Presence of Activated Airway T Lymphocytes in Human Puumala Hantavirus Disease

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1 CHEST Presence of Activted Airwy T Lymphocytes in Humn Puuml Hntvirus Disese Originl Reserch CHEST INFECTIONS John Rsmuson, MD ; Jmshid Pourzr, PhD ; Mts Linderholm, MD, PhD ; Thoms Sndström, MD, PhD ; Anders Blomberg, MD, PhD; nd Cls Ahlm, MD, PhD Bckground: Hntviruses cuse two clinicl syndromes: hemorrhgic fever with renl syndrome (HFRS) nd hntvirus pulmonry syndrome (HPS). The clinicl spectrum in HFRS lso often involves respirtory symptoms. As informtion bout the pulmonry pthogenesis in HFRS is limited, we imed to further study the locl irwy immune response in the lower irwys. Methods: Bronchoscopy ws performed in 5 hospitlized ptients with HFRS, with smpling of endobronchil mucosl biopsies nd BAL fluid. Biopsies were stined for leukocytes, lymphocyte subsets, nd vsculr endothelil dhesion molecules. BAL fluid nd blood lymphocyte subsets were determined using flow cytometry. Fourteen helthy volunteers cted s control group. Results: Compred with control subjects, endobronchil mucosl biopsies from ptients with HFRS reveled incresed numbers of CD8 T cells in both epithelium nd submucos ( P.00), long with n increse in submucosl CD4 T cells ( P 5.00). In contrst, ptients submucosl neutrophil nd eosinophil numbers were reduced ( P,.00). The expression of vsculr cell dhesion molecule- (VCAM-) ws enhnced in ptients with HFRS ( P,.00). In ptients with HFRS, nlyses of T-cell subsets in BAL fluid showed higher proportions of CD3 nd CD8 T cells ( P 5.0 nd P 5.025) nd nturl killer cells ( P,.00), together with n incresed expression of ctivtion mrkers humn leukocyte ntigen-dr (HLA-DR) nd CD25 on T cells ( P,.00 nd P,.00). Conclusions: The present findings indicte locl immune response in terms of ctivted T lymphocytes in the lungs of ptients with HFRS. The elevted expression of ctivtion mrkers nd VCAM- further implies the importnce of cytotoxic lymphocytes in the pthogenesis of pulmonry involvement in HFRS. CHEST 20; 40(3): Abbrevitions: Dlco 5 diffusion cpcity of the lung for crbon monoxide; HFRS 5 hemorrhgic fever with renl syndrome; HLA-DR 5 humn leukocyte ntigen-dr; HPS 5 hntvirus pulmonry syndrome; ICAM- 5 intercellulr dhesion molecule-; IQR 5 interqurtile rnge; NK 5 nturl killer; PUUV 5 Puuml hntvirus; SBP 5 systolic BP; TLC 5 totl lung cpcity; VC 5 vitl cpcity; VCAM- 5 vsculr cell dhesion molecule- Hntviruses re emerging viruses cusing severe nd often ftl diseses in humns. The most common route of infection is inhltion of n erosol contining virus tht hs been shed in excret by the hntvirus nturl rodent hosts. 2,3 Two syndromes relted to hntvirus hve been identified: hemorrhgic fever with renl syndrome (HFRS) in Eursi nd hntvirus pulmonry syndrome (HPS) in the Americs. 4-6 HFRS is typiclly chrcterized by fever, hemorrhgi, nd cute renl filure, wheres cute noncrdiogenic pulmonry edem nd circultory shock re hllmrk findings in HPS. Puuml hntvirus (PUUV) cuses milder form of HFRS, previously nmed nephropthi epidemic, in northern nd centrl Europe. 6 Even if renl involvement is the hllmrk of PUUV infection, respirtory symptoms, such s cough or dyspne, re common nd hve been reported in 23% to 50% of the ptients. 7-9 Clinicl studies of pulmonry involvement in HFRS hve reveled lung infiltrtes, pleurl fluid, nd impired pulmonry function. 8,0, Hntviruses infect endothelil cells but re not cytopthic. Immunomedited dysregultion of endothelil permebility is believed to be centrl in the pthogenesis of hntvirus disese. 2,3 A common finding in hntvirus infection is cytotoxic cell response, which, together with irwy inflmmtory cytokines, hs been suggested to ply n importnt role in CHEST / 40 / 3 / SEPTEMBER, Downloded from chestjournl.chestpubs.org by Kimberly Henricks on September 7, Americn College of Chest Physicins

2 pthogenesis.4-20 In vitro studies hve shown tht humn pthogenic hntviruses cn induce cell dhesion molecules in infected endothelil cells. 2 Studies of ftl HPS cses hve reveled mononucler cell infiltrtes in pulmonry tissue consisting lrgely of CD4 nd CD8 T cells. 22,23 There is still no evidence tht this is lso the cse for ptients with HFRS. However, in study of PUUV-infected cynomolgus mcques, focl lymphocyte infiltrtes of minly CD8 T cells were found in smples from kidney, lung, nd hert. 24 In ptients with HFRS, CD8 T cells hve been found in kidney biopsies, further implying the generl importnce of cytotoxic immune response in the pthogenesis.25 Bronchoscopy nd BAL hve been widely used to dignose respirtory infections Although this pproch provides unique opportunities to explore the locl irwy immune response in vivo, studies on humn respirtory virl infections re limited To our best knowledge, there is only one previous study of hntvirus infection, in which the locl immune response in terms of incresed numbers of CD8 T cells, ctivted mcrophges, nd nturl killer (NK) cells were found in irwy lvge fluid in smll number of ptients with HFRS. 4 However, the locl immune response in pulmonry tissue hs not been evluted. Thus, our im ws to further investigte the irwy immune response in ptients with cute HFRS to improve the understnding of the pulmonry involvement nd pthogenesis of the disese. Ptients Mterils nd Methods Fifteen hospitlized ptients with HFRS (six women, medin ge 48 yers; rnge yers) were included in prospective study t the Deprtment of Infectious Disese t the University Mnuscript received October 28, 200; revision ccepted Februry 9, 20. Affilitions: From Infectious Diseses, Deprtment of Clinicl Microbiology (Drs Rsmuson, Linderholm, nd Ahlm), nd Respirtory Medicine, Deprtment of Public Helth nd Clinicl Medicine (Drs Pourzr, Sndström, nd Blomberg), Umeå University, Umeå, Sweden. Funding/Support: This work ws supported with grnts from the Swedish Hert Lung Foundtion, the Hert Foundtion of Northern Sweden, the County Councils of Northern Sweden, the County Council of Västerbotten, nd the Medicl Fculty of Umeå University. Dr Blomberg is the holder of the Lrs Werkö Distinguished Reserch Fellowship from the Swedish Hert Lung Foundtion. Correspondence to: John Rsmuson, MD, Infectious Diseses, Deprtment of Clinicl Microbiology, Umeå University, SE-90 85, Umeå, Sweden; e-mil: john.rsmuson@climi.umu.se 20 Americn College of Chest Physicins. Reproduction of this rticle is prohibited without written permission from the Americn College of Chest Physicins ( site/misc/reprints.xhtml ). DOI: 0.378/chest Hospitl, Umeå, Sweden. Ech cse ws verified by the presence of PUUV-specific IgM nd IgG in serum. 35 There ws no evidence of concurrent bcteril infection or history of chronic pulmonry or systemic inflmmtory disese in ny ptient. In ll ptients, bronchoscopy ws performed 5 to 0 dys (medin 8 dys) fter onset of symptoms. Peripherl blood smples for flow cytometry nlysis were tken during the cute phse fter 5 to 0 dys (medin 8 dys), nd t follow-up dy 39 to 77 (n 5 7, medin 67). Rdil rtery blood ws smpled from 2 ptients brething room ir, within the first 2 dys fter dmission, nd ws nlyzed ccording to hospitl routine. Pulmonry function tests were performed 4 to 9 dys (medin 7 dys, n 5 5), nd t follow-up fter 5 to 768 dys (medin 59 dys, n 5 3) fter outbrek of disese. As control group, 4 helthy nonsmokers (four femle, medin ge 23 yers; rnge 2-27 yers) were lso exmined with the sme procedures. The project ws pproved by the Reserch Ethics Committee of Umeå University (245/93); ll ptients gve their informed written consent, nd the study ws crried out ccording to the declrtion of Helsinki. Bronchoscopy, Biopsy Smpling, nd BAL Premediction with morphine-scopolmine ws given subcutneously 30 min before strt of bronchoscopy. Topicl nesthesi ws obtined with lidocine. Additionl lidocine ws dministered in the lrynx nd bronchi during the procedure. A flexible video bronchoscope (Olympus BF IT200; Tokyo, Jpn) ws inserted through the mouth vi plstic mouthpiece, with the ptient in the supine position. At ech bronchoscopy, four to six endobronchil biopsies were tken from the min crin nd the min bronchil divisions on the left side using fenestrted forceps (Olympus FB-2C). BAL fluid ws retrieved from the right middle lobe fter infusion of ml liquots of phosphte-buffered sline (PBS). Differentil cell counts were performed on cells obtined from recovered fluid, s previously described. 36 Biopsy Processing nd Quntifiction of Cells by Immunohistochemistry Endobronchil mucosl biopsies obtined during bronchoscopy were processed into glycol methcrylte resin (Polyscience; Northmpton, Englnd), s previously described. 37 Sections from biopsies were cut t 2-μm thicknesses nd stined with monoclonl ntibodies, which were purchsed from Dko (Glostrup, Denmrk), except humn nti-vsculr cell dhesion molecule- (VCAM-) nd pnendothelil ntibody (EN4), which were purchsed from Serotec (Oxford, Englnd) nd Monosn (Uden, The Netherlnds), respectively. Immunostining ws performed s previously described. 36 Briefly, endogenous peroxidses were inhibited using sodium zide nd hydrogen peroxide. After wshing in Tris-buffered sline, nonspecific ntibody binding ws blocked with undiluted culture medium, Dulbecco modified Egle medium (Sigm; St. Louis, Missouri ). Primry ntibodies were then pplied nd incubted overnight. After rinsing with Tris-buffered sline, the biotinylted rbbit nti-mouse (IgG F[b 9 ] 2 ; Dko) ws used s the secondry ntibody nd incubted for 2 h. The streptvidin-biotin horserdish-peroxidse complex (Dko) ws dded s the enzyme link nd incubted for nother 2 h. The sections were then visulized with minoethylcrbzole nd counterstined with Myer hemtoxylin. The immunostining nlysis ws performed by one investigtor (J. P.) blinded to the subject groups. The number of positive cells ws expressed s cells/mm nd cells/mm 2 of epithelium nd submucos, respectively. Quntifiction of vsculr endothelil dhesion molecules in the submucosl blood vessels ws crried out by expressing the number of vessels stined with the specific ntibodies s percentge of 76 Originl Reserch Downloded from chestjournl.chestpubs.org by Kimberly Henricks on September 7, Americn College of Chest Physicins

3 the totl number of vessels reveled by stining with the pnendothelil ntibody. 36 Flow Cytometry Anlysis Lymphocyte subsets in blood nd BAL were determined by flow cytometry s performed previously. 4 In short, BAL cell numbers were djusted to finl concentrtion of 0 6 cells/ml. Dul-color fluorescence conjugted ntibodies (Becton Dickinson; Sn Jose, Cliforni) were dded to ech test tube contining BAL cells or blood nd llowed to bind for 30 min t 4 C in drkness. After lysing red cells, the remining cells were wshed twice by dding PBS to the tubes nd centrifuged t 4 C for 0 min, g. Cells were then fixed before nlysis using FACScn (Becton Dickinson) flow cytometer. A totl of 0,000 events were collected per smple. The lymphocyte popultion ws gted bsed on their physicl chrcteristic forwrd sctter nd side sctter profiles. The lymphocyte subsets dt were expressed s percentge of gted lymphocytes. Lung Function The lung function of ptients with HFRS ws investigted in cute nd convlescent phse. Vitl cpcity (VC), totl lung cpcity (TLC), FEV, nd diffusion cpcity of the lung for crbon monoxide (D lco ), using the single breth method, were evluted using computerized Jeger equipment (Würzburg, Germny). 8 Sttisticl Anlysis SPSS, version 9.0 (IBM; Somers, New York) ws used for ll sttisticl nlysis. Results re expressed s medin nd interqurtile rnge (IQR), unless otherwise stted. As most vribles involved smll smple numbers, nonprmetric tests were used. Mnn-Whitney U test ws used for group comprison nd Wilcoxon signed rnks test for pired within-group observtions. All tests were two-tiled, nd P vlue, 0.05 ws considered significnt. Clinicl Dt Results All 5 ptients experienced symptoms typicl for HFRS. Almost ll ptients hd thrombocytopeni nd renl impirment during the course of infection, nd clinicl chrcteristics re summrized in Tble. Seven ptients suffered from respirtory symptoms, such s dry cough nd dyspne. Arteril blood gs nlysis reveled subnorml results for P o 2, 9.53 kp ( kp) nd oxygen sturtion, 95.6% (93.6%-96.4%), with other prmeters principlly within norml rnge. Seven out of 2 ptients were hypoxemic (P o 2, 0 kp), out of which two ptients required supplementl nsl oxygen tretment. Four ptients hd pthologic chest rdiogrph with pulmonry infiltrtes. Three ptients were hypotensive (systolic BP [SBP], 00 mm Hg) on dmission, two were treted t the ICU for hypotension (SBP 70 nd 65 mm Hg), none required dilysis, nd ll ptients survived. Lung Function In cute phse, ptients with HFRS showed subnorml (, 80% predicted) results for D lco (75.4%, 63.%-95.9%), wheres VC (05%, 83%-20.3%), FEV (99.7%, 75.3%-20%), nd TLC (02%, 88.3%-9%) were within norml rnges. In convlescent phse there ws significnt increse in D lco (94.3%, 88.%-0.6%, P 5.007), but lso in VC (8%, 05.3%-28%, P 5.005) nd FEV (8%, Tble Clinicl Bsic Chrcteristics of 5 Ptients With HFRS Ptient No. Sex-Age, y Smoker Dys in Hospitl Respirtory Symptoms Blood Gs P o 2 b Pek Cretinine Levelc Ndir TPCd Pek WBC Count e F-50 ES 5 No M-66 Yes 8 Yes M-47 No 7 Yes F-59 No 4 No F-23 No 3 No M-53 Yes 6 Yes M-44 Yes 6 No M-39 ND 5 No F-22 No 8 Yes M-66 No 5 Yes M-55 Yes 5 No M-35 Yes 6 Yes F-52 No 0 No, F-48 No 6 Yes M-45 Yes 7 No ES 5 ex-smoker; F 5 femle; HFRS 5 hemorrhgic fever with renl syndrome; M 5 mle; ND 5 no dt; TPC 5 thrombocyte prticle concentrtion. Ptients experiencing respirtory symptoms (n 5 7), dry cough (n 5 5), nd dyspne (n 5 3). b Blood gs results for P o 2, kp, when brething room ir; reference. 0 kp. c Serum cretinine, reference, 0 m mol/l for women,, 5 m mol/l for men; ptient medin 347 mmol/l. d TPC, norml rnge /L; ptient medin /L. e WBC count; norml rnge /L; ptient medin /L. CHEST / 40 / 3 / SEPTEMBER, Downloded from chestjournl.chestpubs.org by Kimberly Henricks on September 7, Americn College of Chest Physicins

4 98.9%-26.%, P 5.006), compred with the cute phse. There ws no significnt chnge in TLC (06.4%, 98.3%-2.5%, P 5.308). Bronchil Biopsies Compred with control subjects, the bronchil biopsies of ptients with HFRS reveled elevted numbers of CD8 T cells ( Fig ) both in the submucos nd the epithelium ( P 5.00 nd P,.00), wheres CD4 T cells were only incresed in the submucosl region ( P 5.00) ( Tble 2 ). Neutrophil nd eosinophil numbers were reduced in the submucos ( P,.00 nd P 5.009, respectively) when compring ptients with HFRS nd control subjects. Compred with control subjects, there ws n increse in the vsculr endothelil expression of VCAM- ( P,.00) but not of intercellulr dhesion molecule- (ICAM-) nd P-selectin, in ptients with HFRS ( Tble 2 ). In ptient No. 2, no biopsy smples were tken becuse of ptient discomfort during bronchoscopy. In ptient No. 6, biopsies were not possible to nlyze. There were no significnt ssocitions between the numbers of cells or vsculr dhesion molecule expression nd clinicl severity expressed s number of dys in hospitl, lowest SBP, mximl cretinine nd leukocyte count, ndir pltelet count, D lco, or blood gs P o 2 (Spermn r, dt not shown). Also, there ws no significnt difference in the inflmmtory irwy response between ptients with nd without respirtory symptoms (dt not shown). BAL Fluid Cell Count Compred with control subjects, ptients with HFRS hd lower numbers of totl leukocytes ( P 5.050), mostly explined by reduced numbers of mcrophges ( P 5.036) nd neutrophils ( P,.00). Also, the neutrophil proportion ws reduced in ptients with HFRS compred with control subjects ( P,.00), s other cell subsets did not differ significntly between the groups ( Tble 3 ). Flow Cytometry of BAL Fluid nd Peripherl Blood In BAL fluid of ptients with HFRS, there were incresed proportions of NK cells ( P,.00), CD3 nd CD8 T cells ( P 5.0 nd P 5.040), long with T lymphocytes expressing ctivtion mrkers CD25 ( P,.00) nd humn leukocyte ntigen-dr (HLA-DR) ( P,.00), compred with control subjects ( Tble 4 ). A similr pttern, except for the proportion of T lymphocytes, ws demonstrted in peripherl blood ( Tble 4 ). When compring the flow cytometry findings of peripherl blood between the cute nd convlescent phses of HFRS, there ws significnt decrese in the proportion of T lymphocytes expressing CD25 ( P 5.04) nd HLA-DR ( P 5.08), together with trend towrd significnt decrese in CD8 cells expressing HLA-DR ( P 5.08). NK cells nd other lymphocyte subsets displyed similr proportions between the two different phses ( Tble 5 ). Surprisingly, ptients with HFRS in convlescent phse still expressed n incresed proportion of blood NK cells (36.5, vs 6.4, ; P 5.020), s well s ctivted T lymphocytes, CD25 (6, 2-9 vs.2, ; P 5.004) nd HLA-DR (6, 3-7 vs, ; P 5.00), when compred with helthy control subjects. Discussion The respirtory trct is the min route of hntvirus entry, nd ptients with both HPS nd HFRS my develop moderte to severe pulmonry symptoms. 4,5,38 In the North nd South Americn forms of hntvirus infection, overt lung filure is common cuse of deth, nd so fr there is no efficient specific Figure. Bronchil biopsy specimen reveling high numbers of CD8 T cells in bronchil epithelil (E) nd submucosl (S) regions in ptient with HFRS, s compred with helthy control subject. Visuliztion ws performed using the immunohistochemicl method, nd positive cells were determined by red ring stining pttern. Br represents 50 m m. HFRS 5 hemorrhgic fever with renl syndrome. 78 Originl Reserch Downloded from chestjournl.chestpubs.org by Kimberly Henricks on September 7, Americn College of Chest Physicins

5 Tble 2 Leukocyte Subsets nd Adhesion Molecules Determined by Immunohistochemistry of Bronchil Biopsies in 3 Ptients With HFRS, Compred With 4 Helthy Control Subjects Cell Subset-Mrker (Locliztion) Ptients With HFRS Helthy Control Subjects P Vlue T cells-cd3 (epithelium) 6 (2-9) 0.5 (0-.3).00 T cells-cd3 (submucos) 30 (8.5-79) 8 ( ).089 Th cells-cd4 (epithelium) 0 (0-0.5) 0 (0-0).062 Th cells-cd4 (submucos) 0 ( ) 0 (0-3.5).00 Tc cells-cd8 (epithelium) 5 (2-8) 0 (0-0),.00 Tc cells-cd8 (submucos) 7 (2.5-4) 4 (0-8).00 Neutrophils-NE (epithelium) 0 (0-2).2 (0-4).089 Neutrophils-NE (submucos) 27 (7-64.5) 0. ( ),.00 Eosinophils-ECP (epithelium) 0 (0-0) 0 (0-0).000 Eosinophils-ECP (submucos) 0 (0-0.5) 5.2 (0-.7).009 VCAM- 9 (3-27.6) 4.6 (.-7.9),.00 ICAM- 50 ( ) 55. ( ).942 P-selectin 47 ( ) 39.9 (3-60.3).482 Vlues re given s the medin (IQR) number of positive cells per mm 2 of submucos or mm of epithelil length for ech mrker. For VCAM-, ICAM-, nd P-selectin, vlues re medin (IQR) proportion (%) of submucosl vsculr endothelium expressing the respective molecule. ECP 5 eosinophil ction protein; ICAM- 5 intercellulr dhesion molecule-; IQR 5 interqurtile rnge; NE 5 neutrophil elstse; Tc 5 T cytotoxic; Th 5 T helper; VCAM- 5 vsculr cell dhesion molecule-. See Tble legend for expnsion of other bbrevitions. Determined by Mnn-Whitney U test. tretment vilble. Consequently, incresed knowledge bout the locl immune response in the lungs is importnt to understnd the pthogenesis of the disese in order to develop new therpeutic strtegies. In the present study, pproximtely one-hlf of the ptients suffered from respirtory symptoms. Blood gs nlysis reveled tht. 50% were hypoxemic, nd compred with the convlescent phse, the mjority hd impired pulmonry gs diffusion cpcity, which is in concordnce with previous study. 8 Studies of the pulmonry immune response in other humn respirtory virl infections re few nd often include subjects with chronic irwy inflmmtion, such s sthm To the best of our knowledge, this is the first report to describe the locl irwy immune response in hntvirus infection using endobronchil mucosl biopsies. Our most pertinent findings were the mrked increse in T lymphocyte numbers, minly the CD8 subset tht ws found in the pulmonry tissue of ptients with HFRS, together with the expnsion of NK cells nd ctivted T lymphocytes found in BAL fluid nd blood. This suggests tht locl cytotoxic response is induced by the hntvirl infection. Even though presence of Puuml virus in lung tissue ws not determined in the study, this hs been shown in recent report in which virus ws detected in cpillry endothelil cells nd mononucler cells long with infiltrtes of CD8 T cells in the lungs of two ftl HFRS cses. 39 A similr pttern hs lso been found in n niml model 24 nd in the lungs of ftl HPS cses. 22,23 Furthermore, n experimentl study reveled tht virus-specific CD8 cells my induce lekge in hntvirus-infected endotheli, 2 nd the proportion of such cells cn predict disese severity in HPS. 8 Cytotoxic CD8 T cells exert their effect by secretion of proinflmmtory cytokines nd cytotoxins to induce poptosis in infected cells, 40 which hs been shown in ptients with HFRS. 20 Tble 3 Cell Count nd Differentil Proportion of Leukocytes in BAL Fluid in 5 Ptients With HFRS, Compred With 3 Helthy Control Subjects Mesure Ptients With HFRS Helthy Control Subjects P Vlue Cell count Totl leukocytes 9.8 ( ) 2.0 ( ).050 Neutrophils 0.06 ( ) 0.29 ( ),.00 Mcrophges 6.96 ( ) 0.2 ( ).036 Lymphocytes 0.85 ( ).50 ( ).222 Eosinophils 0 (0-0.) 0.02 (0-0.05).307 Differentil percentge Neutrophils (-) 3 (.8-3.6),.00 Mcrophges 79 (74-9) 85 ( ).49 Lymphocytes 20 (6-23) 2.4 ( ).22 Eosinophils 0 (0-) 0.2 (0-0.4).304 Vlues re expressed s medin (IQR). Cell count vlues re /L. See Tble legend for expnsion of other bbrevitions. Determined by Mnn-Whitney U test. CHEST / 40 / 3 / SEPTEMBER, Downloded from chestjournl.chestpubs.org by Kimberly Henricks on September 7, Americn College of Chest Physicins

6 Tble 4 Flow Cytometry Results in BAL Fluid nd Peripherl Blood in 5 Ptients With Acute HFRS, Compred With 3 Helthy Control Subjects Cell Subset Mrkers Ptients With HFRS Helthy Control Subjects P Vlue BAL fluid T cells CD3 73 (68-87) 48. ( ).0 Th cells CD3 CD4 29 (20-52) 20.5 ( ).279 Tc cells CD3 CD8 36 (8-49) 3.7 (.7-3.2).040 NK cells CD3 2 CD6 CD56 5 (3-7) 0.5 (0.2-.3),.00 B cells CD9 0 (0-) 0.5 (0.-).373 Activted T cells CD3 CD25 8 (4-0).2 ( ),.00 Activted T cells CD3 HLA-DR 32 (2-36) 0.6 (0.4-),.00 Activted Th cells CD4 HLA-DR 22.5 ( ) b ND N/A Activted Tc cells CD8 HLA-DR 26.5 ( ) b ND N/A Peripherl blood T cells CD3 67 (6-74) 69.2 ( ).549 Th cells CD3 CD4 35 (30-43) 36.9 ( ).836 Tc cells CD3 CD8 28 (23-34) 29. ( ).420 NK cells CD3 2 CD6 CD56 20 (0-27) 6.4 (4.3-7.),.00 B cells CD9 4 (0-6) 9.9 ( ).042 Activted T cells CD3 CD25 (7-6).2 (0.7-2.),.00 Activted T cells CD3 HLA-DR 3 (9-5) (0.7-2.),.00 Activted Th cells CD4 HLA-DR 4 (3.8-6) b ND N/A Activted Tc cells CD8 HLA-DR 2.5 (5.8-9) b ND N/A Vlues re percentge of cells within the lymphocyte popultion positive for the respective mrkers, nd re expressed s medin (IQR). HLA-DR 5 humn leukocyte ntigen-dr; N/A 5 not pplicble; NK 5 nturl killer. See Tble nd 2 legends for expnsion of other bbrevitions. Determined by Mnn-Whitney U test. b No. of ptients 54. In mesles virl pneumoni, incresed numbers of CD8 T cells hve been found in BAL fluid nd believed to be importnt in the pthogenesis. 29 For nother respirtory virus, influenz A, the importnce of pulmonry cytotoxic T-cell response hs been well demonstrted in niml models; however, this is scrcely evluted in vivo in humns I n 2009 influenz A(HN) experimentlly infected pigs, Khtri et l 43 demonstrted virl repliction in the respirtory trct, cusing secretion of proinflmmtory cytokines nd ctivted cytotoxic T-cell response in the irwys. In contrst to hntvirus, influenz virus commonly cuses lesions in the respirtory trct, which were found in the nimls. Endothelil cell dhesion molecules recruit leukocytes to sites of infection, nd hve been demonstrted to ply n importnt role in the pthogenesis of vrious hemorrhgic fevers Levels of soluble VCAM- in plsm were incresed in ptients with Hntn virus-cused HFRS nd ssocited with disese severity. 45 Similrly, in PUUV-infected ptients, we demonstrte notble increse in the pulmonry vsculr endothelil expression of VCAM-, without ny significnt ssocition with disese severity or the mgnitude of the T lymphocyte response, however, possibly due to the reltively smll number of ptients. The findings of reduced neutrophil nd eosinophil numbers in the lungs of the studied ptients Tble 5 Flow Cytometry Results in Peripherl Blood in Ptients With Acute-Phse HFRS, Compred With the Convlescent Phse Cell Subset Mrkers Acute Phse (n 5 5) Convlescent Phse (n 5 7) P Vlue T cells CD3 67 (6-74) 68 (50-78).672 Th cells CD3 CD4 35 (30-43) 33 (28-46).499 Tc cells CD3 CD8 28 (23-34) 24 (8-30).686 NK cells CD3 2 CD6 CD56 20 (0-27) 36.5 ( ).6 B cells CD9 4 (0-6) 3 (9-8).075 Activted T cells CD3 CD25 (7-6) 6 (2-9).04 Activted T cells CD3 HLA-DR 3 (9-5) 6 (3-7).08 Activted Th cells CD4 HLA-DR 4 (3.8-6) b 3.5 (2.8-5).29 Activted Tc cells CD8 HLA-DR 2.5 (5.8-9) b 5.5 (3.3-7).080 Vlues re percentge of cells within the lymphocyte popultion positive for the respective mrkers, nd re expressed s medin (IQR). See Tble, 2, nd 4 legends for expnsion of bbrevitions. Determined by Wilcoxon signed rnks test. b Number of ptients Originl Reserch Downloded from chestjournl.chestpubs.org by Kimberly Henricks on September 7, Americn College of Chest Physicins

7 compred with the helthy control subjects re intriguing, nd could not esily be explined. Interestingly, in contrst to the immune response in the lungs, but in greement with previous study, we could not detect ny expnsion of CD8 T cells in peripherl blood. 4 One could speculte bout potentil lymphocyte homing to sites of infection s consequence of the high expression of VCAM- in the irwy vsculr endothelium. In n niml model for influenz A, Mrshll et l 47 demonstrted pulmonry locliztion of ctivted effector CD8 T cells. CD8 T cells hve been suggested to ply n importnt role in the pthogenesis of hntvirus disese, possibly by cytotoxic effect ginst infected endothelium resulting in endothelil cell dmge nd vsculr lekge syndrome tht is the hllmrk finding in hntvirus disese. 2,45 Our findings of n ctivted locl immune response with minly cytotoxic profile, together with the upregultion of VCAM-, my be n dditionl indiction of this phenomenon. In conclusion, this study demonstrtes the presence of ctivted T lymphocytes in the irwys of ptients with HFRS, minly chrcterized by the expnsion of CD8 cells nd NK cells, indicting locl cytotoxic immune response. We believe tht bronchoscopy is useful tool to explore the locl immune response to gin further understnding of the pthogenesis lso in other importnt virl respirtory infections. Acknowledgments Author contributions: Drs Rsmuson nd Pourzr hd ccess to nd tke responsibility for the integrity of the dt nd the ccurcy of the dt nlysis. Dr Rsmuson: contributed to collection, nlysis, nd interprettion of dt nd ws principlly involved in the preprtion of the mnuscript. Dr Pourzr: contributed to the collection of smples, nlysis nd interprettion of dt, nd reviewed the mnuscript. Dr Linderholm: contributed to study design, inclusion of ptients, collection of smples, nd finl review of the mnuscript. 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