Antigens up the Nose: Identification of Putative ... - ACS Publications

Keywords: immunotherapy • mucosa • antigen presentation • nose draining lymph node • haptoglobin • MRP8 • nonintegrin 67 kDa laminin recep...
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Antigens up the Nose: Identification of Putative Biomarkers for Nasal Tolerance Induction Functional Studies Combined with Proteomics Annemieke M. Boots, Peter D. Verhaert,* Rezie J. te Poele, Sabine Evers, Christina J. Coenen-de Roo, Jenny Cleven, and Ebo S. Bos Departments of Target Discovery and Pharmacology at NV Organon, Oss, The Netherlands Received May 25, 2004

Intranasal autoantigen delivery is the most effective means of inducing mucosal tolerance and suppression of autoimmune disease. In an effort to identify markers of the “tolerant state”, we employed proteomics technology at the level of the cervical lymph node. The analysis revealed that nasal antigen administration (without adiuvant) led to modulation of various proteins among which the most prominent were haptoglobin, nonintegrin 67 kDa laminin receptor, and MRP8. The immunoregulatory haptoglobin may qualify as (bio)marker for effective immunotherapy. Keywords: immunotherapy • mucosa • antigen presentation • nose draining lymph node • haptoglobin • MRP8 • nonintegrin 67 kDa laminin receptor • in vivo study • 2D-based proteome analysis

Introduction The mucosal surfaces of the body are in continuous contact with environmental antigens and infectious agents. Whereas infectious agents generally elicit productive immune responses, the predominant response to soluble, nonpathogenic, Tdependent antigens is one of tolerance.1,2 The introduction of antigen to the nasal mucosa is considered one of the most efficacious routes for effective tolerance induction.3,4 More importantly, it is seen as an effective means of immunotherapy of autoimmune disease (AID). In several experimental autoimmune models, nasal application of antigen has led to prevention or amelioration of clinical symptoms.5-7 Notably, most protocols that we are currently aware of employ antigen delivery in the absence of adiuvant and prior to antigen sensitization or disease induction. There is ample evidence that antigen presentation to naive T-cells in vivo takes place in the lymph nodes draining the injection or application site.8 For successful nasal tolerance induction, however, the cervical lymph node, one of the lymph nodes draining the head and neck region, is required. A series of transplantation experiments revealed not only that the presence of cervical lymph nodes was necessary for nasal tolerance induction but also that this function could not be restored by peripheral lymph nodes when transplanted to this site.9 Thus, the cervical lymph node is key in governing the nasal tolerance induction process. It is envisioned that antigen (protein or peptide) applied to the nasal mucosa drains to, or is transported to the local lymph nodes by specialized antigen presenting cells (APC), dendritic cells (DC), where it is presented to naive antigen-specific

T-cells.8 The mucosal tolerization process eventually leads to suppression of T-cell responses via a mechanism of active suppression (regulatory T-cells), clonal inactivation, or clonal anergy.10,11 Although several putative mechanisms have been proposed, the exact mechanism underlying nasal tolerance induction is unknown. Also, there is a need for identification of markers of the tolerant state. In an effort to identify protein constituents of the lymph node microenvironment contributing to the tolerization process, we performed a proteomics study in which we focused on the nose draining lymph node representing a functional, secluded, and accessible organ for this study. Previously, it was shown that nasally administered OVA induces tolerance in Balb/c and in DO11.10 TCR transgenic (trg) mice.9,12-15 Here, we first addressed the sequence of events in vivo following nasal administration of an I-Ad binding OVAderived epitope (AA 323-339) in these strains. We were able to confirm and to extend the previous studies showing that nasally applied antigen traffics to the cervical lymph node (CLN). In an effort to focus at the cognate APC-OVA323-339TCR interaction events in a controlled setting and in order to maximize reactivity, we transferred our studies to the DO11.10 TCR transgenic mouse. CLN from OVA323-339-sniffed mice and controls were obtained for a tissue protein profile comparison at 24-72 h after nasal antigen administration, a time frame found to cover the bi-directional APC-TCR interaction events. The study identified a restricted number of proteins associated with nasal antigen presentation to T-cells (in the absence of adiuvant). The implication of these findings for our understanding of mucosal tolerance induction is discussed.

Materials and Methods * To whom correspondence should be addressed. Laboratory of Neuroplasticity and Neuroproteomics, Naamestraat 59, B-3000 Leuven (Belgium). Phone: 32-495 16 81 20. Fax: 31-16 32 42 63. E-mail: peter.verhaert@ bio.kuleuven.ac.be.

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Journal of Proteome Research 2004, 3, 1056-1062

Published on Web 09/17/2004

Antigens. One peptide derived from chicken ovalbumin (OVA, Swiss-Prot Accession # P01012) and one from human cartilage glycoprotein-39 (HC gp-3916, Swiss-Prot Accession # 10.1021/pr049907c CCC: $27.50

 2004 American Chemical Society

Antigens up the Nose

P36222) were selected as antigens: OVA323-339, sequence ISQAVHAAHAEINEAGR, an MHC class II/I-Ad binding epitope of ovalbumin with a purity >80% was obtained from Isogen Bioscience BV, Maarssen, The Netherlands. HC gp-39263-275 [RSFTLASSETGVG], an immunodominant epitope in the context of HLA-DR4, was obtained by in-house synthesis [solid-phase peptide synthesis on an automated Milligen 9050 peptide synthesizer] and RP-HPLC purification (purity >90%) and was used as a control for peptide loading.7 All antigen preparations used in this study were endotoxinfree. Animals and Nasal Antigen Administration. Balb/c mice (H2d) were purchased from Charles River (Sulzfeld, Germany). The mutant mouse strain, transgenic (trg) for the DO11.10 T-cell receptor (TCR) and recombinase activating gene 2 (RAG2) deficient, (B10.D2/AiTac-TgN(DO11.10)-RAG2tm1) was obtained from Taconic17. This mouse bears T-cells of the transgene specificity only, recognizing OVA323-339 in the context of H-2d. It does not develop endogenous mature T or B cells. The nasal administration of antigen was performed under light isoflurane aneasthesia. Antigen was administered intranasally in two 10 µL aliquots per dose of 100 µg in saline. Animals were housed in filter-top cages with (OVA-free) food and water ad libitum. Generation and Characterization of an OVA323-339 Specific T-Cell Line. For detection of OVA323-339 peptide presentation by APC from different sources, an OVA323-339 specific T-cell line was used. The T-cell line was generated following peptide stimulation (10 and 0.5 µg/mL) of spleen cells from DO11.10 TCR trg mice. Six days after antigen stimulation, cells were fed with IL-2 (20 U/mL) and IL-4 (5 U/mL). Cells were maintained in a standard 14 day restimulation/expansion cycle. The established T-cell line was found to express CD4 and TCR Vβ8 which is in agreement with the reported phenotype.17,18 To procure an invariable source of T-cells, the OVA-specific T-cell line was expanded and cells were frozen at day seven after the last antigen-specific stimulation in medium containing 10% DMSO. Cells were stored at -70 °C and thawed 7 days prior to use. Cell culture was in Dulbecco’s modified Eagle’s mediumHam’s F-12 (Gibco, BRL, Paisley, U.K.) supplemented with 10% foetal calf serum (Greiner Labortechnik, Germany), IL-2 (20 U/mL), and IL-4 (5 U/mL). All assays were performed with T-cells 14 days after the last antigen stimulation. OVA-specific T-cell responses were assayed by co-culturing T-cells (1 × 104) with APC (2.5 × 105 cells, irradiated at 30 GY) in round-bottomed 96 well plates (Costar; 200 uL medium). Spleen cell suspensions or lymph node cell suspensions from Balb/c mice were used as APC. Cell suspensions were obtained by manual homogenization of spleens using filter chambers (NPBI, Emmer-Compascuum, The Netherlands) or of lymph nodes using a 70 µm cell strainer (Falcon, Le Pont De Claix, France). Spleen cell suspensions were depleted for erythrocytes by lysis on ice (in 0.14 M NH4Cl, 0.017 M Tris, pH 7.2). Splenocytes were harvested by centrifugation and collected in medium with 10% foetal calf serum. In the APC potency experiments, exogenous antigen (OVA323-339) was added to the cultures (0.02, 0.1, 0.5, 2.5, and 12.5 µg/mL final concentration). Cultures were incubated for 3 days (37°C, 5% CO2) and for the last 18 h of culture pulsed with 0.5 µCi (1.85 × 104 Bq) [3H]thymidine ([3H]TdR). Cells were harvested on glass fiber filters and [3H]TdR incorporation was measured by gas scintillation. Since gas scintillation measurements are 5-fold less efficient compared to liquid scintillation, filters were measured for 5

research articles min (Packard Matrix 96 β counter; Meriden, CT). Cytokine production by the OVA-specific T-cells was assessed in a culture setup as described above. At day 3 of culture, supernatants were harvested and assayed for IL-4, IL-10, and IFNγ using cytokinespecific ELISAs (Pharmingen). Antigen Trafficking following Nasal Antigen Administration. Given that antigen application to the nasal mucosa induces tolerance in Balb/c mice, we determined the anatomic location of Ag presentation to T-cells following in vivo nasal OVA peptide administration. To assess this, the OVA323-339specific, I-Ad restricted T-cell line (see above) was used as a functional read-out for antigen-specific presentation at the level of the local lymph nodes. For this purpose, OVA peptide specific proliferation and cytokine production were measured (the T-cell line was found to produce detectable levels of IL-4, IL10 and IFNγ). Thus, 100, 200, 300, and 400 µg dosages of OVA peptide were applied to the nasal mucosa. After 18-24 h, the spleen and thymus, as well as the lymph nodes of the head and neck region, i.e., the cervical, internal jugular, and facial lymph nodes (CLN, IJLN and FLN), were tested for the presence of OVA323-339-bearing APC using the OVA-specific T-cell line. Cell suspensions were made as described. Antigen-specific proliferation and cytokine production were assessed as described above. Notably, in the trafficking studies no antigen was added to the cultures. Transient T-Cell Activation after Nasal Antigen Administration in TCR trg Mice. T-cell responses to OVA or its immunodominant peptide can usually not be detected in nonimmunized mice due to a low T-cell precursor frequency in vivo. Since our studies focus at OVA responses in the absence of adiuvant, we transferred our studies to a mutant mouse strain, transgenic for the DO11.10 (OVA323-339 in context of I-Ad specific) TCR and deficient for RAG-2. This mouse bears T-cells of the transgene specificity only. The innate immune system of this mouse, however, is fully intact: DO11.10 mice are healthy and can be kept under standard housing conditions. To identify the kinetics of the local, OVA-specific response in these mice, two groups of mice (n ) 10) were allowed to sniff saline (background) or specific OVA peptide (100 µg). At 4, 18, and 72 h after in vivo antigen administration, CLN and IJLN were obtained, cell suspensions were made and assayed for functional T-cell responses (proliferation and cytokine production) essentially as described above. Protein Profile Comparison in CLN from TCR trg Mice. After establishing the CLN as the very site of APC-T-cell interaction following nasal antigen administration in the DO11.10 TCR trg, RAG-2KO mice, the protein expression profile was investigated. Three groups of mice (n ) 6) were allowed to sniff saline (background), an irrelevant peptide (HCgp39263-275, as a control for peptide loading), or specific OVA323-339 peptide as the cognate stimulus. A total dose of 200 µg per mouse was achieved by administering 100 µg of peptide at t ) 0 and 100 µg at t ) 8 h. At 16/24, 40/48, and 64/72 h [for clarity reasons these time points will be referred to as 24, 48, and 72 hr, respectively] following nasal antigen administration, the nose draining cervical lymph nodes were obtained and processed for 2D gel based proteome comparison. After dissection, lymph nodes of the individual groups were pooled (10-15 lymph nodes per group). They were rinsed in a salt-free isotonic sugar solution [250 mM sucrose, 10 mM Tris-HCl pH 7.0] and subsequently homogenized [150 µL of 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 50 mM Tris-HCl pH 7.3 supplemented with a complete cocktail of protease inhibitors (Roche)] Journal of Proteome Research • Vol. 3, No. 5, 2004 1057

research articles using the Ettan sample grinding kit (Amersham Biosciences) according to the manufacturer’s instructions. The suspension was centrifuged twice for 2 min at 11.6 × 104 N/kg in order to remove the bulk of lipid and cellular debris. The resulting clear homogenate was subjected to ultracentrifugation at 2 × 106 N/kg for 1 h. Finally, residual low-density material was removed from the supernatant by two consecutive 5 min spins at 11.6 × 104 N/kg. Protein concentration in the samples was determined by the Coomassie Plus Protein Assay, based on Bradford (Pierce, Rockford, IL). CLN (10-15) from saline and irrelevant peptide-sniffed animals gave yield to 400 µg of protein, whereas CLN from OVA peptide-sniffed mice resulted in significantly (2-fold) higher protein yields. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) of lymph node samples was carried out in triplicate per condition (time point/treatment). In an initial, analytical phase, 40 µg of protein (equal to 0.5-1 CLN), dissolved in lysis buffer [9 M urea, 65 mM DTT, 0.5% Triton X-100, 2% Pharmalytes pH 3-10], was loaded on 18 cm immobilized pH gradient (IPG) strips [pH 4-7] by overnight in-strip rehydration. Isoelectric focusing (IEF) was performed at 20 °C [3 h at 500 V and subsequently 17.5 h at 3500 V]. Afterward, proteins in the focused IPG strips were reduced and alkylated [30 min in 65 mM DTT followed by 50 min in 250 mM iodoacetamide at RT]. Second dimension SDS electrophoresis was performed on a 12-14% Excel gel in a cooled (15 °C) Multiphor (Amersham Biosciences) [45 min at 20 mA during IPG strip application and 2.5 h at 40 mA after strip removal]. Gels were fixed overnight [400 mL/L of ethanol, 100 mL/L of acetic acid] and stained by automated silver staining [Hoefer Processor Plus (Amersham Biosciences)] according to Blum et al.19 In the following phase of the study, semipreparative gels were run for protein identification. These were loaded with 150 µg of protein and silver-stained according to Shevchenko et al.20 Gels were scanned on a flat bed scanner, and the resulting image files (TIFF) of the triplicate gels were processed using Z3/Z4000 image analysis software (Compugen, Israel) which included the generation of “raw master gels”,21 to assist the comparison of multiple gels of different conditions. Differentially expressed protein spots were excised from the preparative gel and processed for mass spectrometry (MS) analysis. This involved gel plug washing [2× in H2O], shrinking [2× in MeCN] and drying (in vacuo centrifugation in a SpeedVac system), in gel protein digestion [O/N at 37 °C in a minimal volume (15-30 µL) of 25 mM NH4CO3 pH 8.0 with 12.5 mg/L porcine trypsin (Promega)] and sample acidification [addition of 1 µL HCOOH], concentration and desalting [on self-made reversed phase microcolumns (Eppendorf GELoader tip fitted with a minimal amount of POROS20R2)]. The resulting sample was directly eluted on the stainless steel MS target plate [in 0.5 µL of saturated alpha-cyano-4-hydroxycinnamic acid solution (in 30% MeCN, 0.1% TFA)]. Peptide mass fingerprint (PMF) spectra were recorded by delayed extraction matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS on a Voyager DE Pro (reflectron mode; Applied Biosystems). Mass spectra were processed using a two point external calibration (MH+ ions 927.4935 and 2045.0279 of a bovine serum albumin-tryptic digest) and monoisotopic peptide ion masses were listed and used for database searching [MS-Fit and Mascot on SwissProt and NCBI nrDB]. Protein identifications were validated through independent analysis of the protein digests by tandem MS (MS/ 1058

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Boots et al. Table 1. Antigen-Specific Reactivity of the OVA323-339 Specific T-Cell Linea OVA323-339 [µg/mL]

proliferation [SI]

IL-4 [pg/mL]

IL-10 [pg/mL]

IFNγ [pg/mL]

BG 0.02 0.1 0.5 2.5 12.5 APC alone

1 39.6 153.4 265.9 140.0 14.6 ND

36 682 1700 2342 3003 2050 0

24 1099 6308 >10000 >10000 >10000 0

0 313 902 2156 6688 15792 0

a BG ) background (264 counts/ 5 min). T-cells (1 × 104/well) were added to (2 × 105 cells/well) APC (Balb/c spleen cells). Proliferation (SI ) stimulation index calculated as antigen specific cp5m/background cp5m) and cytokine production was determined at day 3 of culture. ND ) not determined. Results are representative of >3 experiments. Proliferation was measured in triplicate wells. Standard deviations did not exceed 30%. Values in bold are regarded positive (SI values >3). Detection level of cytokine ELISA; detection level for IL-4 was 4 pg/mL, for IL-10 was 6 pg/mL and for IFNγ was 20 pg/mL. Values in bold are regarded positive, i.e., >2× background value or >2× lowest detection level of the specific cytokine.

MS) sequence analysis in an oMALDI QSTAR Pulsari (Applied Biosystems) the source of which elegantly accommodates the very same MALDI sample plate used for the Voyager instrument based PMF analyses.

Results Antigen Trafficking following Nasal Antigen Administration. In 1999, it was shown by Wolvers and co-workers9 that nasally administered OVA induces tolerance in Balb/c mice. In addition, we were able to show that nasal OVA administration leads to a profound suppression of the OVA-specific DTH response when challenged in the footpad in the same mouse strain (data not shown). In this study, we characterized the in vivo process of antigen trafficking via the nasal mucosa. Balb/c mice that had sniffed OVA323-339 peptide were analyzed for the presence of APC presenting OVA peptide in various lymph nodes from the head and neck region, as well as in spleen and thymus. For detection of OVA-bearing APC, an OVA-specific, I-Ad-restricted T-cell line was employed. The proliferative response and cytokine secretion of this newly generated T-cell line were investigated at day 1, 3, 5, and 7 after stimulation with OVA323-339 (Table 1, read-out at day 3 after OVA peptide stimulation). The cell line showed an OVA-specific, dosedependent proliferative response following OVA323-339 stimulation in the presence of I-Ad expressing APC (Balb/c spleen cells). Optimal responsiveness for these parameters was found at day 3 after antigen stimulation, the highest proliferative responses being observed with 0.5 µg of peptide. Also, antigeninduced production of IL-10, IL-4, and IFNγ (in this order of sensitivity) were seen. All parameters mentioned were used as read-out in the tracking studies. In a first phase of the study where 100 µg of peptide was administered to the nasal mucosa, low numbers of OVA323-339 peptide-bearing APC were detected at the optimum time point (18-24 h) in CLN and in IJLN but not in FLN (nor in spleen or thymus). Since the in vivo loaded APC generated only a modest response of the highly sensitive T-cell line, possibly related to peptide degradation events, higher dosages of peptide (100 and also 200, 300, and 400 µg) were applied for loading the nasal mucosa in the follow-up phase of the study. Again, the only tissues showing significant numbers of OVA323-339 presenting APC (18 h after nasal antigen instillation) were the CLN and the IJLN (Table 2) as evidenced by prolifera-

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Antigens up the Nose Table 2. Antigen Tracking; Detection of OVA323-339 Presentation in Lymph Nodes of the Head and Neck Region, 18 h Following Nasal Antigen Administration in Balb/c Mice, Using an OVA-Specific T-Cell Line as a Read-Outa APC source

CLN

FLN

IJLN

spleen

i.n. OVA323-339

proliferation [SI]

IL-4 [pg/mL]

IL-10 [pg/mL]

IFNγ [pg/mL]

control (saline) 100 µg 200 µg 300 µg 400 µg control (saline) 100 µg 200 µg 300 µg 400 µg control (saline) 100 µg 200 µg 300 µg 400 µg control (saline) 100 µg 200 µg 300 µg 400 µg

1 6.8 10.6 32.4 23.9 1 2.8 1.4 1.8 1.0 1 2.4 10.3 24.8 15.2 1 0.6 0.5 0.8 0.7

9 20 50 44 13 49 66 41 -

7 75 150 120 139 107 -

-

a CLN ) cervical lymph node, FLN ) facial lymph node, IJLN ) internal jugular lymph node. SI ) stimulation index; incorporation of tritiated thymidine by OVA specific T-cell line (counts/5 min) incubated with APC from OVA323-339 sniffed animals/incorporation of tritiated thymidine by T-cells (counts/5 min.) incubated with APC from saline sniffed animals. Proliferation was measured in triplicate wells. Values in bold are regarded positive (SI values >3). Detection level of cytokine ELISA; detection level for IL-4 was 4 pg/mL, for IL-10 was 6 pg/mL and for IFNγ was 20 pg/mL. ) below detection level of cytokine ELISA Values in bold are regarded positive, i.e., >2× lowest detection level of the specific cytokine. Results are based on 4 antigen tracking experiments.

tion as well as IL-4 and IL-10 production. Notably, the in vivo loaded APC did not induce detectable levels of IFNγ by the cell line, a phenomenon which may be related to the number of functional MHC-peptide complexes required for induction of IFNγ. Optimal reactivity of the line was seen after in vivo loading of 300 µg of peptide. No OVA-bearing APC were detected, at any dose, in FLN, spleen or thymus (thymus data not shown). When tested for the capacity to present exogenous OVA323-339 to the OVA-specific T-cell line, APC from CLN, FLN and IJLN isolated from non-peptide-treated mice of the same experiment, were equally capable of inducing a response. This indicates that there is no difference in antigen presenting potency between the APC from these tissues (Figure 1, comparison with spleen cells). The tracking studies, therefore, demonstrate that antigen applied to the nasal mucosa specifically drains to the CLN and the IJLN, identifying these lymph nodes as the major sites of functional antigen presentation. Transient T-Cell Activation after Nasal Antigen Administration in TCR trg Mice. After having established the CLN and the IJLN as the major sites of in vivo antigen presentation following nasal antigen administration, we described the events in the DO11.10 TCR trg, RAG-2KO, mouse, a model which offers the possibility to focus at the cognate APC-OVA323-339-TCR interaction in a controlled setting, as other antigen reactivities are not expected and do not dilute or hamper the OVA-induced events. To determine the effect of nasal antigen administration on T-cell activation, mice were allowed to sniff saline (control for treatment) or the cognate OVA323-339 peptide. CLN and IJLN were obtained at 4, 18, and 72 h time points, and cell suspensions were assayed for OVA-specific proliferation and

Figure 1. APC potency: proliferative response of the OVA323339-specific T-cell line when stimulated with (added) peptide presented by I-Ad -carrying, irradiated APC from different sources (Balb/c spleen, CLN ) cervical lymph node, FLN ) facial lymph node, IJLN ) internal jugular lymph node). Proliferative responses were analyzed as described in the legend of Table 2. Results are based on 4 different experiments.

cytokine (IL-4 and IFNγ) production. The functional data show an increased incorporation of 3H-thymidine by CLN cells from mice nasally treated with OVA323-339 and a concomitantly increased IFNγ production at 18 h (801 pg/mL, Figure 2). IL-4 was not detected in this mouse under these conditions. This increased proliferation and cytokine production was only transient; no evidence of T-cell activation was seen either at 4 h or at 72 h. Notably, transient activation of CD4+ T-cells is known to occur prior to the development of tolerance in vivo.22 The IJLN in this strain, however, appeared particularly elusive in this mutant mouse strain. This was confirmed by the minimal amounts of cells isolated from the IJLN (thereby impeding further study of this organ in this strain). Hence, the rest of our studies focused on the CLN as the major site involved in antigen presentation to T-cells in this strain. Differential Protein Expression Profiles following Intranasal Antigen Administration in CLN from TCR trg Mice. Based on the above findings, a protein profile analysis on the CLN in DO11.10 TCR trg, RAG-2 KO mice was done at 24, 48 and 72 h after nasal antigen application. These time points cover a time frame which was functionally established as being suited for studying the events in bi-directional APC-OVA323-339TCR interaction. A typical 2D PAGE analysis of an equivalent of 0.5-1 CLN reveals approximately 1200 protein spots. Within this subset of the CLN proteome, no consistent differences were noted when comparing the protein profiles of the saline-treated with the irrelevant peptide-treated CLN tissue. However, few proteins (eleven to be precise) were found to be consistently changed (more than 2-fold) in the protein profile of OVA323-339sniffed CLN tissue (Table 3, Figure 3). Most changes in the 2D protein patterns analyzed were seen at the 24 h time point. Nine out of eleven modulated protein spots were successfully identified by PMF (Table 3). All these protein identities were unequivocally verified with MS/MS sequence analysis. Spot # 6 and 11 remained unidentified (poor PMF spectra, not allowing MS/MS sequence confirmation). Our data show an upregulation (24 h after OVA323-339 administration) of four different isoforms of haptoglobin, two different isoforms of MRP8 (Calgranulin A), and poly (rC) binding protein (bp)1. At 48 h, all but one (unidentified spot # 6) protein level returned to control (i.e., the 0 h control condition). Spot # 6 expression was downmodulated at 72 h. In contrast, nonintegrin 67 kDa laminin receptor (LR, also known as P40-8) and fragment of tubulin beta 5 chain (TUBB5) show a downmodulation at 24 Journal of Proteome Research • Vol. 3, No. 5, 2004 1059

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Boots et al.

Figure 2. Nasal antigen administration leads to transient T-cell activation in cervical lymph nodes (CLN) from DO11.10 TCR transgenic mice (results obtained in 2 experiments). Table 3. Overview of Consistently Modulated Protein Spots in CLN Form OVA-Sniffed Animals Compared to Controlsa spot #

1 2 3 4 5 6 7 8 9 10 11

PMF result (database accession #)

Trembl Q61646

Swiss-Prot P14206 Swiss-Prot P05218 Swiss-Prot P27005 NCBI 6754994

Protein ID

24 h [OVAp vs control]

48 h [OVAp vs control]

72 h [OVAp vs control]

haptoglobin β-isoform haptoglobin β-isoform haptoglobin β-isoform haptoglobin β-isoform 67 kDa LR not identified; pI 4.3, Mw 39 kDa TUBB5 MRP8 isoform MRP8 isoform poly(rC) bp1 not identified; pI 5.6, Mw 30 kDa

+ + + + + + + + +

) ) ) ) ) + ) ) ) ) )

) ) ) ) ) ) ) ) ) ) )

a Eleven protein spots were consistently modulated in CLN from OVA323-339 peptide (OVAp) sniffed animals compared to HC gp-39 peptide sniffed animals. Conclusions are based on results obtained from 3 different in vivo experiments each involving the running of triplicate gels. Only proteins showing consistent modulation in, a least, 7 gels are listed. 67 kDa Laminin Receptor is also known as P40-8, MRP8 is also known as Calgranulin A, TUBB5 is the tubulin beta-5 chain. +, upregulated; ), unchanged; -, downregulated.

h. Likewise, inhibition was short-lasting; levels returned to control at 48 h. Modulation of haptoglobin and 67 kDa LR are visualized at 24 and 48 h after peptide sniffing (Figure 4).

Discussion Delivery of peptide or protein antigens intranasally is an effective way of inducing antigen specific peripheral tolerance, a process exploited for immunotherapy of AID.3-7 Moreover, it was found that the nose draining lymph nodes are required for successful nasal tolerance induction.9 The particular reasons why the mucosal environment may be especially conducive to the development of tolerance, being mediated either by clonal inactivation or by induction of regulatory T-cells (active suppression), is unclear. Several studies, highlighting the importance of the lymph node microenvironment, ascribe this intrinsic capacity to (1) differential expression of homing markers, (2) differences of stromal cells forming the basic structure of the node, (3) the presence of distinct APC capable of skewing the T-cell repertoire, and (4) the presence of natural immune regulatory proteins (feedback mediators) contributing to the lymph node micro-environment. Few studies have addressed antigen presentation to naive MHC class II restricted T-cells in vivo.8 Here, we employed proteomics technology to approach the issue of nasal tolerance from a distinct perspective in an experimental immunotherapeutic setting closely resembling the clinical situation. Pro1060

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teome alterations in entire, local draining lymph nodes were studied at functionally selected time points after nasal antigen administration in vivo, in an effort to identify protein markers involved in the local, micro-environment determined, antigen presentation process. For this purpose, we chose to work with the DO11.10 TCR transgenic mouse for the following reasons; (1) it is a broadly used, well-described mouse model with an intact innate immune system, (2) functional studies of mucosal tolerance induction had been reported indicating that mucosal antigen administration leads to functional suppression, (3) the mouse bears only one TCR specific for OVA323-339 in the context of I-Ad, thereby providing a model system with amplified T-cell responses, and (4) the discrimination between specific activation and loading with a control (non cognate) peptide allows for a considerable window of defined reactivity (no other T-cell specificities are involved). This latter aspect enabled the focus on a defined, in vivo interaction between APC and T-cell within an entire, functionally involved lymph node for an ex vivo analysis of the proteome. The type of proteome analysis employed here shows the differential display of abundant proteins (with molecular size of >10 kDa) rather than proteins of low abundance. In addition, most proteins visualized in the 2D gels are of intracellular/ cytoplasmic protein origin as membrane proteins will be underrepresented because of technological limitations of the first dimension. Any differences found, however, either reflect direct

Antigens up the Nose

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Figure 3. 2D images showing protein profiles of cervical lymph nodes at 24 (magenta) and 48 h (green) after OVA sniffing. Z3 generated overlay of two raw master gels of 3 individual silver stained gels, pseudocolored in silico. Protein spots present in both raw master gels show up in black. Protein spots encircled in red were excised from corresponding semipreparative gels (stained with Coomassie) for protein identification analysis (numbers correspond with those in Table 3). The gel area marked in yellow represents the areas of the gels shown in 3D view in Figure 4.

functional involvement of the proteins in the immunological process or merely present a (bio)marker thereof. The results of the proteome analysis in this study revealed modulation of only a small subset of proteins. This is not unexpected in view of the characteristics of the model used, zooming in on the seclusion of the cervical lymph nodes in a time frame covering the APC-OVA323-339-TCR interaction process and in view of the single TCR specificity. In this context, we found four isoforms of haptoglobin, two isoforms of MRP8 and poly (rC) bp1 to be upregulated, and nonintegrin 67kDa LR as well as the tubulin beta-5 chain to be downregulated for a short period (at 24 and 48 h after antigen administration). It is unclear how modulation of a protein like poly (rC) bp1 (a nucleic acid binding protein) or a fragment of tubulin beta 5 chain should be functionally interpreted in the context of the immunological process under investigation. On the other hand, in view of literature data, proteins such as MRP8, 67 kDa LR, and haptoglobin are particularly interesting in relation to the process studied. MRP8 is a member of calcium-binding proteins of the S100 family predominantly localized in the cytoplasm.23 MRP8 and its S100 family member MRP14 are regarded as marker proteins for activated or recruited phagocytes. In addition, MRP8 recruited phagocytes have a distinct phenotype expressing CD11b/CD18 and represent a population of early infiltrating cells. Important in relation to the mouse study reported here is that, prior to cognate APC-T-cell interaction, recruitment and migration of APC to the lymph node is required. This agrees with chemotactic properties ascribed to murine MRP8. The nonintegrin 67 kDa LR is a well-known protein in oncology where its expression is correlated with the metastatic potential of cancer cells.24 Furthermore, it is expressed on vascular endothelial cells where it may have a role in angiogenesis.25 Also, most peripheral blood cells including monocytes/macrophages, neutrophils, and subsets of T lymphocytes

express 67 kDa LR. The receptor binds to laminin, the predominant glycoprotein of the basement membrane and hence plays a role in cellular trafficking across blood vessels and tissue basement membrane barriers. The fact that activated T-cells express the 67 kDa LR suggests a role in lymphocyte migration.26 The present data show a temporary down-modulation of the receptor at 24 h after sniffing. This temporary downmodulation is likely to result in suppression of adhesion to the ECM surfaces of the T-cell cortex of the lymph node, eventually leading to increased mobility of T-cells. Also, reduced expression of the receptor may be followed by integrin mediated cellcell interactions such as those between APC and T-cells during antigen presentation. Besides the modulation of the above proteins, our study shows a marked transient upregulation of four isoforms of haptoglobin in the process of danger-free antigen exposure. Haptoglobin is an acute phase plasma protein for which opposing activities have been described. Whereas it is frequently found upregulated and as such associated with acute and chronic inflammatory disorders,27,28 several reports have ascribed antiinflammatory activities to the protein as well.29-31 For instance, it has been suggested that haptoglobin plays a role in maternal-fetal tolerance induction.30,31 Also, fetal haptoglobin is analogous with “suppressive E-receptor” (SER), a potent immune suppressive factor isolated from cancer patients.32 In view of the study presented here and of more recent data, the association of haptoglobin with an antiinflammatory function is of particular interest. Intriguingly, haptoglobin can regulate Langerhans cell function by preventing the maturation of this DC subset, a phenomenon reported to be involved in T-cell skewing or the induction of regulatory T-cells.33 Also, immune suppressive activities on leukocytes in general have recently been extended to suppressive effects on T-cells.34 How haptoglobin exerts its suppressive function is unknown but the reported affinity of haptoglobin for CD11b which is expressed Journal of Proteome Research • Vol. 3, No. 5, 2004 1061

research articles

Boots et al.

Prof. Dr. Lutgarde Arckens and Dr. Patrick Matthys (respectively at the Laboratory of Neuroplasticity and Neuroproteomics and at the Immunology Department of the REGA Institute, both at the University of Leuven) are gratefully acknowledged for their priceless critical suggestions.

References

Figure 4. Image analysis “in 3D” of the protein modulation at 24 h (a) and 48 h (b) after nasal antigen administration at the level of the CLN. The selected area (see yellow frame in Figure 3) shows haptoglobin in yellow, upregulated at 24 h and back to control level at 48 h, and 67kDa LR (circled in red) downmodulated at 24 h and back to control level at 48 h.

on the majority of lymph node DC subsets (myeloid and plamacytoid DC) is particularly suggestive in this respect.8,35,36 The present functional kinetics study shows a transient local upregulation of haptoglobin during antigen presentation under tolerising conditions (without adiuvant). This emphasizes a role for haptoglobin as mediator in nasal tolerance induction. It may be part of a natural feedback regulation, rather than an epiphenomenon associated with inflammatory conditions. In light of this, haptoglobin may qualify as a (bio)marker for effective immunotherapy. In conclusion, in vivo administered antigen is drained from the nasal mucosa to the local cervical lymph node where it is presented by APC to naive antigen-specific T-cells. Here, transient T-cell activation will eventually lead to a T-cell hyporesponsive state (tolerance). The data presented suggest involvement of MRP8 and non integrin 67 kDa LR in migration/ mobility of APC and/or T-cells and imply an immunoregulatory role for haptoglobin in nasal tolerance.

Acknowledgment. We thank Wouter Hoeberichts and Alain van Gool for their contributions to the proteomics studies. 1062

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