representative of three experiments.

 
In 16-day i.n. immunized mice, iLP contained a unique {alpha}E+ B220+ population that was not found in RT or NP, and 6 wk after primary immunization, this population remained. However, in L-Sel-/- mice, the percentage of B lymphocytes expressing {alpha}E decreased to ~9.5% (Fig. 5GoD), whereas, at 16 days, this same population consisted of 23% of the total B220+ cells (20). In contrast, ~28% of L-Sel+/+ iLP B lymphocytes expressed {alpha}E (Fig. 5GoC) as opposed to 13% at 16 days. In addition, most {alpha}E+ lymphocytes expressed a lower level of {alpha}4{beta}7 than {alpha}E- lymphocytes. {alpha}E{beta}7 expression was increased slightly at 6 wk in L-Sel+/+ and L-Sel-/- NP and RT, but remained {alpha}Elow. The majority of the {alpha}E{beta}7 expression remained {alpha}Elow in 6 wk postprimary immunization of L-Sel+/+ and L-Sel-/- NP and RT. This {alpha}E{beta}7low population also expressed a slightly lower level of {alpha}4{beta}7. These results indicated that there was a restoration of the immune response in L-Sel-/- NP and RT at 6 wk postimmunization, and this was not the result of an up-regulation of {alpha}4{beta}7 or {alpha}E{beta}7 in these tissues. In fact, the majority of the B220+ lymphocytes in NP and RT maintained a L-Sellow/{alpha}4{beta}7+/{alpha}E- phenotype, similar to that observed at 16 days, while the iLP contained L-Sellow/{alpha}4{beta}7+/{alpha}E- and L-Sellow/{alpha}4{beta}7low/{alpha}E+ lymphocyte populations.

Together, these results suggested that although L-Sel-/- NP and RT effector Ab responses are restored in 6-wk immunized mice, these responses are not due to an increase in {alpha}4{beta}7 or {alpha}E{beta}7 expression by B lymphocytes in these tissues. Although L-Sel did not appear to be necessary for the induction of immunity at 6 wk postimmunization, it is possible that compensatory homing by {alpha}4{beta}7 interactions might be responsible for the restoration of immune response in the nonintestinal effector sites at this time point. To understand more fully the mechanisms that allow for the recovery of Ag-specific immune response in L-Sel-/- effector sites, we next investigated Ab responses in the inductive NALT and CONALT.

CT-B-specific and total Ab responses are increased in L-Sel-/- CONALT

Investigation of responses in inductive sites at 6 wk postprimary immunization revealed that CT-B-specific, as total IgG and IgA responses, were maintained in both L-Sel+/+ and L-Sel-/- NALT (Fig. 6Go, A and B). An increase in the number of NALT AFC produced in these tissues was also observed when compared with those obtained at 16 days. In contrast, CT-B-specific AFC was absent from PP at this time point, although strong total IgA and IgG AFC responses remained in both L-Sel+/+ and L-Sel-/- PP.



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  		FIGURE 6. CT-B-specific and total AFC/106 lymphocytes are increased in 6 wk i.n. CT-immunized L-Sel-/- CONALT. ELISPOT analysis was performed to determine CT-B-specific IgA (A) and IgG (B) in L-Sel+/+ and L-Sel-/- NALT and PP, and CT-B-specific IgA (C) and IgG (D) in L-Sel+/+ and L-Sel-/- CONALT. Results are the mean of four experiments ± SEM. *, p < 0.001; **, p < 0.01.

 
We previously showed that L-Sel deficiency does not affect immune responses in CONALT at 16 days post-i.n. immunization and may in fact increase total IgA response in these tissues (Fig. 1Go, C and D). To determine whether the CT-B-specific Ab responses in CONALT at 16 days were maintained at 6 wk, a B cell ELISPOT analysis on CONALT was conducted (Fig. 6Go, C and D). The results showed increased total IgA responses in CONALT at 6 wk. L-Sel-/- SMLN contained 8.2-fold more total IgA AFC than L-Sel+/+, while L-Sel-/- SMLN and CLN contained 6.7-fold more AFC than L-Sel+/+ CLN. Again, the most striking difference between IgA AFC in L-Sel+/+ and L-Sel-/- mice was observed in the PRLN, in which L-Sel-/- AFC responses were 41-fold greater than L-Sel+/+ AFC responses (average number of AFC/106 lymphocytes of 19 vs 782; p < 0.01). In addition, CT-B-specific IgA responses were increased in L-Sel-/- CONALT, as well, with AFC responses of 13.7-, 41-, and 4-fold greater than L-Sel+/+ SMLN, PRLN, and CLN, respectively. Studies of IgG AFC responses in L-Sel-/- CONALT also revealed a significant increase in CT-B-specific and total response in SMLN and PRLN. Again, response in PRLN showed the greatest change in L-Sel-/- mice, with a 40-fold increase in CT-B-specific and 10-fold increase in total IgG response (p < 0.001), while total IgG response in SMLN increased 3-fold (p < 0.01). However, no significant increases in the number of CT-B-specific or total IgG in the CLN were observed. Finally, the magnitude of all CT-B-specific and total AFC responses in CONALT was as great as or greater than those observed at 16 days postprimary immunization, indicating that Ab responses in L-Sel-/- CONALT are long-lived. This is especially true when the data were analyzed as total response/LN (Table IIIGo). There were no differences in total IgA and IgG CT-B-specific AFC/LN in the SMLN or CLN. Rather, there were significant increases by 7.2- and 4.1-fold, respectively, in the PRLN IgA and IgG CT-B-specific AFC/LN (Table IIIGo). Comparison of 16-day (Table IGo) and 42-day (Table IIIGo) results shows the magnitude of the SMLN and CLN IgA and IgG and the PRLN IgA responses did not change in the L-Sel-/- mice over the course of 6 wk. This was in striking contrast to the L-Sel+/+ CONALT, in which the magnitude of the responses decreased over time. The L-Sel-/- CONALT were much smaller and contained fewer lymphocytes than did L-Sel+/+ CONALT (Table IIIGo); however, the number of recovered lymphocytes, in both L-Sel+/+ and L-Sel-/- CONALT, was still less than those recovered at 16 days (Table IGo). These results indicated that trafficking to these LN, but not response to CT, might indeed be disrupted by the loss of L-Sel.


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  		Table III. Total CT-B-specific AFC responses in the CONALT at 6 wk post-i.n. CT immunizationa

 
An {alpha}E{beta}7 lymphocyte population may provide increased AFC in L-Sel-/- CONALT

To determine whether the significant increase in CONALT AFC production in L-Sel-/- CONALT was due to unique populations of B lymphocytes, we performed a FACS analysis for {alpha}E, {alpha}4{beta}7, and L-Sel expression on CONALT lymphocytes. Our results showed that at 6 wk, nearly 50% of SMLN, PRLN, and CLN B lymphocytes expressed L-Sel (Fig. 7GoA), a clear increase from the percentages observed at the 16-day time point. Expression of {alpha}4{beta}7 on lymphocytes, isolated from 6-wk immunized L-Sel+/+ CONALT, revealed that the majority expressed this integrin at a MFI of ~100. However, some lymphocytes isolated from L-Sel-/- CONALT at 6 wk appeared to express lower levels of {alpha}4{beta}7, which was similar to the results observed in effector sites at this time point. In addition, very few B220+ lymphocytes were recovered from L-Sel-/- CLN, as the majority of cell population in these nodes was found to be mostly T cells (unpublished observation). Finally, the L-Sel+/+ and L-Sel-/- CONALT were analyzed for the expression of {alpha}E{beta}7. It was found that 10% of SMLN B lymphocytes, 17% of PRLN B lymphocytes, and 9% of CLN B lymphocytes expressed {alpha}E in L-Sel-/- mice (Fig. 7GoB). In contrast, 6% or fewer CONALT B lymphocytes from L-Sel+/+ mice at the same time point expressed {alpha}E.



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  		FIGURE 7. {alpha}4{beta}7 expression is not increased, but {alpha}E{beta}7 is increased on L-Sel-/- CONALT 6 wk post-CT i.n. immunization. Lymphocytes from NALT, PRLN, SMLN, and CLN were stained with anti-L-Sel mAb MEL 14, anti-{alpha}4{beta}7 mAb DATK 32, and anti-B220 mAb. L-Sel- vs {alpha}4{beta}7-staining profiles (A) and {alpha}E- vs {alpha}4{beta}7-staining profiles (B) are shown for B220+ L-Sel+/+ and L-Sel-/- lymphocytes. Results are representative of three experiments.

 
Together, these results indicated that although the number of lymphocytes recovered from L-Sel-/- CONALT are still less than lymphocytes recovered from CONALT of L-Sel+/+ mice, these mice are capable of mounting an enhanced, sustained Ab response subsequent to i.n. immunization. This response is not reliant upon L-Sel expression, although 50% of lymphocytes in L-Sel+/+ CONALT express this homing receptor. In addition, L-Sel-/- CONALT B lymphocytes might actually lose some expression of {alpha}4{beta}7, but increase expression of {alpha}E. Clearly, immune response in CONALT is not dependent upon L-Sel interactions, and lack of L-Sel may actually enhance Ab response in these tissues.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results have clearly shown that loss of NP and RT immunity in L-Sel-/- mice at 16 days postprimary i.n. immunization with CT is due to the delay of the L-Sellow/{alpha}4{beta}7low (double-low) B cells entering into these sites, rather than the complete abatement of AFC responses. Kinetic studies indicate that Ab responses are restored in L-Sel-/- NP and RT ~21–26 days postprimary immunization, and the restoration of effector site immunity in L-Sel-/- mice does not appear to be the result of a compensatory influx of {alpha}4{beta}7- or {alpha}E{beta}7-bearing lymphocytes. Instead, the delay in effector site immunity appears to be caused by increased lymphocyte retention in L-Sel-/- CONALT.

Our previous studies (20) showed that L-Sel-/- NP and RT AFC were negligible 16 days after primary i.n. immunization with CT, and that immune response in these sites in L-Sel+/+ and L-Sel-/- mice was primarily contained within the double-low B cell subset. In contrast, AFC in iLP were primarily contained within the unique subset of L-Sellow/{alpha}4{beta}7low/{alpha}E+ B lymphocytes and, to a lesser extent, double-low B cell subset. At this same time point, lymphocyte trafficking by the double-low subset to L-Sel-/- NP and RT was affected, as fewer numbers of lymphocytes were recovered from these sites. In L-Sel-/- iLP, this decreased presence of {beta}7low population resulted in a diminished total IgA response.

By 6 wk postprimary immunization of L-Sel-/- mice with CT, the numbers of lymphocytes recovered from L-Sel+/+ and L-Sel-/- NP and RT were similar, indicating lymphocyte trafficking can occur to theses sites in the absence of L-Sel. To determine whether the restoration of responses at nonintestinal mucosal effector sites was due to the influx of AFC that express higher levels of {alpha}4{beta}7 or {alpha}E{beta}7, we investigated the profiles of B lymphocytes in NP and RT at 6 wk postprimary immunization. We found that subsets of lymphocytes in NP, RT, as well as iLP, remained the same as those observed at 16 days postprimary immunization (20), with the double-low population of B lymphocytes dominant in all three tissues, and the {alpha}E+ population remained in the iLP. However, the percentage of B lymphocytes expressing {alpha}E was decreased in L-Sel-/- iLP. These results indicated that lymphocyte trafficking to the nonintestinal L-Sel-/- mucosal effector sites is most likely mediated, in part, through {alpha}4{beta}7-binding interactions with MAdCAM-1. It is possible that immunization with CT will induce TNF-{alpha}-driven up-regulation of MAdCAM-1 in effector sites (44, 45, 46). In future studies, we will undertake immunohistochemical staining studies to determine the expression of MAdCAM-1 in NP and RT, as well as determine the effect of nasal immunization of Ags other than CT on mucosal immune responses in L-Sel-/- mice at this time point.

Because we previously determined that L-Sel deficiency results in a lack of effector site immunity in the nonintestinal effector sites, but not in the intestine, we questioned whether the loss of L-Sel would have a deleterious effect on the AFC response in the nasal inductive site, NALT, when compared with the PP. Although L-Sel deficiency has little effect on lymphocyte trafficking to PP (36), maintenance of Ab response in L-Sel-/- NALT is surprising, given that the majority of lymphocyte binding to the naive NALT is mediated by L-Sel-PNAd interactions (21). {alpha}4{beta}7 interactions might compensate for the loss of L-Sel in immunized NALT because we observed increased MAdCAM-1 expression and function in the NALT after i.n. immunization with CT (unpublished observation). As immunized L-Sel-/- NALT remain smaller than immunized L-Sel+/+ NALT, it is also possible that the immune response in this site will be the result of stimulation of existing lymphocyte populations. Like the PP, the NALT contains specialized epithelial M cells, which could facilitate transport of CT to the underlying dome region dendritic cells, which can then present CT Ag to the existing populations of B and T cells in the lymphocyte compartment. Furthermore, the number of AFC found in L-Sel-/- NALT and PP at 6 wk post-i.n. immunization was not significantly lower than L-Sel+/+ NALT and PP. However, by 6 wk, the NALT maintained a robust Ag-specific immune response, while this response was abated in both L-Sel+/+ and L-Sel-/- PP. These results indicated that long-term stimulation of immune responses in the local, but not the distal immune inductive site, occurs following i.n. immunization.

To further elucidate the inductive immune response in i.n. immunized L-Sel-/- mice, we also examined the AFC response in the draining CONALT. As observed in NALT, the loss of L-Sel had no effect on induction of immunity in these sites. In fact, total IgA response was increased in L-Sel-/- CONALT. Again, these results were surprising, given that the majority of lymphocyte binding to these LN is mediated through L-Sel-PNAd interactions (22). Indeed, cell numbers were significantly reduced in CONALT at 16 days postprimary immunization, indicating a loss of lymphocyte trafficking to these sites. Again, it is possible that MAdCAM-1 might be up-regulated in these LN, following CT immunization, or that CT-draining or CT-presenting dendritic cell migration to these sites might stimulate resident lymphocyte populations.

It is also possible that the increased AFC responses observed in L-Sel-/- CONALT might not represent an increased number of Ag-specific B cells retained in these sites. Previous studies have shown that i.p. and s.c. immunization induces elevated humoral immune responses in L-Sel-/- PLN, although these LN, like the CONALT, remained smaller in L-Sel-/- mice. However, germinal centers in L-Sel-/- PLN were found to be markedly larger and more organized than germinal centers in L-Sel+/+ PLN (47). Although little evidence was observed for lymphocyte trafficking in these LN, the increased size of the germinal centers in L-Sel-/- PLN indicated possible expansion of existing B cell populations. Alternatively, there might be a decreased ability of non-Ag-specific lymphocytes to traffic to the CONALT, leaving only Ag-specific populations to respond. In PRLN, which displays the most striking difference in L-Sel-/- and L-Sel+/+ AFC response, 0.05% of lymphocytes recovered from this tissue produced IgA at 16 days post-i.n. CT immunizations, compared with 0.004% of L-Sel+/+ lymphocytes. This 10% increase in Ag-specific AFC was not reflective of a dearth of nonresponsive cells that were unable to traffic to the LN, because the LN are 17% smaller than L-Sel+/+ counterparts. At 6 wk, this increased percentage of IgA-producing cells was even more pronounced, as 0.09% of L-Sel-/- vs 0.002% of L-Sel+/+ lymphocytes produced IgA, with the L-Sel-/- PRLN being only 10% in size of the L-Sel+/+ LN. In addition, flow cytometry analysis showed no difference in the overall percentage of B lymphocytes found in L-Sel-/- vs L-Sel+/+ CONALT (~30% in each). Therefore, it seems likely that the increased Ag-specific and total AFC responses observed in L-Sel-/- mice are a direct result of increased retention of Ag-specific B lymphocytes.

It is also possible the immune B cells cannot exit the L-Sel-/- CONALT. It was surprising to observe the lack of change in the number of immune CONALT B cells between days 16 and 42, whereas reductions in total IgA and IgG CT-B-specific AFC were noted for L-Sel+/+ CONALT. Because it would be unexpected to observe a particular bias for L-Sel-/- CONALT B cells to undergo cell division more so than L-Sel+/+ B cells, the data do suggest that B cells are unable to exit the CONALT. Alternatively, it may be that the L-Sel deficiency might affect the ability of lymphocytes to migrate out of LN by way of efferent lymphatics, as well as traffic via HEV binding. Recent studies have shown that L-Sel is important in the binding of lymphocytes to the efferent lymphatics in the LN, and they may play a role in the migration of memory lymphocytes from the initial site of stimulation (48). It has also been shown that while activated B lymphocytes shed L-Sel (49), re-expression of L-Sel is important for the trafficking of memory B lymphocytes (50). Finally, L-Sel-/- mice retain increased numbers of memory T cells in their PLN, although these LN remain smaller than their L-Sel+/+ counterparts (51). Therefore, increased Ab responses in L-Sel-/- CONALT might be the result of an inability of B lymphocytes to traffic from the LN, and the delay in response in L-Sel-/- effector sites might represent a decreased ability of memory lymphocytes to migrate from the inductive sites. Future histochemical studies will address the issue of the retention of naive, activated, and memory B lymphocytes in L-Sel+/ and L-Sel-/- CONALT.

These AFC responses also indicated a role for the PRLN in immune response to i.n. introduced Ags. This LN has the most peripheral phenotype of the CONALT (22) and has previously been shown to produce immune response to Ags that drain from the skin of the head and neck, rather than drain from the nasal mucosa (52, 53). However, these results indicated a role for this tissue in immune response to i.n. immunization, especially in L-Sel-/- mice, indicating possible draining of the nasal mucosa or selective trafficking of Ag-specific B lymphocytes.

Finally, studies of lymphocyte-homing receptor profiles on L-Sel-/- CONALT indicated while there was not an increase in {alpha}4{beta}7 expression, there may be an increased number of {alpha}E{beta}7-expressing lymphocytes. This result is particularly intriguing in context of the decrease in {alpha}E{beta}7+ lymphocytes in the L-Sel-/- iLP, indicating that this population might actually migrate from the intestine to other mucosal sites. {alpha}E{beta}7 has not previously been shown to mediate T lymphocyte trafficking, but rather it appears to play a role in lymphocyte retention in the iLP (54, 55). However, recently, a novel ligand for {alpha}E{beta}7 has been observed (56), and little is known about the role of {alpha}E{beta}7 on B, rather than T lymphocytes.

In conclusion, our results indicate that restoration of the nonintestinal mucosal immune response in L-Sel-/- mice might be a function of delay in lymphocyte trafficking from the mucosal inductive tissues. In addition, these data show a continued important role of the double-low B lymphocyte subset in Ab response of the NP and RT, and suggest a novel role for {alpha}E{beta}7 expression on B lymphocytes. Finally, these results further define the role of L-Sel in the induction and dissemination of Ab responses following i.n. immunization.


   Acknowledgments
 
Special thanks go to Carol Riccardi for her expert technical assistance and to Nancy Kommers for her assistance in preparing this manuscript.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grant DE-13812 and in part by the Montana Agricultural Station and U.S. Department of Agriculture Formula Funds. This is Montana Agricultural Station Journal Series 2002-26. Back

2 Address correspondence and reprint requests to Dr. David W. Pascual, Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717-3610. E-mail address: dpascual{at}montana.edu Back

3 Abbreviations used in this paper: i.n., intranasal; AFC, Ab-forming cell; CLN, cervical LN; CM, complete medium; CONALT, cranial-, oral-, nasal-associated lymphoid tissue; CT, cholera toxin; CT-B, CT B subunit; HEV, high endothelial venule; iLP, intestinal lamina propria; L-Sel, L-selectin; LN, lymph node; MAdCAM-1, mucosal addressin cell adhesion molecule-1; MFI, mean fluorescence intensity; NALT, nasal-associated lymphoid tissue; NP, nasal passage; PLN, peripheral LN; PNAd, peripheral node addressin; PP, Peyer’s patch; PRLN, parotid gland LN; RT, reproductive tract; SMLN, submaxillary gland LN. Back

Received for publication June 5, 2002. Accepted for publication September 17, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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