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Lymphoid neogenesis in the giant papillae of patients with chronic allergic conjunctivitis

2010; Elsevier BV; Volume: 126; Issue: 6 Linguagem: Inglês

10.1016/j.jaci.2010.06.001

1097-6825

Akira Matsuda, Nobuyuki Ebihara, Norihiko Yokoi, Kazuichi Maruyama, Junji Hamuro, Shigeru Kinoshita, Akira Murakami,

T-cell and B-cell Immunology

To the Editor: Lymphoid neogenesis (tertiary lymphoid organ [TLO]) resembles a secondary lymphoid organ (eg, the lymph node) and is observed in patients with chronic inflammatory diseases.1Aloisi F. Pujol-Borrell R. Lymphoid neogenesis in chronic inflammatory diseases.Nat Rev Immunol. 2006; 6: 205-217Crossref PubMed Scopus (713) Google Scholar TLOs play roles in immune responses against persistent antigens; however, they can also induce severe tissue damage.1Aloisi F. Pujol-Borrell R. Lymphoid neogenesis in chronic inflammatory diseases.Nat Rev Immunol. 2006; 6: 205-217Crossref PubMed Scopus (713) Google Scholar It has also been reported that the germinal centers and follicular dendritic cell (FDC) networks in TLOs relate to class-switch recombination.2Gould H.J. Takhar P. Harries H.E. Durham S.R. Corrigan C.J. Germinal-centre reactions in allergic inflammation.Trends Immunol. 2006; 27: 446-452Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar In this study we examined the lymphoid neogenesis and lymphatic vessel formation in patients with atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC),3Barney N.P. Vernal and atopic keratoconjunctivitis.in: Kratchmer J.H. Mannis M.J. Holland E.J. Cornea. 2nd ed. Mosby, Philadelphia2005: 667-674Google Scholar severe chronic forms of allergic conjunctivitis. AKC and VKC are often accompanied by giant papillae formation in tarsal conjunctivae and/or marked inflammatory cell infiltration in the limbal region, with prominent TH2 cytokine profiles.3Barney N.P. Vernal and atopic keratoconjunctivitis.in: Kratchmer J.H. Mannis M.J. Holland E.J. Cornea. 2nd ed. Mosby, Philadelphia2005: 667-674Google Scholar We found various degrees of lymphoid neogenesis in the giant papillae with B-cell clusters, FDCs, and a marginal T-cell zone essential for TLO formation.1Aloisi F. Pujol-Borrell R. Lymphoid neogenesis in chronic inflammatory diseases.Nat Rev Immunol. 2006; 6: 205-217Crossref PubMed Scopus (713) Google Scholar The IgE class of immunoglobulin is produced by B cells and plasma cells through class-switch recombination in the germinal center of lymphoid tissues.2Gould H.J. Takhar P. Harries H.E. Durham S.R. Corrigan C.J. Germinal-centre reactions in allergic inflammation.Trends Immunol. 2006; 27: 446-452Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Preferential expression of the FcεRI β-chain protein, an amplifier of allergic responses, has been observed in the giant papillae of patients with AKC/VKC.4Matsuda A. Okayama Y. Ebihara N. Yokoi N. Hamuro J. Walls A.F. et al.Hyperexpression of the high-affinity IgE receptor-beta chain in chronic allergic keratoconjunctivitis.Invest Ophthalmol Vis Sci. 2009; 50: 2871-2877Crossref PubMed Scopus (17) Google Scholar These findings suggested the role of an IgE–FcεRI β cascade in the pathophysiology of severe chronic allergic conjunctivitis. The IgE might originate from tears5Ebihara N. Okumura K. Nakayasu K. Kanai A. Ra C. High level of Fc epsilon receptor I-bindable immunoglobulin E in the tear fluid and increased immunoglobulin E-saturated cells in the giant papillae of vernal keratoconjunctivitis patients.Jpn J Ophthalmol. 2002; 46: 357-363Crossref PubMed Scopus (8) Google Scholar or from blood flow; however, local IgE class-switch recombination in the giant papillae itself, as well as in the nasal polyps,6Takhar P. Smurthwaite L. Coker H.A. Fear D.J. Banfield G.K. Carr V.A. et al.Allergen drives class switching to IgE in the nasal mucosa in allergic rhinitis.J Immunol. 2005; 174: 5024-5032Crossref PubMed Scopus (172) Google Scholar might also be possible. Regional IgE expression at the mast cells, dendritic cells, and Langerhans cells of the giant papillae was reported previously.7Montan P.G. Biberfeld P.J. Scheynius A. IgE, IgE receptors, and other immunocytochemical markers in atopic and nonatopic patients with vernal keratoconjunctivitis.Ophthalmology. 1995; 102: 725-732Abstract Full Text PDF PubMed Scopus (41) Google Scholar Therefore it is important to clarify the origin of IgE production in patients with AKC/VKC because anti-IgE antibody therapy has recently been introduced for the treatment of severe atopic asthma, and it might also be effective for AKC/VKC. Ten giant papillae obtained from patients with AKC/VKC were resected for a therapeutic purpose, as previously described.4Matsuda A. Okayama Y. Ebihara N. Yokoi N. Hamuro J. Walls A.F. et al.Hyperexpression of the high-affinity IgE receptor-beta chain in chronic allergic keratoconjunctivitis.Invest Ophthalmol Vis Sci. 2009; 50: 2871-2877Crossref PubMed Scopus (17) Google Scholar The clinical information of the patients is summarized in Table I. All the patients had chronic allergic conjunctivitis and were treated with topical dexamethasone eye drops for at least 4 weeks before surgery. Total IgE concentrations and specific IgE titers against 26 common antigens were measured by SRL, Inc (Tokyo, Japan), using enzyme immunoassay methods. All procedures were approved by the ethics committee of Juntendo University School of Medicine and of Kyoto Prefectural University of Medicine. This study was conducted in accordance with the tenets of the Declaration of Helsinki, and written informed consent was obtained from all patients before their participation in the study. Immunohistochemical staining was performed according to the previously described methods,4Matsuda A. Okayama Y. Ebihara N. Yokoi N. Hamuro J. Walls A.F. et al.Hyperexpression of the high-affinity IgE receptor-beta chain in chronic allergic keratoconjunctivitis.Invest Ophthalmol Vis Sci. 2009; 50: 2871-2877Crossref PubMed Scopus (17) Google Scholar and the list of antibodies is shown in Table II. To examine ε-chain mRNA expression and activation-induced cytidine deaminase (AID) mRNA expression in giant papillae, total RNA was isolated from biopsy specimens of the giant papillae obtained from 5 patients with VKC (see patients 1, 3, 5, 8, and 10 in Table I). cDNA was prepared according to a previously described method.8Matsuda A. Ebihara N. Yokoi N. Kawasaki S. Tanioka H. Inatomi T. et al.Functional roles of thymic stromal lymphopoietin for chronic allergic keratoconjunctivitis.Invest Ophthalmol Vis Sci. 2010; 51: 151-155Crossref PubMed Scopus (42) Google Scholar ε-Chain mRNA and AID mRNA were amplified by using nested PCR methods, as previously described, with modification for the second-round PCR.6Takhar P. Smurthwaite L. Coker H.A. Fear D.J. Banfield G.K. Carr V.A. et al.Allergen drives class switching to IgE in the nasal mucosa in allergic rhinitis.J Immunol. 2005; 174: 5024-5032Crossref PubMed Scopus (172) Google Scholar Touchdown PCR methods were used for the second-round PCR. The primers used for PCR reactions are shown in Table III. The PCR products were verified by means of direct sequencing with VF2 and CεR2 primers (for ε-chain mRNA) and AID2F and AID2R primers (for AID mRNA), respectively. The sequence of the VH region of immunoglobulin was verified by information obtained at the IgBLAST immunoglobulin analysis Web site (http://www.ncbi.nlm.nih.gov/igblast/).Table IClinical information of the patients and summary of the resultsPatient no.Age (y)SexTotal IgE (IU/mL)Specific IgEDiagnosis∗AKC was defined as a bilateral chronic inflammation of the conjunctiva and lids associated with atopic dermatitis. VKC was defined as a bilateral, chronic, conjunctival inflammatory condition of the conjuctiva found in subjects predisposed by their atopic background. Patients who had atopic dermatitis or corneal stromal neovascularization were excluded from the VKC diagnosis.CD20+ cell cluster†Large lymphocyte clusters were defined as those having more than 10 lymphocytes in length/diameter.FDC116F509PositiveVKCLarge clusters+222M89PositiveVKCDiffuse+313M2,319PositiveVKCLarge clusters+418M375PositiveAKCDiffuse and large clusters+517M17,260PositiveAKCLarge clusters+621M1,904PositiveAKCSmall clusters+716M3,763PositiveAKCLarge clusters+834M22,800PositiveAKCLarge clusters+945F28NegativeAKCSmall clusters+1029M56PositiveVKCSmall clusters+∗ AKC was defined as a bilateral chronic inflammation of the conjunctiva and lids associated with atopic dermatitis. VKC was defined as a bilateral, chronic, conjunctival inflammatory condition of the conjuctiva found in subjects predisposed by their atopic background. Patients who had atopic dermatitis or corneal stromal neovascularization were excluded from the VKC diagnosis.† Large lymphocyte clusters were defined as those having more than 10 lymphocytes in length/diameter. Open table in a new tab Table IIList of antibodies used in this studyAntigenClassDilutionSupplierAnti–LYVE-1Rabbit polyclonal1:400RELIATech, Wolfenbüttel, GermanyAnti-CD3Murine monoclonal1:100DAKO, Glostrup, DenmarkAnti-CD20Rabbit monoclonal1:200Epitomics, South San Francisco, CalifAnti-CD35Murine monoclonal1:50DakoAnti-PNAdRat monoclonal (IgM)1:25Becton Dickinson, Franklin Lakes, NJAlexa 488–anti-mouse IgGDonkey polyclonal1:1,000Invitrogen, Carlsbad, CalifAlexa 594–anti-rabbit IgGDonkey polyclonal1:1,000InvitrogenAlexa 488–anti-rabbit IgGGoat polyclonal1:1,000InvitrogenAlexa 594–anti-rat IgMGoat polyclonal1:1,000Invitrogen Open table in a new tab Table IIIOligonucleotide primers used to amplify ε-chain mRNA and AID mRNAPrimer nameSequenceVF25′-AGTCTGGAGCAGAGGTG-3′CεR15′-CAGGACGACTGTAAGATCTTCACG-3′CεR25′-GGGGTGAAGTCCCTGGAGC-3′AIDF15′-GAGGCAAGAAGACACTCTGG-3′AIDR15′-GTGACATTCCTGGAAGTTGC-3′AIDF25′-TAGACCCTGGCCGCTGCTACC-3′AIDR25′-CAAAAGGATGCGCCGAAGCTGTCTGGAG-3′ Open table in a new tab Lymphatic vessel endothelial hyaluronan receptor 1+ (LYVE-1+) lymphatic vessels were observed in the substantia propria of the giant papillae (Fig 1, A), and CD3/CD20 double immunostaining of the adjacent section showed nearby localization of the lymphatic vessels and TLOs (Fig 1, A and B). The TLOs were accompanied by CD35+ FDCs (Fig 1, C). Peripheral node addressin (PNAd)-positive high endothelial venules existed near the LYVE-1+ lymphatic endothelial cells (Fig 1, D). Various degrees of TLOs were observed in all of the AKC/VKC tissues (summarized in Table 1). ε-Chain mRNA and AID mRNA were detected in the cDNA sample obtained from the giant papilla of a patient with VKC (see patient 1 in Table I) by means of nested RT-PCR (Fig 2), and the sequence of the PCR products was verified by means of PCR direct sequencing. No ε-chain mRNA or AID mRNA transcripts were detected in 2 control conjunctivae cDNA samples (samples were obtained during cataract surgery, data not shown).Fig 2RT-PCR analysis of the ε-chain mRNA and AID mRNA expression in the giant papillae tissues. Nested RT-PCR was carried out with cDNA specimens obtained from patients with AKC/VKC (patients 1, 3, 5, 8, and 10 in Table I). The 2% agarose gel electrophoresis showed positive bands (789 bp for ε-chain mRNA and 355 bp for AID mRNA).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Ectopic lymphoid neogenesis has been observed in patients with many chronic inflammatory disorders,1Aloisi F. Pujol-Borrell R. Lymphoid neogenesis in chronic inflammatory diseases.Nat Rev Immunol. 2006; 6: 205-217Crossref PubMed Scopus (713) Google Scholar including rheumatoid synovitis, Sjogren syndrome, and a murine model of asthma. We found the essential structures for ectopic lymphoid neogenesis (clusters of B cells, the T-cell zone, and FDCs) in the giant papillae. PNAd+ high endothelial venules around the TLOs, which are known to supply naive T cells, further suggested ongoing immune responses at the giant papillae of patients with chronic allergic conjunctivitis. The TLOs in the giant papillae might also contribute to regional immune responses against persistent antigens at the ocular surface and thus promote chronic inflammation.1Aloisi F. Pujol-Borrell R. Lymphoid neogenesis in chronic inflammatory diseases.Nat Rev Immunol. 2006; 6: 205-217Crossref PubMed Scopus (713) Google Scholar Another function of lymphoid neogenesis is immunoglobulin class switching in the B-cell clusters.2Gould H.J. Takhar P. Harries H.E. Durham S.R. Corrigan C.J. Germinal-centre reactions in allergic inflammation.Trends Immunol. 2006; 27: 446-452Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar In addition to ε-chain mRNA expression in the giant papillae, we also detected AID mRNA, which encodes an essential enzyme that catalyzes the class-switch recombination process and is expressed only in germinal-center B cells. We also showed IgE plasma cells and IgE+ B cells in the substantia propria of giant papillae (Fig E1). Although we could not exclude the possibility that class-switch recombination in B cells might occur in regional secondary lymphoid organs, our results suggested that direct class-switch recombination in the TLOs of the giant papillae is highly probable, as reported in cases involving nasal polyps.6Takhar P. Smurthwaite L. Coker H.A. Fear D.J. Banfield G.K. Carr V.A. et al.Allergen drives class switching to IgE in the nasal mucosa in allergic rhinitis.J Immunol. 2005; 174: 5024-5032Crossref PubMed Scopus (172) Google Scholar It also should be noted that all of the giant papillae analyzed in this study were treated with topical glucocorticoid eye drops. A previous report9Jabara H.H. Ahern D.J. Vercelli D. Geha R.S. Hydrocortisone and IL-4 induce IgE isotype switching in human B cells.J Immunol. 1991; 147: 1557-1560PubMed Google Scholar showed that glucocorticoid could induce IgE isotype switching in human B cells, and therefore further studies are essential to elucidate the effect of glucocorticoids in giant papillae. In conclusion, we showed the formation of TLOs and some evidence of local IgE production in patients with severe chronic allergic conjunctivitis (AKC/VKC). Regional therapy, including anti-IgE therapy with eye drops, might be effective for the treatment of AKC/VKC.