B -Derived IL-16 Attracts Dendritic Cells and Th Cells Arthur Kaser, Stefan Dunzendorfer, Felix A. Offner, Othmar Ludwiczek, Barbara Enrich, Robert O. Koch, William W. This information is current as Cruikshank, Christian J. Wiedermann and Herbert Tilg of September 30, 2021. J Immunol 2000; 165:2474-2480; ; doi: 10.4049/jimmunol.165.5.2474 http://www.jimmunol.org/content/165/5/2474 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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. B Lymphocyte-Derived IL-16 Attracts Dendritic Cells and Th Cells1

Arthur Kaser,* Stefan Dunzendorfer,† Felix A. Offner,‡ Othmar Ludwiczek,* Barbara Enrich,* Robert O. Koch,* William W. Cruikshank,§ Christian J. Wiedermann,† and Herbert Tilg2*

Interaction of B with Th cells is a fundamental step in the establishment of humoral immunity, and recent evidence suggests that direct interaction between B lymphocytes and dendritic cells (DCs) is also an important prerequisite. Factors involved in the selective recruitment of Th cells and DCs by B lymphocytes are insufficiently defined. We set out to delineate the role of IL-16, the soluble ligand of CD4, which is expressed on Th cells and DCs. B lymphocytes express IL-16 mRNA and synthesize bioactive IL-16 , and IL-16 is expressed in lymph node follicles in situ. B lymphocyte supernatant efficiently .induces migration of CD4؉ Th cells, -derived DCs, and circulating blood DCs in nitrocellulose filter-based assays

Neutralization of IL-16 bioactivity strongly inhibits this migratory response, suggesting that IL-16 might be a major chemotactic Downloaded from factor derived from B cells. The present data further support the idea that IL-16 might have a role in the initiation of cellular as well as humoral immunity by mediating the cellular cross-talk among T lymphocytes, B cells, and DCs, leading to recruitment of these cell types at common anatomical sites. The Journal of Immunology, 2000, 165: 2474–2480.

nterleukin-16 is a polypeptide that was originally latory signals to B cells, resulting in their activation, proliferation,

described as a -specific chemoattractant factor (1). It is and ultimately in Ab formation (8). Much is known about T cell http://www.jimmunol.org/ I synthesized by CD4ϩ and CD8ϩ T cells and is released in trafficking in lymphoid tissue, but few reports have focused on response to Ag, mitogen, histamine, and serotonin (1); further- factors directing the chemoattraction of T lymphocytes toward B more, IL-16 is produced by , mast cells, airway epithe- lymphocytes and vice versa (9). Recent reports suggest an impor- lial cells of asthmatics, keratinocytes, and monocyte-derived den- tant role of direct B cell/DC interaction in B cell proliferation and dritic cells (DCs)3 (1, 2). -3 cleaves pro-IL-16, and the isotype switching (10–16). Because chemoattraction of CD4ϩ 20-kDa IL-16 released is biologically active as a multimer (3, 4). cells is a fundamental property of IL-16, we investigated whether As the natural soluble ligand for CD4, it has been shown to induce this cytokine is derived from B cells and whether it might have a in CD4ϩ T cells as well as in , eosinophils, role in the attraction of CD4ϩ Th lymphocytes and CD4ϩ DCs. and monocyte-derived DCs (1, 2). Besides its chemotactic prop- by guest on September 30, 2021 erties IL-16 induces the expression of IL-2R␣ (CD25) and acti- ϩ Materials and Methods vates CD4 T cells synergistically with IL-2 and IL-15 (5, 6). In Media, reagents, and Abs contrast, preincubation of CD4ϩ T cells with IL-16 reduces the proliferative response to CD3/TCR ligation, probably via steric The culture medium (CM) used in this study was RPMI 1640 (Schoeller Pharma, Vienna, Austria) supplemented with 10% heat-inactivated (30 interaction with CD4-MHC binding (7). min, 56°C) FCS (Life Technologies, Vienna, Austria), 100 U/ml penicillin Foreign Ag is captured by DCs, processed to antigenic peptides, G, and 100 ␮g/ml streptomycin (Schoeller Pharma). Recombinant human and presented to CD4ϩ and CD8ϩ T lymphocytes in the groves of (rhu) IL-4 and rhuIL-10 were supplied by Schering-Plough Research In- MHC II and MHC I molecules, respectively. Subsequently, CD4ϩ stitute (Kenilworth, NJ), and rhuIL-6 was purchased from PeproTech (Lon- don, U.K.). Recombinant human IL-16 was obtained from PharMingen Th cells migrate to the edge of the B cell-rich follicles in lymphoid (Hamburg, Germany), as were mAbs directed against CD3 (HIT3a), CD4 organs, recognize Ag presented in the grove of MHC II on B (RPA-T4), CD14 (M5E2), CD19 (B43) and CD56 (B159), CD69 (FN50), lymphocytes by virtue of their TCR, and provide several costimu- and IL-16 (14.1). Biotinylated polyclonal Abs (pAbs) against IL-16 were purchased from R&D Systems (Minneapolis, MN; for Western analysis and ELISA) and PharMingen (for intracellular FACS staining). Anti-CD20 *Division of Gastroenterology and Hepatology and †Division of General Internal mAb was purchased from Dako (Carpinteria, CA). Mouse IgG1 and mouse Medicine, Department of Medicine, and ‡Department of Pathology, University Hos- IgG2a were obtained from Sigma (Vienna, Austria). Biotinylated rabbit pital Innsbruck, Innsbruck, Austria; and §Pulmonary Center, Boston University IgG was obtained from Pierce (Biotrade, Vienna, Austria), FITC-anti- School of Medicine, Boston, MA 01226 mouse IgG, human IgG, rabbit IgG, and saponin were purchased from Received for publication April 28, 2000. Accepted for publication June 14, 2000. Sigma, and streptavidin-PE was obtained from Becton Dickinson (Vienna, Austria). Recombinant human monocyte chemoattractant protein-3 The costs of publication of this article were defrayed in part by the payment of page (MCP-3) and rhuRANTES were obtained from R&D Systems. PMA and charges. This article must therefore be hereby marked advertisement in accordance LPS (Escherichia coli 055:B5) were purchased from Sigma. Ionomycin with 18 U.S.C. Section 1734 solely to indicate this fact. and Staphylococcus aureus Cowan I strain (SACS) were obtained from 1 This work was supported by Grants P12790 (to H.T.) and P09977 (to C.J.W.) from Calbiochem (Biotrade). the Austrian Science Fund and National Institutes of Health Grant AI35680 (to W.W.C.). Cell culture 2 Address correspondence and reprint requests to Dr. Herbert Tilg, Department of Medicine, University Hospital Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria. PBMC were obtained by density gradient centrifugation (Histopaque 1077, E-mail address: [email protected] Sigma) of buffy coats obtained from the local blood bank. PBMC were 3 Abbreviations used in this paper: DC, dendritic cells; CM, complete medium; CI, washed and incubated for 45 min at 4°C with an appropriate amount of magnetic beads coated with anti-CD19 (Dynabeads, Dynal, Oslo, Norway). chemotaxis index; GC, germinal center; rhu, recombinant human; MCP-3, monocyte ϩ chemoattractant protein-3; pAb, polyclonal Ab; SACS, Staphylococcus aureus After positive selection of CD19 cells, Dynabeads were detached by ad- Cowan I strain. dition of Detach-A-Bead and further incubation for 45 min. Subsequently,

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 2475 pure B cells (Ͼ99%, as determined by cytofluorometry) were intensely at a 1/1500 dilution for 60 min at 22°C. After washing several times with washed and cultured at a density of 1 ϫ 106 (for ELISA experiments) or the above-described buffer, the blots were developed in 100 mM Tris-HCl ϫ 7 1 10 (for Northern and Western experiments)/ml in CM for the indi- (pH 9.5), 100 mM NaCl, 5 mM MgCl2, 0.033% nitro blue tetrazolium, and cated period of time. 0.017% 5-bromo-4-chloro-3-indolyl phosphate. For the generation of B cell supernatant for chemotaxis assays, 1 ϫ 106 purified B cells/ml were cultured in RPMI 1640/0.5% BSA as follows: 1) ELISA for pro-IL-16 and cleaved IL-16 for 24 h without addition of activating agents; 2) for 12 h on anti-CD40 mAb-coated (2 ␮g/ml, overnight) 48-well plates and addition of IL-4 (1000 Rabbit pAb generated to recombinant pro-IL-16 was used to establish an U/ml), subsequent intense washing, and further culture for 24 h without affinity column on cyanogen bromide-coupled Sepharose 4B beads. Super- activating agents; and 3) for 24 h with addition of anti-IgM-coated mi- natants were incubated on the column for1hatroom temperature before crobeads (5 ␮g/ml; Irvine Scientific, Santa Ana, CA) (17). elution with PBS. Cleaved, mature IL-16 would be eluted, whereas pro- Purified CD4ϩ T lymphocytes were obtained by depletion of monocytes IL-16 (cleaved and uncleaved) would remain on the column. ELISA read- by plastic adherence, followed by incubation with anti-CD14, anti-CD16, ings were taken before and after column chromatography. The efficiency of anti-CD19, and anti-CD56 mAbs. Ab-binding cells were removed by the column was checked by applying T cell lysates, and for these samples means of anti-mouse Ig-coated magnetic beads (Dynabeads M-450). In the 75–80% of the total IL-16 was pro-IL-16. A sandwich ELISA for IL-16 next step, CD8ϩ T lymphocytes were depleted by CD8-coated Dynabeads was performed according to standard protocols with immobilized anti- as recommended by the manufacturer. The resulting CD4ϩ Th cell popu- IL-16 14.1 mAb (3 ␮g/ml in carbonate buffer) and biotinylated IL-16 pAb lation had a purity of Ͼ95% as determined by cytofluorometry. (200 ng/ml; R&D Systems) as detection Ab. The secondary reagent, CD8ϩ T lymphocytes were obtained as described above for CD19ϩ B streptavidin-peroxidase (Roche), was used according to the manufacturer’s cells, with anti-CD8-coated magnetic beads instead of anti-CD19 beads. instructions. Tetramethylbenzidine (BM Blue POD substrate, Roche) was Purity was Ͼ99% as determined by FACS analysis. used as substrate and measured at 450 nm in an ELISA reader. The stan- Monocyte-derived DCs were generated from PBMC as previously de- dard curve was prepared with rhuIL-16 in a concentration range from 15.63 7 scribed (18, 19). In brief, PBMC were resuspended in CM, and 5 ϫ 10 to 2000 pg/ml. Downloaded from PBMCs were allowed to adhere in 75-cm2 cell culture flasks for 45 min in a 37°C humidified 5% CO2 atmosphere. Nonadherent cells were removed, Cytofluorometric analysis of B lymphocytes and adherent cells were cultured in 10 ml of CM supplemented with 1 ϫ PBMC (1 ϫ 106) were washed in PBS/2% FCS, resuspended in 250 ␮g/ml 103 U/ml IL-4 and 1 ϫ 103 U/ml GM-CSF. On day 2, 5 ml of CM sup- hIgG/PBS/2% FCS, and incubated for 20 min at 4°C. After pelleting, plemented with IL-4 and GM-CSF (both 1 ϫ 103 U/ml), and on day 5, 5 PBMC were incubated with anti-CD19 at 10 ␮g/ml for 30 min at 4°C. ml of CM supplemented with 2 ϫ 103 U/ml IL-4 and 2 ϫ 103 U/ml After washing in PBS/2% FCS, a 1/40 dilution of FITC-anti-mouse IgG in GM-CSF were added. On day 6 of culture, DCs were harvested and puri- PBS/2% FCS was incubated for 30 min at 4°C. Subsequently, PBMC were http://www.jimmunol.org/ fied by magnetic cell separation, employing magnetic beads as previously washed twice in PBS and resuspended in 2% formaldehyde/PBS for 10 described (2). The resulting DC population had a purity of Ͼ97% as de- min, washed once in PBS, and incubated with 0.1% saponin/10% goat termined by cytofluorometry and CD1a, CD3, CD14, CD19, CD56, and serum/250 ␮g/ml rabbit IgG/PBS for 10 min at room temperature for per- CD40 staining. Following this procedure, DCs were subjected to chemo- meabilization. After pelleting, 10 ␮g/ml biotinylated IL-16 pAb or the taxis assay. respective biotinylated control Ig in PBS/2% FCS/0.1% saponin was in- Purification of circulating blood DCs was essentially performed as pre- cubated for 30 min at room temperature, followed by washing in PBS/2% viously described (20). In brief, PBMC were depleted of T cells, NK cells, FCS/0.1% saponin. Subsequently, PBMC were incubated with a 1/25 di- and monocytic cells by means of immunomagnetic beads directed against lution of streptavidin-PE in PBS/2% FCS/0.1% saponin for 30 min at room CD3, CD11b, and CD16 following the manufacturer’s instructions (Mil- temperature, washed twice in PBS/2% FCS, and immediately analyzed on tenyi Biotec, Bergisch Gladbach, Germany). Subsequently, cells were fur- a FACScan (Becton Dickinson, Mountain View, CA). For analysis of the ther depleted of B cells by CD19 beads (Miltenyi Biotec). The resulting by guest on September 30, 2021 activation marker CD69, purified B cells were blocked with hIgG as de- cell population was subjected to chemotaxis assay. A purity of Ͼ95% was scribed above, followed by anti-CD69-FITC (10 ␮g/ml) or IgG1-FITC (10 ascertained by cytofluorometry and staining for CD3, CD14, CD19, CD56, ␮g/ml), washed, and analyzed. Data analysis was performed with and HLA-DR. CellQuest software (Becton Dickinson). Northern analysis IL-16 immunohistochemistry Total RNA was prepared from 1 ϫ 107 purified B cells either immediately after positive selection from PBMC or alternatively from B lymphocytes Tissue sections of human tonsils were acquired from excess tissue obtained cultured with the indicated agents for 5 h. Ten micrograms of total RNA during elective surgery. Serial sections were fixed in 2% paraformaldehyde per lane was gel-electrophoresed and blotted onto nylon membranes as in Tris buffer for 8 min at 4°C. Sections were incubated overnight at 4°C recently described (21). The IL-16 probe was a 909-bp PstI fragment span- with biotinylated IL-16 pAb (dilution 1/50 in 1% BSA/0.1% saponin in ning nucleotides 930-1839 (GenBank M90391). The probe was radioac- Tris buffer). Blocking of specific binding sites of the pAb was performed tively labeled with [32P]dCTP employing the random primed labeling with 10 ␮g/ml rhuIL-16. Subsequently, streptavidin-peroxidase was added method according to the manufacturer’s (Roche, Vienna, Austria) instruc- at a dilution of 1/800 and incubated for 30 min, and the signal was devel- tions and hybridized as previously described (21). Control hybridizations oped with 3,3Ј-diaminobenzidine (Sigma). Slides were counterstained with were performed with rat cDNA of the housekeeping GAPDH to en- hemalun. Identification of B lymphocytes was performed by anti-CD20 sure equal loading of RNA. mAb according to standard protocols. Western analysis Chemotaxis assay Cell extracts of 1 ϫ 107 purified B cells were prepared by lysis in RIPA Migration of cells into cellulose nitrate along gradients of soluble attract- buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, ants was measured using a 48-well microchemotaxis chamber (Neuro- 50 mM Tris-HCl (pH 8.0), 0.2 mM PMSF, 1 ␮g/ml pepstatin, and 0.5 probe, Bethesda, MD) in which a 5-␮m (for lymphocytes) or an 8-␮m (for ␮g/ml leupeptin) for 30 min at 4°C and subsequent centrifugation for 5 min DCs) pore size filter (Sartorius, Gottingen, Germany) separates the upper at 16,000 ϫ g. Protein content was estimated by a commercial Bradford and lower chamber. Thirty-microliter aliquots of chemoattractant solution assay (Bio-Rad, Vienna, Austria) with BSA standards, and 60 ␮g of cell or control medium (RPMI 1640/0.5% BSA) were added to the lower wells lysates were electrophoresed per lane. were transferred under con- of the chamber, then the filter was carefully layered onto the wells and stant cooling in 25 mM Tris, 192 mM glycine, and 10% (v/v) methanol covered with a silicon gasket and the top plate. Fifty microliters of cell from 10% polyacrylamide gel onto polyvinylidene difluoride membranes suspension (1ϫ106 cells/ml) were seeded in the upper chamber. Purified (Schleicher & Schuell, Vienna, Austria) for 60 min at a 100-V constant CD4ϩ T lymphocytes, CD8ϩ T lymphocytes, monocyte-derived DCs, and voltage. After protein transfer, the polyvinylidene difluoride membrane circulating blood DCs were allowed to migrate toward attractants in the was incubated with 10% skim milk powder, 0.5% (w/v) Triton X-100, and lower chambers for 2 h (CD4ϩ/CD8ϩ T lymphocytes) or 4 h (DCs) at 37°C

0.1% Tween 20 dissolved in 20 mM Tris-HCl (pH 7.4) and 150 mM NaCl in humidified atmosphere (5% CO2). After this incubation time, the nitro- for 12–18 h at 4°C. Subsequently, the immunoblot was incubated with 14.1 cellulose filters were dehydrated, fixed, and stained with hematoxylin-eo- anti-IL-16 mAb at a dilution of 1/2000 in the same buffer for 12 h at 22°C. sin. Migration depth of cells into the filter was quantified by microscopy, After washing three times with 0.5% (w/v) Triton X-100 and 0.1% Tween measuring the distance (microns) from the surface of the filter to the lead- 20 in 20 mM Tris-HCl (pH 7.4) and 150 mM NaCl, blots were incubated ing front of cells before any cells had reached the lower surface (leading with affinity-purified alkaline phosphatase-conjugated goat anti-mouse IgG front assay). Data are expressed as the chemotaxis index (CI), which is the 2476 B CELL-DERIVED IL-16 ATTRACTS Th CELLS AND DCs ratio between the distances of directed and undirected migration of DCs and T lymphocytes into the nitrocellulose filters.

Statistical analysis ELISA data and chemotaxis data are expressed as the mean Ϯ SEM. The significance of ELISA data was determined by Student’s t test for paired samples using the SPSS software package (SPSS, Chicago, IL). Means of the CI were compared by Kruskal-Wallis ANOVA and Mann-Whitney U test. A difference with p Ͻ 0.05 was considered significant. Calculation was performed using the StatView software package (Abacus Concepts, Berkeley, CA).

Results B lymphocytes are a source of IL-16 Highly purified B lymphocytes were obtained by positive selection of CD19ϩ cells employing magnetic beads. Obtained B cells were Ͼ99% pure as determined by cytofluorometry and staining for CD3, CD14, CD16, CD19, and CD56 (data not shown). B lym-

phocytes did not express the activation Ag CD69 either immedi- Downloaded from FIGURE 2. Western analysis. Sixty micrograms of cellular extracts de- ately after isolation or after 24 h of culture in medium alone (data ϩ not shown), thus showing that the isolation procedure does not rived from highly purified CD19 B lymphocytes were separated by SDS- induce cell activation. mRNA analysis revealed constitutive ex- PAGE, blotted, and probed with 14.1 anti-IL-16 mAb. Molecular mass standards (kilodaltons) are indicated by arrows. The blot shows pro-IL-16 pression of IL-16 in freshly isolated B cells (Fig. 1). Western anal- as an ϳ80-kDa band. Cleaved, ϳ20-kDa, bioactive IL-16 was not detected ysis of cell extracts derived from freshly isolated purified B lym- in cell lysates. The figure is representative of three independent phocytes showed a single band at ϳ80 kDa, representing experiments. http://www.jimmunol.org/ premature IL-16, but no band at ϳ20 kDa as would be expected for bioactive cleaved IL-16 (Fig. 2), suggesting that cleaved IL-16 is readily secreted, rather than stored, in B lymphocytes. Correspond- ing to these data, intracellular FACS analysis showed preformed B lymphocytes in tonsil lymphatic tissue express IL-16 protein IL-16 in the CD19ϩ subpopulation (i.e., B cells) of freshly isolated Immunohistochemistry was performed on serial tissue sections of PBMC (Fig. 3). Stimulation of purified B cells for 5 h with various tonsils obtained during elective surgery. B lymphocytes were iden- agents showed that IL-4, IFN-␥, and SACS down-regulate IL-16 tified by CD20 staining (Fig. 4A). IL-16 staining was most pro- mRNA expression. IL-10, IL-6, and anti-CD40 had no effect (Fig. nounced in the mantle zone and less intense in the germinal center 1). Twenty-four-hour culture of unstimulated purified B cells at a by guest on September 30, 2021 (GC; Fig. 4, B and C). Fig. 4D proves specificity of IL-16 staining density of 1ϫ106/ml resulted in IL-16 synthesis of 506 Ϯ 61 pg/ by preincubation of the IL-16 mAb with rhuIL-16. ml. Analysis of IL-16 protein synthesis after culture with various for various time periods did not show statistically sig- IL-16 is a major constituent of chemotactic activity of B cell nificant changes compared with unstimulated B cells, thus dem- supernatant toward CD4ϩ Th lymphocytes onstrating no correlation between IL-16 mRNA regulation and Supernatants of unstimulated B lymphocytes (1ϫ106/ml) and IL- IL-16 protein synthesis (Table I). ELISA performed after depletion 4/anti-CD40-stimulated B cells were tested for chemotactic activ- of supernatants from pro-IL-16 with anti-pro-IL-16 pAb by affinity ity toward CD4ϩ Th lymphocytes. Migration data are expressed as chromatography yielded identical results, showing that all IL-16 the depth of migration into nitrocellulose filters induced by B cell detected in the supernatant is bioactive, mature IL-16 (data not supernatant compared with migration induced by medium alone shown). In addition, a dose response for IL-16 chemotaxis was not (CI). Undiluted supernatant from unstimulated B cells resulted in altered by the column (data not shown). a CI of 1.615 Ϯ 0.077 (Fig. 5). Even a 1000-fold dilution of this supernatant produced significant CD4ϩ T cell migration (CI, 1.295 Ϯ 0,093; Fig. 5). To evaluate the relative contribution of

FIGURE 1. Northern analysis. Total RNA was extracted from purified CD19ϩ B lymphocytes immediately after isolation (lane 0) or from puri- fied B cells cultured with indicated agents for5h(lane 1, medium; lane 2, FIGURE 3. Intracellular FACS analysis. Freshly isolated PBMC were IL-4 (1000 U/ml); lane 3, IFN-␥ (100 U/ml); lane 4, IL-10 (100 U/ml); stained with anti-CD19 mAb detected by FITC-anti-mouse IgG, fixed in lane 5, IL-6 (100 U/ml); lane 6, anti-CD40 mAb (1 ␮g/ml); lane 7, SACS formaldehyde, permeabilized with saponin, intracellularly stained with bi- (1/5000)) and analyzed for IL-16 mRNA expression by Northern analysis. otinylated anti-IL-16 pAb (full line) or biotinylated control Ig (dotted line), Equal loading was ascertained by hybridization with the housekeeping and detected by streptavidin-PE. The histogram (right) shows IL-16 stain- gene GAPDH. This figure is representative of three experiments. ing pattern gated on CD19ϩ cells (left). The Journal of Immunology 2477

Table I. IL-16 synthesis in B lymphocytesa induction of CD69 expression on anti-CD40/IL-4 treated B cells (data not shown). Concentration Mean Ϯ SEM p Value Ϯ Medium alone 506 61 IL-16 is a major constituent of chemotactic activity of B cell PMA 10 ng/ml 552 Ϯ 101 0.674 Ionomycin 500 ng/ml 708 Ϯ 144 0.273 supernatant toward DCs PMA/ionomycin 593 Ϯ 98 0.480 To gain clues as to whether B cells secrete factors attracting DCs, ␮ Ϯ LPS 10 g/ml 638 131 0.481 B cell supernatant was tested for chemotactic activity on highly SACS 1Ϻ5000 590 Ϯ 147 0.920 IL-4 1000 U/ml 651 Ϯ 147 0.425 purified monocyte-derived DCs. Supernatant from unstimulated B IL-6 100 U/ml 478 Ϯ 117 0.427 cells efficiently attracted monocyte-derived DCs, with a maximum IL-10 100 U/ml 533 Ϯ 60 0.779 CI of 1.634 Ϯ 0.060 (Fig. 5). Significant migration was even noted ␥ Ϯ IFN- 100 U/ml 769 231 0.301 at a 100-fold dilution (CI, 1.325 Ϯ 0.058). To identify the role of Anti-CD40 1 ␮g/ml 711 Ϯ 119 0.148 Anti-CD40 ϩ IL-4 1171 Ϯ 345 0.127 IL-16, we added neutralizing anti-IL-16 mAb and found that mi- Anti-CD40 ϩ IFN-␥ 826 Ϯ 164 0.098 gration was almost completely inhibited (CI, 1.099 Ϯ 0.037 with undiluted supernatant). As shown in Fig. 5, the inhibitory effect of a Purified B cells (1 ϫ 106 per ml) were cultured for 24 h with addition of indi- cated agents. Supernatants were assayed for total IL-16 (pg/ml) by ELISA. Pro-IL-16 the mAb was specific for IL-16, because migration toward recom- was not detected in the same samples, suggesting that all IL-16 detected is cleaved binant MCP-3 was not inhibited. IL-16. Significance was calculated by Student’s t test for paired samples for the comparison of B cells stimulated with indicated agents compared to B cells cultured Furthermore, we tested supernatant from anti-CD40/IL-4- in medium alone. Various other time points (2, 4, 6, 12, 48, and 72 h) were evaluated, stimulated B cells as shown in Table II. The relative contribution Downloaded from but not statistically significant regulation was observed (n ϭ 7). of IL-16 to the chemotactic response was 61% as determined by neutralization experiments. Similar results were obtained with su- pernatant from B lymphocytes stimulated with anti-IgM-coated microbeads (data not shown). IL-16 in chemotactic activity of supernatant from unstimulated B Because CD4ϩCD11cϩ circulating blood DCs have been sug- cells, we added neutralizing IL-16 mAb and found that CI was gested as the DC population migrating into lymph node follicles http://www.jimmunol.org/ reduced almost to background levels (Fig. 5). The inhibitory ac- subsequently constituting germinal center DCs (20), we tested B tivity of the mAb was specific for IL-16, because migration of Th cell supernatant for its chemotactic activity on circulating blood cells toward recombinant RANTES was not inhibited (Fig. 5). DCs. Circulating blood DCs were obtained by depletion of CD3ϩ, To evaluate the influence of cellular activation on the chemo- CD11bϩ, CD16ϩ, and CD19ϩ cells employing immunomagnetic tactic activity of B cell supernatant, we tested supernatant from microbeads. The resulting cell population had a purity of Ͼ95% as anti-CD40/IL-4-stimulated B cells. This supernatant resulted in determined by cytofluorometry for lineage markers and HLA-DR substantial directed migration of CD4ϩ T lymphocytes, as dem- (data not shown). Extensive chemotaxis analysis was impossible onstrated in Table II. Addition of neutralizing anti-IL-16 mAb re- due to the very low frequency of this cell type in circulating blood. sulted in a 62% inhibition of migration (Table II). Similar results However, undiluted B cell supernatant efficiently induced migra- by guest on September 30, 2021 were obtained with supernatant from B lymphocytes stimulated tion of circulating blood DCs (CI, 1.64 Ϯ 0.08), which could sub- with anti-IgM-coated microbeads (data not shown). To prove the stantially be blocked by adding neutralizing anti-IL-16 mAb (CI, activation state of B lymphocytes, we analyzed CD69 expression 1.26 Ϯ 0.04; Table III). As shown in Table III, rhuIL-16 induced on anti-CD40/IL-4-stimulated B cells and B cells cultured without significant migration of DCs, which could be blocked by anti- addition of activating agents for 24 h. As expected, we observed IL-16 mAb as well.

FIGURE 4. IL-16 immunohisto- chemistry. IL-16 synthesis was studied in serial tissue sections of human tonsil lymphatic tissue ob- tained routinely during elective sur- gery. A, Identification of B lympho- cytes within lymph follicles by CD20 staining. Magnification, ϫ200. B, IL-16 reactivity in lym- phatic tissue detected by anti-IL-16 pAb. IL-16 staining is most pro- nounced in the mantle zone and less intense in the GC. Magnifica- tion, ϫ200. C, IL-16 reactivity in the GC. Magnification, ϫ400. D, Blocking of IL-16 reactivity by preincubation of IL-16 pAb with rhuIL-16. Magnification, ϫ200. 2478 B CELL-DERIVED IL-16 ATTRACTS Th CELLS AND DCs Downloaded from FIGURE 5. Left, CD4ϩ T lymphocyte chemotaxis. Dilutions of B cell supernatants with (Ⅺ) or without (E) mAb against IL-16 (10 ␮g/ml) were added to the lower wells of a Boyden microchemotaxis chamber. Thereafter, 50-␮l aliquots of purified CD4ϩ T lymphocytes (1 ϫ 106 cells/ml) were seeded in the upper wells and allowed to migrate into 5-␮m pore size nitrocellulose filters for 120 min. Recombinant human IL-16 (10 ng/ml) and RANTES (100 ng/ml) served as positive controls (F). f, Medium alone. Right, Monocyte-derived DC chemotaxis. Day 6 DCs migrated into 8-␮m pore size filters for 4 h toward pure medium (f), rhuIL-16 (10 ng/ml; F), B cell supernatant (E), or MCP-3 (10 nM; positive control; F) or medium supplemented with an Ab against rhuIL-16 (10 ␮g/ml; Ⅺ). Migration depth was quantified microscopically (leading front assay), and data are expressed as the mean Ϯ SEM of Ͻ ء ϭ ϭ Ͻ the CI. n 6(n 3 for experiments using Abs). Statistical analyses: Mann-Whitney U test vs medium after Kruskal Wallis ANOVA (p 0.001): , p http://www.jimmunol.org/ .p Ͻ 0.01 ,ءء ;0.05

B cell supernatant is chemotactic for CD8ϩ T cells leased into supernatant rather than stored in B lymphocytes. Intra- independently of IL-16 cellular FACS staining of PBMC confirmed that the CD19ϩ B cell To provide further evidence of the specificity of IL-16 we tested B population stains positively for IL-16. Culture of highly purified B cell supernatant for its chemotactic properties for CD8ϩ T lym- cells for various time periods revealed constitutive synthesis and phocytes (e.g., a CD4Ϫ cell type). As shown in Table II, superna- release into supernatant of cleaved, biologically active IL-16. This tant derived from unstimulated B cells as well as from anti-CD40/ was not due to cellular activation during the isolation procedure, by guest on September 30, 2021 IL-4-stimulated B cells was chemotactic for CD8ϩ T lymphocytes. because B cells did not express the activation Ag CD69 immedi- However, addition of neutralizing IL-16 mAb did not inhibit the ately after isolation or after 24 h in culture in medium alone, while migratory response (Table II). addition of PMA/ionomycin efficiently up-regulated CD69 (data not shown). Investigating modulation of IL-16 mRNA expression Discussion and protein synthesis by various cytokines, we noted that some Herein we report that human B cells are a source of IL-16. We regulation occurs at the mRNA level, while no significant changes provide evidence that freshly isolated B cells contain IL-16 mRNA in IL-16 released into supernatant was noted. This underscores the as determined by Northern analysis. Western analysis of cellular importance of post-transcriptional and post-translational mecha- extracts derived from freshly isolated B cells revealed an ϳ80-kDa nisms in regulating IL-16 release (3). band, representing pro-IL-16. In accordance with previous reports Immunohistochemistry of tonsil lymphatic tissue demonstrated (2, 3, 22), we suggest that bioactive 20-kDa IL-16 is readily re- IL-16 staining in CD20ϩ B lymphocytes in the mantle zone as well

Table II. Chemotaxis with supernatant derived from activated B cellsa

Migrating Cell Type B Cell Stimulation CI Ϯ SEM p % Inhibition

CD4ϩ T cells Anti-CD40/IL-4-stimulated 1.584 Ϯ 0.070 Ͻ0.05 62 plus IL-16 mAb 1.222 Ϯ 0.043 Monocyte-derived DC Anti-CD40/IL-4-stimulated 1.540 Ϯ 0.082 Ͻ0.05 61 plus IL-16 mAb 1.212 Ϯ 0.075 CD8ϩ T cells Unstimulated 1.280 Ϯ 0.057 NS No inhibition plus IL-16 mAb 1.345 Ϯ 0.040 Anti-CD40/IL-4-stimulated 1.408 Ϯ 0.063 NS No inhibition plus anti-IL-16 mAb 1.442 Ϯ 0.048 rhuRANTES 1.625 Ϯ 1.673 NS No inhibition plus anti-IL-16 mAb 1.673 Ϯ 0.062

a Purified monocyte-derived DCs, CD4ϩ, and CD8ϩ T lymphocytes were subjected to chemotaxis into nitrocellulose filters toward undiluted supernatant derived from anti-CD40/IL-4-stimulated B lymphocytes as described in Materials and Methods. For CD8ϩ T lymphocytes, migration toward unstimulated B cell supernatant and rhuRANTES (20 ng/ml) is additionally included. Results are expressed as CI Ϯ SEM, and the effect of IL-16 neutralization by mAbs (10 ␮g/ml) is expressed as percent inhibition of the migratory response (n ϭ 3). Statistical analysis evaluates the significance of addition of neutralizing anti-IL-16 mAb. A value of p Ͻ 0.05 was considered statistically significant. Similar results were obtained with supernatant derived from anti-huIgM-coated microbead-stimulated B cells. The Journal of Immunology 2479

Table III. Circulating DC chemotaxisa to the edge of the B cell-rich follicles and establish cognate T-B cell interactions (26), leading to the formation of GCs (29–31). Chemoattractant Mean SEM p Value Therefore, maturation of the B cell response depends on the avail- ability of primed T cells and the ability of Ag-specific B cells to Medium 1.000 0.000 Ͼ0.999 ϩ mAb (10 ␮g/ml) 1.096 0.038 selectively recruit them. Recent evidence suggests that direct B IL-16 (10 ng/ml) 1.612 0.079 0.029 cell-DC interactions are also a prerequisite for establishing hu- ϩ mAb (10 ␮g/ml) 1.149 0.051 moral immunity (10–16, 32, 33). However, the accurate sequence Supernatant (1Ϻ1) 1.637 0.082 0.029 of events in DC-B cell-T cell cooperation in vivo has yet to be ϩ ␮ mAb (10 g/ml) 1.256 0.038 established. This includes another distinct cell population impli- a Circulating blood DCs were purified and subjected to chemotaxis assay as de- cated in B cell maturation, namely follicular DCs (34–37). The scribed in Materials and Methods. Migration of DCs into nitrocellulose filters toward presence of GC DCs within primary follicles and the fact that they rhuIL-16 and B cell supernatant is compared to migration induced by medium and ϩ ϩ expressed as CI Ϯ SEM. Statistical analysis evaluates the significance of addition of display a phenotype similar to CD4 CD11c blood DCs (20) sug- neutralizing anti-IL-16 mAb. A value of p Ͻ 0.05 was considered statistically gest that DCs originating from blood colonize primary follicles. In significant. line with this model are recent data showing the progressive lo- calization of DCs to B cell follicles during a primary immune response (32). However, in that study migrating DCs where char- as in the GC. The weaker IL-16 staining in the GC compared with acterized as CD4Ϫ by immunohistochemistry, but this could be that in the mantle zone might reflect enhanced release of IL-16 due to the low sensitivity of this method compared with that of from B cells and consecutive depletion of intracellular stores. Al- cytofluorometric analysis. The elucidation of factors involved in Downloaded from though we did not observe up-regulation of the activation marker the migratory pathway of these cell populations is of critical in- CD69 on unstimulated B cells cultured for 24 h as described in the terest (38). Our in vitro migration data are further corroborated by previous paragraph, we cannot definitely exclude the possibility demonstrating IL-16 expression in B cells in lymph follicles by that B cells release IL-16 spontaneously only in the in vitro culture immunohistochemistry, suggesting an important role in vivo. system and not in quiescent state in vivo. This might explain the Although it has been long established that Th/B cell interaction ϩ absence of CD4 T lymphocytes and DCs in the mantle zone, in is required for the efficient elucidation of a humoral immune re- http://www.jimmunol.org/ contrast to the GC, where both cell types are found and probably sponse, few reports have focused on the discovery of factors de- recruited by IL-16 derived from B lymphocytes. rived from B cells mediating the selective attraction of T cells (9). To determine factors involved in the selective attraction of Th Schaniel et al. reported a novel , ABCD-1, derived from cells and DCs toward B cells, we tested supernatant derived by activated murine B lymphocytes and DCs that selectively acts on 24-h culture of highly purified B lymphocytes for its ability to activated T cells (39). The human orthologue, STCP-1 (MDC), induce the migration of Th cells and DCs into nitrocellulose filters. acts specifically on chronically activated Th2 lymphocytes (23, This assay has previously been employed in the evaluation of 24). ABCD-2, which acts via CCR4, as does ABCD-1, has also IL-16 as a chemotactic factor for DCs (2). For reasons of compar- been proposed as a factor attracting T lymphocytes toward acti- ison, we demonstrated IL-16-induced migration of DCs through vated B cells (25). Macrophage inflammatory protein-1␤ has been by guest on September 30, 2021 polycarbonate filters, showing similar results and confirming the reported to be derived from Ag -triggered B lymphocytes reliability of our method (2). We furthermore provided evidence and to partly account for T cell chemotaxis toward activated B that the migratory activity of IL-16 toward monocyte-derived DCs cells (17). IL-16 is theoretically a well-suited cytokine to meet the is chemotactic rather than chemokinetic (2). Herein we demon- biological requirement of selectively attracting CD4ϩ Th cells, strate that B cell supernatant efficiently attracts CD4ϩ T lympho- because it acts via CD4. Regarding the recently discovered direct cytes, monocyte-derived DCs, and circulating blood DCs. To de- interaction between DCs and B cells, no factors involved in DC lineate the role of IL-16 in chemotactic activity of B cell attraction by B cells besides the herein reported IL-16 have been supernatant, we neutralized IL-16 bioactivity by adding anti-IL-16 identified to date to our knowledge. We conclude that IL-16 could mAb and found the migratory response almost completely abro- orchestrate the interaction of Th cells, B cells, and DCs during the gated with supernatant derived from unstimulated B cells. 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