Feb 1, 2014 - Ioannis Morianos, Ph.D. Biomedical Research Foundation of the Academy of Athens. Athens, Greece. Aikaterini Tsoutsa, Ph.D. Panagiota Kara ...
CORRESPONDENCE fibers) was unaffected (6). Direct contacts have been observed between reticular fibers and migrating lymphocytes (1). Strikingly, we indeed observed a strong expression of CXCL13 lining the reticular fiber network of tertiary lymphoid follicles in lung sections from patients with PAH, but no detectable expression in B lymphocytes (4). Further, here we provide evidence that the same pattern of CXCL13 expression on reticular fibers can be observed in lung sections from patients with COPD (Figure 1). Considering the image provided by Litsiou and colleagues, it is difficult to conclude that CXCL13 is produced by B cells in vivo. The staining pattern observed could also well be attributed to cell types other than B cells, such as FDC (4), that are found in the same area as B cells in lymphoid follicles. The difference in the staining/interpretation of CXCL13 pattern of expression (cell-like vs. fiber-like staining) could be explained by the treatment of the tissue (formalin fixation and paraffin embedding vs. frozen tissue), difference in antibodies used, or difference in the staining technique (immunohistochemistry vs. immunofluorescence). Thus, whatever the source of CXCL13, which might be produced by many cell types as underlined above (FDCs, B cells, etc.), CXCL13 is likely to be trapped on reticular fibers through binding to collagen IV, to mediate its function, and this has to be integrated in any model discussing the role of CXCL13 in lymphoid neogenesis in COPD (6). n Author disclosures are available with the text of this letter at www.atsjournals.org. Sylvia Cohen-Kaminsky, Ph.D. Benoˆıt Ranchoux, M.Sc. Fred ´ eric ´ Perros, Ph.D. Universite´ Paris-Sud Le Kremlin-Bicetre, ˆ France INSERM UMR-S 999 Le Plessis-Robinson, France Centre Chirurgical Marie Lannelongue Le Plessis-Robinson, France and AP-HP, DHU TORINO, Hopital ˆ Bicetre ˆ Le Kremlin-Bicetre, ˆ France
References 1. Bajenoff ´ M. Stromal cells control soluble material and cellular transport in lymph nodes. Front Immunol 2012;3:304. 2. Breitfeld D, Ohl L, Kremmer E, Ellwart J, Sallusto F, Lipp M, Forster ¨ R. Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J Exp Med 2000;192:1545–1552. 3. Neyt K, Perros F, GeurtsvanKessel CH, Hammad H, Lambrecht BN. Tertiary lymphoid organs in infection and autoimmunity. Trends Immunol 2012;33:297–305. 4. Litsiou E, Semitekolou M, Galani IE, Morianos I, Tsoutsa A, Kara P, Rontogianni D, Bellenis I, Konstantinou M, Potaris K, et al. CXCL13 production in B cells via Toll-like receptor/lymphotoxin receptor signaling is involved in lymphoid neogenesis in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013;187:1194–1202. 5. Perros F, Dorfmuller ¨ P, Montani D, Hammad H, Waelput W, Girerd B, Raymond N, Mercier O, Mussot S, Cohen-Kaminsky S, et al. Pulmonary lymphoid neogenesis in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med 2012;185:311–321. 6. Yang B-G, Tanaka T, Jang MH, Bai Z, Hayasaka H, Miyasaka M. Binding of lymphoid chemokines to collagen IV that accumulates in the basal lamina of high endothelial venules: its implications in lymphocyte trafficking. J Immunol 2007;179:4376–4382. 7. Monin L, Khader SA. B cells produce CXCL13 in lymphoid neogenesis during chronic obstructive pulmonary disease: the new kid on the block? Am J Respir Crit Care Med 2013;187:1162–1164.
Copyright © 2014 by the American Thoracic Society
Reply From the Authors: We thank Cohen-Kaminsky and colleagues for their comments on our study (1). We agree that CXCL13 binds to regions where collagen is strongly expressed, creating the reticular pattern of expression inside the lymphoid follicles that Perros and colleagues show in their studies (2). Having observed a cell-like pattern of CXCL13 expression inside the lymphoid follicles of patients with chronic obstructive pulmonary disease (COPD) using immunohistochemistry staining, we were very keen to show CXCL13 production by lung B cells using immunofluorescence
Figure 1. Sections of 6-mm-thick paraffin-embedded lung samples from patients with chronic obstructive pulmonary disease with lymphoid follicles (LFs) were analyzed by immunofluorescence confocal microscopy with antibodies against B cells (CD79a, red) and CXCL13 (green). All sections were counterstained with 49,6-diamidino-2-phenylindole (DAPI, blue). Insets show a higher magnification of the area marked with the white quadrant. Scale bars = 50 mm.
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CORRESPONDENCE staining. Similarly to Perros and colleagues, we have also detected CXCL13 staining in acellular areas of B cell–rich lymphoid follicles, but we could not detect intracellular staining in any of the cell populations present (Figure 1). This is presumably due to the low intensity of the signal and the relatively low amplification achieved by immunofluorescence staining compared with immunohistochemistry. We have therefore used a much more sensitive method based on fluorescence-activated cell sorting and real-time PCR, and found that in lung samples of patients with COPD, highly purified fluorescence-activated cell sorting–sorted CD191 B cells but not CD452 stromal cells are the dominant populations that express high levels of CXCL13 mRNA in vivo (1). In addition, in culture, purified lung B cells from these patients produce CXCL13 protein that is up-regulated by stimuli relevant to COPD pathogenesis (1). These data combined provide solid evidence for the role of B cells in CXCL13 production. n Author disclosures are available with the text of this letter at www.atsjournals.org. Eleni Litsiou, Ph.D. Evaggelismos General Hospital Athens, Greece Maria Semitekolou, Ph.D. Ioanna Galani, Ph.D. Ioannis Morianos, Ph.D. Biomedical Research Foundation of the Academy of Athens Athens, Greece Aikaterini Tsoutsa, Ph.D. Panagiota Kara, M.D. Dimitra Rontogianni, M.D. Ion Bellenis, M.D. Evaggelismos General Hospital Athens, Greece Maria Konstantinou, B.Sc. Konstantinos Potaris, M.D., Ph.D. Sotiria Chest Hospital Athens, Greece Evangelos Andreakos, Ph.D. Paschalis Sideras, Ph.D. Biomedical Research Foundation of the Academy of Athens Athens, Greece Spyros Zakynthinos, M.D., Ph.D. Maria Tsoumakidou, M.D., Ph.D. Evaggelismos General Hospital Athens, Greece
References 1. Litsiou E, Semitekolou M, Galani IE, Morianos I, Tsoutsa A, Kara P, Rontogianni D, Bellenis I, Konstantinou M, Potaris K, et al. CXCL13 production in B cells via Toll-like receptor/lymphotoxin receptor signaling is involved in lymphoid neogenesis in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013;187: 1194–1202. 2. Perros F, Dorfm uller ¨ P, Montani D, Hammad H, Waelput W, Girerd B, Raymond N, Mercier O, Mussot S, Cohen-Kaminsky S, et al. Pulmonary lymphoid neogenesis in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med 2012;185: 311–321.
Copyright © 2014 by the American Thoracic Society
Correspondence
Surgical Lung Biopsy over Bronchoalveolar Lavage in Chronic Hypersensitivity Pneumonitis To the Editor: We read with great interest the article by Hodnett and Naidich (1), which presents a diagnostic algorithm for delineating among fibrotic interstitial lung diseases (FILDs)—specifically idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia (NSIP), and chronic hypersensitivity pneumonitis (HP)—according to high-resolution computed tomography (HRCT) radiographic pattern(s). HRCT imaging is an integral diagnostic tool in accurate FILD classification, which is essential for prognostication and therapeutic assessment in clinical trials. We agree with the emphasis on lobular air-trapping, centrilobular nodules, and relative basilar sparing as radiographic features inconsistent with a usual interstitial pneumonia pattern that should prompt further diagnostic investigation and consideration of HP. However, the authors de-emphasize the role of surgical lung biopsy in patients with suggested HP in favor of performing bronchoalveolar lavage (BAL). Given the inconsistent and contradictory data on BAL findings in fibrotic HP, the proposed dependence on BAL analysis over surgical lung biopsy is unjustified. Vourlekis and colleagues demonstrated significantly lower mean BAL total lymphocyte count in subjects with HP with fibrosis (15 6 31 3 106 cells/ml) compared with those without fibrosis (46 6 31 3 106 cells/ml) (2). American Thoracic Society guidelines acknowledge that a BAL lymphocyte differential count greater than 50% suggests HP or cellular NSIP (3). However, in unpublished analysis of a previously described HP cohort, we found a lymphocyte differential count greater than 50% in only 25% of patients (2 of 8) (4). Furthermore, Ohshimo and colleagues evaluated BAL utility in patients with suspected idiopathic pulmonary fibrosis, and demonstrated BAL lymphocytosis greater than 30% in 8% (6 of 74) of patients, in which further pathologic analysis ultimately classified three patients as NSIP and three as HP (5). Based upon literature to date, BAL lymphocytosis has an unproven role in differentiating HP from other FILDs due to both poor sensitivity, likely due to variances in HP phenotype, antigen exposure, smoking use, and/or exacerbations, and specificity, due to elevated levels in NSIP. Further studies are needed to evaluate novel BAL biomarkers that could have better diagnostic utility in differentiating HP from other FILDs. For now, we advocate that surgical lung biopsy remain the standard for improving the diagnostic certainty of FILD, specifically HP. n
Author disclosures are available with the text of this letter at www.atsjournals.org. Joshua J. Mooney, M.D. Stanford University Stanford, California Laura L. Koth, M.D. University of California San Francisco San Francisco, California
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