Endothelial Venules in a Mucosal Site in Naive Lymphocyte Adhesion to High Primary Role of Peripheral Node Addressin Phenotypic

Nasal-Associated Lymphoid Tissue: Phenotypic and Functional Evidence for the Primary Role of Peripheral Node Addressin in Naive Lymphocyte Adhesion to High This information is current as Endothelial Venules in a Mucosal Site of October 1, 2021. Keri L. Csencsits, Mark A. Jutila and David W. Pascual J Immunol 1999; 163:1382-1389; ; http://www.jimmunol.org/content/163/3/1382 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Nasal-Associated Lymphoid Tissue: Phenotypic and Functional Evidence for the Primary Role of Peripheral Node Addressin in Naive Lymphocyte Adhesion to High Endothelial Venules in a Mucosal Site1

Keri L. Csencsits, Mark A. Jutila, and David W. Pascual2

Nasal-associated lymphoid tissue (NALT), a mucosal inductive site for the upper respiratory tract, is important for the development of mucosal immunity locally and distally to intranasally introduced Ag. To more fully understand the induction of nasal mucosal immunity, we investigated the addressins that allow for lymphocyte trafﬁcking to this tissue. To investigate the addressins responsible for naive lymphocyte binding, immunoﬂuorescent and immunoperoxidase staining of frozen NALT sections were

performed using anti-mucosal addressin cell adhesion molecule-1 (MAdCAM-1), anti-peripheral node addressin (PNAd), and Downloaded from anti-VCAM-1 mAbs. All NALT high endothelial venules (HEV) expressed PNAd, either associated with MAdCAM-1 or alone, whereas NALT follicular dendritic cells expressed both MAdCAM-1 and VCAM-1. These expression proﬁles were distinct from those of the gut mucosal inductive site, Peyer’s patches (PP). The functionality of NALT HEV was determined using a Stamper- Woodruff ex vivo assay. The anti-L-selectin MEL-14 mAb blocked >90% of naive lymphocyte binding to NALT HEV, whereas the anti-MAdCAM-1 mAb, which blocks almost all naive lymphocyte binding to PP, minimally blocked binding to NALT HEV.

NALT lymphocytes exhibited a unique L-selectin expression proﬁle, differing from both PP and peripheral lymph nodes. Finally, http://www.jimmunol.org/ NALT HEV were found in increased amounts in the B cell zones, unlike PP HEV. These results suggest that NALT is distinct from the intestinal PP, that initial naive lymphocyte binding to NALT HEV involves predominantly L-selectin and PNAd rather than ␣ ␤ 4 7-MAdCAM-1 interactions, and that MAdCAM-1 and VCAM-1 expressed by NALT follicular dendritic cells may play an important role in lymphocyte recruitment and retention. The Journal of Immunology, 1999, 163: 1382–1389.

unique nasal-associated lymphoid tissue (NALT)3 has that expected for the PP. In fact, the relative percentages of B and been identified in rodents (1, 2) that represents the oro- T cells in the NALT approximate those observed in the PP (4–6). pharyngeal lymphoid tissues of the upper respiratory air- Although the NALT may not be the only site that contributes sig- A by guest on October 1, 2021 ways. This structure is believed to be analogous to human Waldey- nificantly to the stimulation of mucosal effector precursors for the er’s ring (tonsils and adenoids) (3) and consists of bilateral upper airways, its importance may be due to its proximity to the lymphoid structures dorsal to the cartilaginous soft palate. It is nasal lamina propria. Collectively, this evidence suggests that this thought, much like the Peyer’s patches (PP), to behave as a mu- unique structure is an important tissue for studying immunity to cosal inductive site. This resemblance is evident in both the orga- nasally introduced Ags. nization and the structure of NALT as well as its cellular compo- Intranasal (i.n.) immunization is an effective route for stimulat- sition. First, there is an epithelial layer overlaying the NALT that ing mucosal immunity to a variety of pathogens (7–11), soluble contains specialized M cells (1, 2), which are thought to mediate proteins including cholera toxin (12, 13), and microparticle-deliv- Ag entry much like that observed in the PP. In addition, there is a ered Ags (14). This route of immunization induces strong Ag- unique organization of both B cell zones and T cell areas much like specific mucosal and systemic IgA and IgG Ab responses (6, 7, 9–13) and stimulates elevated CTL responses to viral and OVA peptides (15–17). Thus, the elevations in humoral and cell-mediated immunity coupled with the ease of administering Ags make Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717 this a favorable route of immunization not only for local respira- Received for publication March 10, 1999. Accepted for publication May 13, 1999. tory immunity, but also for providing immunity at distal mucosal The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance sites (8, 9, 12, 13, 18, 19). In particular, i.n. immunization provides with 18 U.S.C. Section 1734 solely to indicate this fact. immunity in the genito-urinary tract (11, 13, 20–22) where direct 1 This work was supported by U.S. Public Health Service Grants AI-40288, immunization is often hampered by epithelial cell turnover and AI-42673, S10 RR-11877 (to D.W.P.), and AI-47671 (to M.A.J.); in part by the Montana Agricultural Station and U.S. Department of Agriculture Formula Funds; hormonal influences. For instance, mice immunized i.n. with the and in part by the M. J. Murdock Charitable Trust. HIV gp160 protein produced HIV-1-neutralizing IgA and IgG Abs 2 Address correspondence and reprint requests to Dr. David W. Pascual, Veterinary in serum, lung, and vagina (11). Elevations in Ag-specific IgG and Molecular Biology, Montana State University, Bozeman, MT 59717-3610. E-mail IgA were reported in vaginal secretions of mice following i.n. address: [email protected] immunization with plasmid DNA as well as viral and bacterial 3 Abbreviations used in this paper: NALT, nasal-associated lymphoid tissue; FDC, follicular dendritic cell; GALT, gut-associated lymphoreticular tissue; HEV, high- vectors (8, 22, 23). Although i.n. immunization may prove to be a walled endothelial venule; i.n., intranasal; MAdCAM-1, mucosal cell adhesion mol- highly effective method of inducing immunity in the genito-urinary ecule-1; MLN, mesenteric lymph node; PP, Peyer’s patches; PLN, peripheral lymph node; PNAd, peripheral node addressin; DPBS, Dulbecco’s PBS; NGS, normal goat tract, it is unknown how immunization of the NALT leads to im- serum: SA, HRP, streptavidin-HRP; AEC, 3-amino-9-ethylcarbazole. munity at this distal mucosal site.

The ability of NALT to induce an immune response at a distal Tissue isolation and collection mucosal site may lie in part within the specialized ligands ex- MLN, PLN, and PP were isolated from normal BALB/c mice. Each set of pressed by its high-walled endothelial venules (HEV). These li- lymphoid tissue was pooled from five different mice, washed in RPMI gands, or addressins, interact with specific homing receptors ex- 1640 medium (BioWhittaker, Walkersville, MD), and frozen using Tissue- pressed by B and T lymphocytes and allow for trafficking of the Tek O.C.T. compound embedding medium (Miles, Elkhart, IN) in a 15- by Ϫ lymphocytes from blood into lymph tissue. However, the addressin 15-mm Tissue Tek Cryomold. Samples were stored at 80°C until use. NALT tissues were collected by removing the soft palates as previously profiles displayed by these HEV have yet to be identified, although described (5, 14). Briefly, euthanized mice were decapitated, their heads some functional attributes of preferential homing by NALT lym- were immobilized, and the lower jaws, including tongue, were removed. phocytes have been observed. Lymphocytes isolated from rat Palates were scored along the outer edge and removed gently. Palates were NALT preferentially homed back to NALT, to cervical lymph washed in RPMI 1640 medium, blotted dry, and arranged in the cryomolds with their ventral faces (containing the NALT) oriented at the bottom of the nodes, and to mesenteric lymph nodes (MLN) rather than to the PP mold and frozen in O.C.T. as described above. For double immunofluo- (24). This evidence suggests that the NALT addressin profile may rescent and immunoperoxidase staining, 5-␮m frozen sections were cut, differ from the PP profile. air-dried, fixed in acetone at 4°C, and air-dried before rehydration. Naive lymphocyte homing to the PP has been well characterized. It has been shown that the mucosal vascular addressin, Double immunofluorescent staining for tissue addressins MAdCAM-1, plays an important role in the trafficking of naive Lymphoid tissue sections were incubated with MECA 367 supernatant (rat B and T cells into PP (25–27). MAdCAM-1 is expressed in the PP, anti-mouse MAdCAM-1 mAb) or normal rat serum for 30 min at room temperature. Specific detection of MAdCAM-1 was obtained upon incu- MLN, and gut lamina propria (28, 29). Both B and T cells interact ϩ ␣ ␤ bation of a 1/150 dilution of TRITC conjugate goat anti-rat IgG (H L) Ab with MAdCAM-1 through the cellular ligand, 4 7 (30). In some (Southern Biotechnology Associates, Birmingham, AL) in the dark for 30 Downloaded from tissues, MAdCAM-1 can also bind cells expressing L-selectin min at room temperature. Sections were then stained for PNAd using a 1/50 through the expression of the peripheral node addressin (PNAd) dilution of MECA 79 (FITC-conjugated rat IgM anti-mouse PNAd) mAb carbohydrate on the MAdCAM-1 glycoprotein backbone (26, 31). in the dark for 30 min at room temperature. To prevent nonspecific binding of FITC-conjugated MECA 79 mAb to free arms of the TRITC-goat anti- In contrast, naive lymphocyte trafficking into peripheral lymph rat IgG Ab, sections were blocked with 1% rat serum for 30 min before nodes (PLN) is mediated by L-selectin binding to PNAd expressed addition of the FITC-conjugated MECA 79 mAb. After rinsing, slides were on glycoproteins other than MAdCAM-1 (32, 33). Thus, there ap- coverslipped using Vecta-Shield mounting medium (Vector Laboratories, pears to be a definite separation between mucosal and peripheral Burlingame, CA). http://www.jimmunol.org/ type lymphocyte recirculation pathways, and it has yet to be de- Immunohistochemical staining of MAdCAM-1 and VCAM-1 termined whether NALT HEV express an exclusive mucosal or location within NALT peripheral phenotype, or a combination of both. In this study we show that NALT HEV express a unique ad- Serially cut frozen sections were rehydrated in Dulbecco’s PBS (DPBS) dressin profile that resembles neither a strict mucosal nor a pe- containing 0.2% normal goat serum (NGS), and endogenous peroxidase was blocked with Dako peroxidase blocking reagent (Dako, Carpinteria, ripheral phenotype. All NALT HEV express PNAd, either alone or CA). Nonspecific binding was blocked using 10% NGS in DPBS for anti- in conjunction with MAdCAM-1. This profile differs greatly from MAdCAM-1 and VCAM-1 mAb staining or 10% NGS with 2.5% mouse the PP, the inductive site for the gut-associated lymphoreticular serum in DPBS for anti-B220 mAb staining for 30 min at room tempera- by guest on October 1, 2021 tissue (GALT). The functionality of this PNAd expression was ture. Endogenous avidin and biotin were blocked with avidin/biotin blocking solution (Vector Laboratories). Sections were incubated with a 1/200 explored through an ex vivo binding assay, where lymphocytes dilution of biotinylated rat anti-mouse VCAM-1 mAb, a 1/500 dilution of bound to NALT HEV primarily through PNAd-L-selectin interac- biotinylated rat anti-mouse mouse B220 mAb, a MECA 367 mAb super- tions. NALT lymphocytes also displayed unique expression of L- natant, or an isotype-matched rat IgG control for 30 min at room temper- selectin profiles. In addition, NALT displayed both MAdCAM-1 ature. Sections incubated with MECA 367 mAb were then treated with a 1/250 dilution of biotinylated goat F(abЈ) anti-rat Ig (␥ and light chain, and VCAM-1 on the follicular dendritic cells (FDC) within its B 2 adsorbed against mouse Ig; BioSource International, Camarillo, CA) for 30 cell areas. Finally, the relative location of HEV within the NALT min at room temperature. All sections were then incubated with a 1/500 differed greatly from that in the PP. This study suggests that NALT dilution of streptavidin-HRP (SA-HRP; BioSource International), and HRP is a unique mucosal tissue, where lymphocyte binding is primarily was visualized with a precipitate of 3-amino-9-ethylcarbazole (AEC; mediated by PNAd and where the location of MAdCAM-1 and Sigma, St. Louis, MO). After AEC development for 3–5 min, the sections were counterstained with hematoxylin (Richard-Allan Scientific, Kalama- VCAM-1 expressed by FDC may play an important role in lym- zoo, MI) and counterslipped using Immuno-Mount mounting medium phocyte recruitment and retention. (Shandon-Lipshaw, Pittsburgh, PA).

Materials and Methods Double immunohistochemical staining of NALT lymphocytes and Mice addressins Specific pathogen-free BALB/c mice were purchased from the National Frozen sections were rehydrated and blocked as described above. A 10% Cancer Institute at 5–6 wk of age, or BALB/cBY mice were bred and NGS and 2.5% mouse serum block was used for the anti-B220 mAb staining, whereas a 10% NGS block was used for the anti-CD3 mAb staining. maintained in the Animal Resources Center at Montana State University ␮ (Bozeman, MT). All mice were kept under pathogen-free conditions in Sections were incubated with 100 g/ml of MECA 79, MECA 367 super- horizontal laminar flow cabinets and were fed sterile food and water ad natant, or mAb isotype control for 30 min at room temperature, followed libitum. The mice were free of bacterial and viral pathogens as determined by a 1/250 dilution of biotinylated goat anti-rat Ig (BioSource Interna- by Ab screening and by histopathologic analysis of major organs and tis- tional) for 30 min at room temperature, then treated with a 1/500 dilution sues. The mice were used at 6–8 wk of age in these experiments. of SA-HRP (BioSource International) for 20 min. Location of the HRP was visualized with unenhanced diaminobenzidine (Vector Laboratories). Monoclonal Abs After diaminobenzidine development, sections were reblocked for endogenous peroxidase, and 0.2% BSA was added to the PBS wash to pre- The rat anti-mouse mAbs used were anti-PNAd mAb MECA 79 (32), vent nonspecific Ab binding. Sections were blocked with 10% NGS and anti-MAdCAM-1 mAb MECA 367 (28), anti-L-selectin mAb MEL-14 1% rat serum. Sections were then stained with a 1/100 dilution of biotin- (34), anti-VCAM-1 mAb 426 (MVCAM.A; PharMingen, San Diego, CA), ylated hamster anti-mouse CD3 mAb (PharMingen) for 1 h, a 1/750 dilu- ␤ PE-conjugated MEL-14 (PharMingen), anti- 7 mAb FIB 30 (35), anti- tion of biotinylated rat anti-mouse B220 mAb (PharMingen), or isotype- CD45R/B220 mAb RA3-6B2 (PharMingen), anti-CD4 mAb RM4-5 matched mAb control for 30 min, followed by a 1/500 dilution of SA-HRP (PharMingen), and CD8␣ mAb 53-6.7 (PharMingen). Also used was a (BioSource International). SA-HRP was visualized with VIP substrate kit hamster anti-mouse CD3 mAb 500-A2 (PharMingen). (Vector Laboratories). Sections were counterstained with hematoxylin 1384 MURINE NALT EXPRESSES A UNIQUE ADDRESSIN PHENOTYPE

(Richard-Allan Scientific). Slides were coverslipped using Crystal/Mount type of the PLN as well as from the intestinal PP and MLN HEV aqueous mounting medium (Biomeda, Foster City, CA). addressin profiles. Ab blocking of naive lymphocyte adhesion to HEV Staining of NALT sections with MECA 367 mAb revealed diffuse MAdCAM-1 located within the B cell area of the NALT as A modification of the Stamper-Woodruff protocol (36) was performed. well as on HEV (Fig. 1, I and M). Staining for VCAM-1 showed Murine MLN were isolated, suspended in RPMI 1640 medium, and sub- jected to Dounce homogenization. The resulting cell suspension was fil- localization within the B cell areas, but not on HEV within the tered through NITEX fabric (Fairmont Fabrics, Mountain View, CA). Fol- NALT (Fig. 1Q). However, staining for MAdCAM-1 in the NALT lowing centrifugation, the MLN lymphocytes were resuspended in DMEM consistently appeared diffuse, whereas staining for VCAM-1 ap- 7 (Sigma) and 2.5% BSA at a concentration of 1 ϫ 10 cells/ml. peared intense. This staining profile differed greatly from that in Frozen tissue samples were cut to a thickness of 7 ␮m and allowed to air-dry for 30 min. A hydrophobic circle was drawn around each section, the PP, which showed dark staining with the anti-MAdCAM-1 and the sections were then placed on an orbital shaker (GeneMate, Inter- mAb in the B cell areas (Fig. 1, G and K). In addition, NALT FDC mountain Scientific, Bountiful, UT) at 4°C at 80 rpm. MECA 79 or MECA displayed addressin profiles different from those of MLN, which 367 mAb supernatant was added, and the Ab was rotated on the sections for showed very little VCAM-1 staining and a higher incidence of 45–60 min to allow binding to MAdCAM-1 or PNAd and then decanted. MAdCAM-1 (Fig. 1, H, L, and P). In contrast, although PLN dis- One hundred microliters of lymphocytes (1 ϫ 106) were added and rotated over the sections for 30 min to allow binding to HEV. Lymphocytes bound plays VCAM-1 on its FDC, very little MAdCAM-1 staining is to HEV were fixed by placing sections in a cold 1.5% glutaraldehyde observed in the follicles (Fig. 1, J, N, and R). Taken together, these solution. Ab stains provide further evidence for the expression of a distinct ␣ ␤ To block lymphocyte binding to L-selectin or 4 7, cells were treated addressin profile in the NALT. with 50 ␮g/ml MEL-14 (rat anti-mouse L-selectin) mAb or FIB 30 (rat ␣ ␤ anti-mouse 4 7) mAb, respectively, for 30 min. Pretreated cells were then Initial naive lymphocyte binding to NALT HEV is mediated Downloaded from rotated over the sections as described above. primarily by PNAd –L-selectin interaction Comparison of L-selectin expressed on NALT, PP, MLN, and To determine whether the PNAd expressed by NALT HEV was PLN lymphocytes functional, we used a Stamper-Woodruff ex vivo binding assay. Lymphocytes were isolated from NALT using procedures previously de- Frozen tissue sections were treated with mAbs specific for scribed (4, 6). Briefly, dissected NALT was disrupted by Dounce homog- MAdCAM-1 or PNAd or with irrelevant isotype-matched Abs. enization, and released lymphocytes were collected in serum-free HBSS For blocking of cellular homing receptors, naive MLN lympho- http://www.jimmunol.org/ (Sigma). PP and PLN lymphocytes were obtained by Dounce homogeni- ␣ ␤ zation of the tissues as described above, and the resulting cell suspensions cytes were pretreated with mAbs specific for 4 7 or L-selectin or were filtered through NITEX. Lymphocytes were stained with FITC-anti- with a rat IgG2a isotype control Ab. The number of cells bound per B220, FITC-anti-CD4, or FITC-anti-CD8 mAbs and PE-MEL-14 rat anti- HEV per node in the presence of addressin-specific Ab was com- mouse L-selectin mAb. FL1 and FL2 gains and compensation were set by pared with control binding, defined as lymphocytes bound to HEV the analysis of single-color FITC or PE. Two-color analyses were performed using a FACSCalibur (Becton Dickinson, Mountain View, CA). in the presence of isotype-matched control Ab. Blocking of the Ten thousand events per sample were collected. PNAd expressed on the HEV of NALT with the MECA 79 mAb resulted in a 60% reduction of binding compared with that in con- Statistical analysis trols (Fig. 2). This reduction of lymphocyte binding was similar to by guest on October 1, 2021 Results were analyzed using paired Student’s t test. Significant p values are that observed in PLN sections treated with MECA 79 mAb. In indicated. contrast, treatment of PP with MECA 79 mAb resulted in no significant reduction in naive lymphocyte binding ( p Ͻ 0.01). Treat- Results ment of NALT HEV with MECA 367 mAb had little effect Murine NALT HEV express a unique addressin profile (Ͻ20%) on the number of lymphocytes bound per HEV, indicating Lymphocyte trafficking into the PP requires MAdCAM-1 expres- that the expressed MAdCAM-1 has no apparent role in naive lym- sion by its HEV (25, 26). If the NALT is indeed analogous to PP phocyte binding. These results were in direct contrast with those and behaves as a mucosal inductive site, it would be expected that for PP, where treatment of sections with MECA 367 mAb resulted its HEV would also mediate lymphocyte binding primarily through in Ͼ90% reduction in binding. In addition, NALT HEV displayed MAdCAM-1. To assess the NALT addressin phenotype, its HEV no similarities in binding to MLN HEV, as 50% of naive lympho- were examined for expression of both mucosal and peripheral ad- cyte binding to MLN was blocked by MECA 367 mAb. Thus, dressins. NALT HEV were stained simultaneously with mAb for initial naive lymphocyte binding to NALT HEV was most similar MAdCAM-1 (MECA 367 mAb and TRITC-labeled secondary) to naive lymphocyte binding to peripheral lymph node HEV and and PNAd (FITC-conjugated MECA 79 mAb). The number of dissimilar to the binding observed in the PP, again providing ev- HEV expressing MAdCAM-1, PNAd, or both addressins in each idence that NALT acts in a manner unique from other character- NALT, PP, or MLN section was determined. As expected in a ized mucosal tissues. mucosal tissue, the NALT HEV expressed MAdCAM-1; however, Blocking of naive lymphocyte homing receptors provided fur- the total percentage of HEV expressing mucosal addressin was ther evidence that the NALT HEV displayed a more peripheral significantly less than that observed in the PP (Fig. 1, A–F, and addressin phenotype (Fig. 3). Blocking of L-selectin using Table I). In fact, all the MAdCAM-1-positive HEV in the NALT MEL-14 mAb resulted in Ͼ90% reduction in binding of naive also coexpressed PNAd. This coexpression was observed in 62.8% lymphocytes to NALT HEV. Binding of lymphocytes to NALT ␣ ␤ of the NALT HEV. No NALT HEV expressed MAdCAM-1 only. HEV was reduced to a lesser extent by blocking of 4 7 with FIB In contrast, 52.4% of PP HEV expressed MAdCAM-1 only, 30 mAb, which reduced binding by about 60% compared with the whereas the remaining 47.6% of PP HEV expressed MAdCAM-1 control value. MEL-14 mAb blocking of naive lymphocyte bind- and PNAd ( p Ͻ 0.0001). In addition, there was a significant dif- ing to NALT was similar to that observed in the PLN, where this ference in the number of HEV expressing PNAd alone in the mAb blocked nearly all naive lymphocyte binding. However, as NALT (37.2%) compared with that in the MLN (2.5%; p Ͻ 0.01). expected, treatment with FIB 30 mAb had no effect on binding in NALT also differs from the PLN, where nearly 100% of HEV the PLN. In the PP, MEL-14 mAb reduced binding of naive lym- expressed PNAd alone. Thus, NALT HEV express a unique ad- phocytes to HEV by ϳ30% compared with the control value ( p Ͻ dressin profile, one that differs from the strictly peripheral pheno- 0.01), whereas FIB 30 mAb showed a Ͼ90% reduction in naive The Journal of Immunology 1385

FIGURE 1. A–F, Murine NALT HEV display an addressin phenotype different from PP, MLN, and PLN. Fixed tissue sections were treated with MECA 367 (rat anti-mouse MAdCAM-1 mAb) and TRITC-conjugated goat anti-rat IgG (red color) and FITC-conjugated MECA 79 (rat anti-mouse PNAd mAb; green color). Results are shown at ϫ200 magniﬁca- tion, and images were captured using a Spot dig- ital imaging system (Diagnostic Instruments, Sterling Heights, MI). Red and green images were overlaid to produce the double-positive images shown in A–D. Negative isotype controls are shown for the NALT tissue, where the MECA 367 mAb and TRITC-goat anti-rat IgG Ab was followed by treatment with an FITC- labeled irrelevant IgM isotype control (E)oran

IgG2a isotype control, and TRITC-goat anti-rat Downloaded from IgG Ab was followed by treatment with FITC- MECA 79 mAb (F). As shown, the murine NALT contained two types of HEV: those expressing MAdCAM-1 and PNAd, and those expressing PNAd only. Relative percentages of the expressed addressins are quantiﬁed in Table I.

G–V, NALT FDC display both MAdCAM-1 and http://www.jimmunol.org/ VCAM-1. PP, MLN, NALT, and PLN serially cut frozen sections were treated with anti-B220 mAb to visualize the B cell areas (G–J) with an AEC substrate (red color). Results are shown at ϫ200 magniﬁcation. The same areas were then observed for diffuse staining with MECA 367 (K–N) or anti-VCAM-1 (O–R) mAbs. Black arrows denote HEV staining, whereas yellow arrows indicate staining of FDC. NALT FDC by guest on October 1, 2021 displayed diffuse staining of MAdCAM-1 and dark staining of VCAM-1. The speciﬁcity of staining is shown by treatment of the tissues with an irrelevant IgG2a isotype control (S–V).

lymphocyte binding ( p Ͻ 0.0001). In the MLN, naive lympho- results were consistent with the NALT phenotype, in which all cytes exhibit characteristics of binding to both mucosal and pe- HEV coexpress PNAd, unlike PP HEV. NALT HEV mediated ripheral addressin, evidenced by an ϳ80% reduction in binding by initial naive lymphocyte binding through different addressin-recep- both FIB 30 and MEL-14 mAbs ( p Ͻ 0.01). These results suggest tor pairs than did PP HEV. Thus, NALT HEV expressed more of that the NALT HEV express an addressin phenotype distinct from a peripheral phenotype in both form and function. This preferential those of other characterized mucosal sites, and that naive lympho- lymphocyte binding to the NALT HEV via PNAd rather than cyte binding to NALT HEV is primarily mediated by L-selectin- MAdCAM-1 suggested that the L-selectin expression on NALT B ␣ ␤ PNAd interactions. However, 4 7-MAdCAM-1 interactions may and T cells might be different from that on lymphocytes in the PP. also play a role, as evidenced by the reduction in binding observed upon treatment of naive lymphocytes with FIB 30 mAb. These NALT lymphocytes express a distinct L-selectin profile Based upon the observation that naive lymphocyte binding to Table I. Percentages of MAdCAM-1, PNAd, and double positive HEV NALT HEV was primarily mediated by L-selectin-PNAd interac- in NALT, PP, MLN, and PLNa tions, it would be expected that NALT lymphocytes express profiles of L-selectin expression more similar to those of PLN than to Tissue n MAdCAM-1 Positive PNAd Positive Double Positive those of PP lymphocytes. Two-color flow cytometric analyses of lymphocytes isolated from NALT, PP, and PLN were performed to PP 147 52.4 Ϯ 6.4 0 47.6 Ϯ 6.4 ϩ Ϯ Ϯ Ϯ determine the expression of L-selectin on B cells, CD4 T cells, MLN 343 40.9 4.8 2.5 0.9 56.8 4.6 ϩ NALT 921 0 37.2 Ϯ 5.0 62.8 Ϯ 5.0 and CD8 T cells (Fig. 4). As expected for naive lymphocytes, PLN 209 0 95.5 Ϯ 2.0 4.5 Ϯ 2.0 nearly all the NALT B and T cells expressed L-selectin, similar to ϩ a Ϯ the L-selectin populations observed in the PLN. This result dif- Mean values SEM are expressed as the percentage of HEV expressing MAd- ϩ CAM-1, PNAd, or double positive/total number of HEV per node. fered from that in the PP, where a significant portion of the B220 1386 MURINE NALT EXPRESSES A UNIQUE ADDRESSIN PHENOTYPE

FIGURE 2. An anti-PNAd Ab blocks initial naive lymphocyte binding FIGURE 3. An anti-L-selectin Ab blocks initial naive lymphocyte bind- to NALT HEV. Fresh tissue sections were treated with 50 ␮g/ml MECA 79 7 ing to NALT HEV. MLN lymphocytes (10 /ml) were treated with 50 Downloaded from (anti-PNAd) or MECA 367 (anti-MAdCAM-1) mAbs or with 50 ␮g/ml ␮g/ml of purified rat anti-mouse MEL-14 (anti-L-selectin) mAb, FIB 30 purified rat IgG2a or IgM isotype control for 1 h before the addition of ␣ ␤ (anti- 4 7) mAb, or purified rat IgG2a isotype control for 30 min before MLN lymphocytes. The average number of lymphocytes binding per HEV their addition to the tissue sections. The average number of lymphocytes in each node was determined. Lymphocyte binding is expressed as a per- bound per HEV in each node was determined. Ten to twenty HEV per node centage of the number of cells bound per HEV in the presence of a non- were counted in each tissue type, and an average of 6.5 lymphocytes were specific, isotype-matched control Ab. Ten to twenty HEV per node were bound per HEV in the isotype control. Lymphocyte binding is expressed as counted in each tissue type, and an average of 7.5 lymphocytes were bound a percentage of the number of cells bound per HEV with treatment of a http://www.jimmunol.org/ per HEV in the isotype control. MECA 79 mAb blocked Ͼ60% naive nonspecific isotype-matched Ab. MEL-14 mAb blocked Ͼ90% of naive lymphocyte binding to both NALT and PLN HEV, but not to PP HEV. The lymphocyte binding to NALT HEV, but had a negligible effect on naive p Ͻ 0.01. lymphocyte binding to PP HEV. The indicated values represent the mean ,ء .indicated values are the mean of three experiments Ϯ SEM .p Ͻ 0.0001 ,ءء ;p Ͻ 0.01 ,ء .of three experiments Ϯ SEM and CD4ϩ lymphocytes were L-selectin negative (Fig. 4 and Table Discussion II). The NALT L-selectin-positive lymphocyte homing receptor We have shown that murine NALT HEV express a distinct ad- profile appeared to differ greatly from that in the PP, and these data by guest on October 1, 2021 dressin phenotype. All NALT HEV expressed either PNAd alone substantiated the results of the ex vivo binding assays, where initial or coexpressed PNAd with MAdCAM-1. The addressin profile binding was mediated primarily through L-selectin interactions expressed by NALT HEV is considerably different from that ex- with PNAd. pressed by the HEV of the gut mucosal inductive tissue, the PP. In addition, intense staining was obtained in the FDC of NALT with Segregation of double-positive and PNAd-positive NALT HEV in the mAb against VCAM-1. Together, these results suggest that B and T cell areas although NALT behaves as a mucosal inductive site like the PP, it displays a distinct addressin profile, much different from the PP Although van der Ven and Sminia had observed HEV within the expression of MAdCAM-1. T cell areas of murine NALT (2), the expressed mucosal and Our results show that naive lymphocytes from nonimmunized peripheral addressins had not yet been fully characterized. It mice bind to the NALT HEV using a peripheral addressin homing was therefore unknown whether the various HEV phenotypes ␣ ␤ receptor pair. The mucosal ligand-addressin 4 7-MAdCAM-1 in- unique to the NALT segregated to specific B cell zones or T cell teractions have been shown to mediate both naive and memory areas within the tissue. To determine whether the location of HEV lymphocyte homing to the PP and extralymphoid mucosal sites within the NALT affected the type of addressin expressed, a se- such as the gut lamina propria (27, 37). L-selectin can also play a quential double immunoperoxidase staining was performed. Stain- role in naive lymphocyte trafficking into the PP (26), and this can ing of murine NALT with anti-B220 and anti-CD3 mAbs showed be observed in the results of the ex vivo binding assay, where clearly defined B and T cell regions. HEV were localized to both treatment of lymphocytes with MEL-14 mAb reduces binding to the T and B cell regions (Table III); 39.7% of HEV that expressed PP HEV (Fig. 3). Almost all naive lymphocytes display L-selectin PNAd were located in the B cell areas, whereas the remaining on their cell surface and thus are able to traffic throughout many ϩ PNAd HEV (58.6%) were located within the T cell areas. No different lymphoid sites in the body through interactions with significant difference was observed for the distribution of NALT PNAd. NALT HEV may express PNAd to facilitate naive lym- HEV coexpressing PNAd and MAdCAM-1 in the B and T cell phocyte trafficking through the nasal passages. Thus, naive lym- areas. In addition, our evidence suggests that these double-positive phocytes can readily circulate through the lymphoid tissue that first HEV were more likely to be located within the B cell areas of the encounters nasally introduced Ag. NALT, than were the HEV expressing PNAd alone ( p ϭ 0.05). However, this does not readily explain why all NALT HEV However, compared with the PP, in which only 16.2% of double- express PNAd. In this respect, NALT more resembles a PLN than positive HEV were located in the B cell areas, a significantly a PP. Several experiments have shown that lymphocyte homing to greater number of NALT double-positive HEV were located the upper airways of humans and sheep does not appear to be Ͻ ␣ ␤ within the B cell area ( p 0.001; Table III). mediated primarily by 4 7-MAdCAM-1 interaction, (38–40). The Journal of Immunology 1387 Downloaded from http://www.jimmunol.org/

FIGURE 4. Expression proﬁles of L-selectin on NALT lymphocytes more closely resemble PLN. NALT, PP, MLN, or PLN lymphocytes were treated simultaneously with FITC-conjugated anti-B220, anti-CD4, or anti-CD8 mAbs and PE-conjugated anti-L-selectin (MEL-14) mAb. Representative plots are shown for each tissue. As depicted, the majority of the NALT and PLN lymphocytes are L-selectinϩ, in contrast to the PP, which contained both L-selectinϩ and L-selectinϪ lymphocyte populations.

␣ ␤ Table II. Comparison of levels of expression of L-selectin on NALT, that 4 7–MAdCAM-1 interactions do not play a major role in PP, PLN, and MLN B and T lymphocytesa lymphocyte trafﬁcking into the lung or pulmonary tissues (39–42), but do provide a mechanism for protective immunity against gut by guest on October 1, 2021 PPb MLNc NALTb PLNb pathogens (43). Therefore, it has been suggested that this ad-

ϩ dressin/homing receptor pair should be regarded as intestinal B220 71.2 Ϯ 3.7 87.0 Ϯ 2.3 86.7 Ϯ 2.6 96.9 Ϯ 0.4 CD4ϩ 60.2 Ϯ 2.9 88.7 Ϯ 0.6 89.3 Ϯ 1.5 94.8 Ϯ 0.8 rather than mucosal (44). However, our studies in the mouse have ϩ ␣ ␤ CD8 62.7 Ϯ 8.5 92.9 Ϯ 0.2 87.2 Ϯ 4.5 94.3 Ϯ 1.6 shown that 4 7-MAdCAM-1 interactions may appear to play

ϩ ϩ ϩ some role in the recruitment of lymphocytes to the nasal tissues a Percentage of NALT, PP, PLN, and MLN B220 , CD4 , and CD8 lymphocytes expressing L-selectin. and may thus provide a method of dissemination of Ab-producing b Mean values Ϯ SEM from nine experiments. cells to distal mucosal effector sites, including the intestinal and c Ϯ Mean values SEM from three experiments. reproductive tracts. ␣ ␤ 4 7-MAdCAM-1 interactions may also play a role in the initial Also, MECA 79 mAb has been shown to block naive lymphocyte binding of naive lymphocytes to NALT HEV. This is evidenced by ␤ binding to HEV of human tonsils (41). I.n. immunization induces a 60% reduction in binding in lymphocytes treated with the anti- 7 ␣ ␤ B cells that have both L-selectin and 4 7 on their cell surface, in FIB 30 mAb (Fig. 3). These data may suggest a mechanism for the ␣ ␤ contrast to enteric immunization, which induces mainly 4 7-expressing B cells (42). In addition, i.n. immunization has been more effective than intragastric immunization at producing protective Table III. Locations of Double and Single Positive HEV within NALT a immune responses in the lower respiratory tract (18, 19). This may and PP reflect a specific homing pathway of effector cells induced in the NALT rather than the PP. It appears as though L-selectin-PNAd B Cell Areab T Cell Areab interactions may be most important for lymphocyte trafficking into NALT HEV the respiratory tract, and this conclusion is reflective of the ad- Double positive 57.4 Ϯ 8.3 40.9 Ϯ 7.9 dressin profile of the NALT HEV. Because the respiratory tract PNAd positive 39.7 Ϯ 3.8 58.6 Ϯ 3.7 contains both mucosal (upper airways and bronchi) and systemic PP HEV Double positive 16.2 Ϯ 4.4 74.5 Ϯ 8.0 (alveolar spaces) compartments, the coexpression of a peripheral MAdCAM-1 positive 19.1 Ϯ 4.5 81.0 Ϯ 4.5 phenotype with the mucosal addressin may enable trafficking of a lymphocytes that facilitate both systemic and mucosal immune The number of HEV expressing both MAdCAM-1 and PNAd (double positive) or PNAd or MAdCAM-1 alone in the B and T cell areas was determined. The number responses. of HEV expressing either PNAd or MAdCAM-1 (indicated with DAB substrate) and The presence of MAdCAM-1 as well as VCAM-1 and PNAd their relative locations in the B cell zones or T cell areas (indicated with VIP substrate) in the NALT were determined. The number of HEV/phenotype/area was di- within the NALT presents interesting implications for the theory of vided by the total number of HEV/phenotype. the common mucosal immune system. Recent studies have shown b The values represent the mean of 12 sections Ϯ SEM. 1388 MURINE NALT EXPRESSES A UNIQUE ADDRESSIN PHENOTYPE entrance of intestinal lymphocytes, which have been shown to where T and B cells activated in the NALT are able to preferen- ␣ ␤ preferentially express 4 7 and enter lymphoid tissue primarily tially circulate to alternate mucosal tissues. ␣ ␤ through MAdCAM-1- 4 7 interactions (26, 28). Alternatively, the ␣ ␤ 4 7–MAdCAM-1 interaction may play a significant role in tight Acknowledgments adhesion of lymphocytes to NALT HEV. In the three-step model We thank Dr. E. C. Butcher for his critical review of this manuscript as of lymphocyte homing, the initial binding of lymphocytes to well as for his gifts of the MECA 79, MECA 367, and FIB 30 mAbs. We NALT HEV may be primarily mediated by L-selectin-PNAd in- also thank Gayle Callis for her assistance with the immunohistochemical teractions, whereas subsequent tight binding might be mediated studies. ␣ ␤ primarily by MAdCAM-1- 4 7 interactions (26, 27). ␣ ␤ The 4 7-MAdCAM-1 interaction also may play a significant References role in the trafficking of memory lymphocytes to NALT. Some 1. Spit, B. J., E. G. J. Hendriksen, J. P. Bruijntjes, and C. F. Kuper. 1989. Nasal ␣ ␤ memory lymphocytes preferentially express 4 7 and home to lymphoid tissue in the rat. Cell Tissue Res. 255:193. MAdCAM-1 in the mucosal PP and lamina propria (45, 46). In 2. van der Ven, I., and T. Sminia. 1993. The development and structure of mouse nasal associated lymphoid tissue: an immuno- and enzyme-histochemical study. addition, MAdCAM-1 has been shown to be up-regulated in in- Regul. Immunol. 5:69. flamed mucosal tissue (29, 47, 48). It has yet to be determined, 3. Goeringer, G. C., and B. Vidic. 1987. The embryogenesis and anatomy of however, whether MAdCAM-1 is up-regulated on the HEV of Waldeyer’s ring. Otol. Clin. North Am. 20:207. 4. Wu, H.-Y., and Russell, M.W. 1993. Induction of mucosal immunity by intra- inflamed NALT and if such an up-regulation would result in a nasal application of a streptococcal surface protein antigen with the cholera toxin higher percentage of lymphocytes binding through ␣ ␤ B subunit. Infect. Immun. 61:314. 4 7 5. Asanuma, H., A. H. Thompson, T. Iwasaki, Y. Sato, Y. Inaba, C. Aizawa, interaction. T. Kurata, and S. Tamura. 1997. Isolation and characterization of mouse nasal- Downloaded from Another method for naive lymphocyte recruitment and/or reten- associated lymphoid tissue. J. Immunol. Methods 202:123. tion in NALT is suggested by the expression of MAdCAM-1 and 6. Heritage, P. L., B. J. Underdown, A. L. Arsenault, D. P. Snider, and M. R. McDermott. 1997. Comparison of murine nasal-associated lymphoid tissue VCAM-1 by the FDC in the NALT. MAdCAM-1 expression on and Peyer’s patches. Am. J. Respir. Crit. Care Med. 156:1256. PP FDC has been recently characterized (49). MAdCAM-1 ex- 7. Gallichan, W. S., and K. L. Rosenthal. 1995. Specific secretory immune response ␣ ␤ ϩ in the female genital tract following intranasal immunization with a recombinant pression within the PP follicles could mediate binding by 4 7 adenovirus expressing glycoprotein B of herpes simplex virus. Vaccine 13:1589. memory lymphocytes, whereas binding to the follicles of PLN 8. Langermann, S., S. Palaszynski, A. Sadziene, C. K. Stover, and S. Koenig. 1994. ␣ ␤ Systemic and mucosal immunity induced by BCG vector expressing outer-sur- http://www.jimmunol.org/ appeared to be primarily mediated by VCAM-1- 4 1 interaction. ␣ face protein A of Borrelia burgdorferi. Nature 372:552. It has also been shown (50) that VCAM-1 interactions with the 4 9. van Ginkel, F. W., C. Liu, J. W. Simecka, J.-Y. Dong, T. Greenway, subunit can mediate binding to the germinal centers of human R. A. Frizzell, H. Kiyono, J. R. McGhee, and D. W. Pascual. 1995. Intratracheal ␣ ␤ ␣ ␤ gene delivery with adenoviral vector induces elevated systemic IgG and mucosal tonsil. However, VCAM-1 also binds both 4 7- as well as 4 1- IgA antibodies to adenovirus and ␤-galactosidase. Hum. Gene Ther. 6:895. ␣ ␤ ϩ expressing lymphocytes (51), suggesting that the 4 7 lympho- 10. Tamura, S., T. Iwasaki, A. H. Thompson, H. Asanuma, Z. Chen, Y. Suzuki, cytes might bind in the germinal centers through this route as well. C. Aizawa, and T. Kurata. 1998. Antibody-forming cells in the nasal-associated lymphoid tissue during primary influenza virus infection. J. Gen. Virol. 79:291. In addition, adhesion to VCAM-1 may prevent apoptosis and allow 11. VanCott, T. C., R. W. Kaminski, J. R. Mascola, V. S. Kalyanaraman, positive selection of B cells within the follicle (52). Although bind- N. M. Wassef, C. R. Alving, J. T. Ulrich, G. H. Lowell, and D. L. Birx. 1998. HIV-1 neutralizing antibodies in the genital and respiratory tracts of mice intra- by guest on October 1, 2021 ing studies of lymphocytes to the NALT follicles have yet to be nasally immunized with oligomeric gp160. J. Immunol. 160:2000. undertaken, intense staining with anti-VCAM-1 mAb in the NALT 12. Wu, H.-Y., E. B. Nikolova, K. W. Beagley, and M. W. Russell. 1996. Induction suggests that lymphocytes expressing both ␣ ␤ and ␣ ␤ inte- of antibody secreting cells and T-helper and memory cells in murine nasal lym- 4 7 4 1 phoid tissue. Immunology 88:493. grins are able to traffic into these germinal centers, where they then 13. Bergquist, C., E.-L. Johansson, T. Lagergard, J. Holmgren, and A. Rudin. 1997. mature and differentiate. Intranasal vaccination of humans with recombinant cholera toxin B subunit induces systemic and local antibody responses in the upper respiratory tract and the Finally, our results indicated that compared with PP, a greater vagina. Infect. Immun. 65:2676. percentage of the NALT HEV were located within B cell areas. It 14. Heritage, P. L., M. A. Brook, B. J. Underdown, and M. R. McDermott. 1998. appeared as though the double-positive HEV would more likely be Intranasal immunization with polymer-grafted microparticles activates the nasal- associated lymphoid tissue and draining lymph nodes. Immunology 93:249. located within the B cell areas of the NALT. The reason for this 15. Ennis, F. A., Q. Yi-Hua, and G. C. Schild. 1982. Antibody and cytotoxic T cell apparent localization of the MAdCAM-1ϩ HEV is unclear, but this responses of humans to live and inactivated influenza vaccines. J. Gen. Virol. ␣ ␤ ϩ 58:273. localization of HEV that can support binding of 4 7 in the B 16. Gallichan, W. S., and K. L. Rosenthal. 1996. Long-lived cytotoxic T lymphocyte cell areas might provide a new mechanism for B cell localization. memory in mucosal tissues after mucosal but not systemic immunization. J. Exp. Rather than entering the lymphoid tissue in the paracortex and Med. 184:1879. 17. Porgador, A., H. F. Staats, B. Faiola, E. Gilboa, and T. J. Parker. 1997. Intranasal migrating to the B cell areas in response to chemotactic factors, as immunization with CTL epitope peptides from HIV-1 or ovalbumin and the mu- has been recently reported (53), these lymphocytes might simply cosal adjuvant cholera toxin induces peptide specific CTLs and protection against enter the B cell areas directly, where MAdCAM-1 and VCAM-1 tumor development in vivo. J. Immunol. 158:834. 18. Kanesaki, T., B. R. Murphy, P. L. Collins, and P. L. Ogra. 1991. Effectiveness of expression on dendritic cells might also enable B cell survival and enteric immunization in the development of secretory immunoglobulin A re- differentiation. However, if T lymphocytes enter these B cell areas sponse and the outcome of infection with respiratory syncytial virus. J. Virol. 65:657. as well, some type of migration would still occur. 19. Shahin, R. D., D. F. Amsbaugh, and M. F. Leef. 1992. Mucosal immunization In summary, characterization of addressin expression revealed with filamentous hemagglutinin protects against Bordetella pertussis respiratory that NALT HEV were phenotypically and functionally distinct. infection. Infect. Immun. 60:1482. 20. Gallichan, W. S., D. C. Johnson, F. L. Graham, and K. L. Rosenthal. 1993. This addressin profile was demonstrated to be important for initial Mucosal immunity and protection after intranasal immunization with recombi- naive lymphocyte binding mediated by PNAd. Lymphocytes iso- nant adenovirus expressing herpes simplex virus glycoprotein B. J. Infect. Dis. 168:622. lated from the NALT also displayed a homing receptor profile that 21. Staats, H. F., W. G. Nichols, and T. J. Parker. 1996. Mucosal immunity to HIV-1: more resembled lymphocytes derived from peripheral tissues. Fi- systemic and vaginal antibody responses after intranasal immunization with the nally, the location of the HEV within the NALT differed dramat- HIV-1 C4/V3 peptide T1SP10 MN(A). J. Immunol. 157:462. 22. Hordnes, K., I. Tynning, T. A. Brown, B. Haneberg, and R. Jonsson. 1997. Nasal ically from that observed in the PP. Collectively, these results sug- immunization with group B streptococci can induce high levels of specific IgA gest that NALT is a truly unique inductive mucosal tissue that antibodies in cervicovaginal secretions of mice. Vaccine 15:1244. 23. Kuklin, N., M. Daheshia, K. Karem, E. Manickan, and B. T. Rouse. 1997. In- cannot be equated with the intestinal PP. 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