of September 30, 2014.This information is current as

Fibrosis PatientsDifferentiation in Idiopathic Pulmonary Plasma B Lymphocyte Stimulator and B Cell

Sciurba and Steven R. DuncanKaminski, Joseph M. Pilewski, Michael Donahoe, Frank C. Soejima, Marc C. Levesque, Kevin F. Gibson, NaftaliJiangning Tan, Eva Csizmadia, Leo Otterbein, Makoto Jianmin Xue, Daniel J. Kass, Jessica Bon, Louis Vuga,

http://www.jimmunol.org/content/191/5/2089doi: 10.4049/jimmunol.1203476July 2013;

2013; 191:2089-2095; Prepublished online 19J Immunol 

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DC1.htmlhttp://www.jimmunol.org/content/suppl/2013/07/22/content.1203476.

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The Journal of Immunology

Plasma B Lymphocyte Stimulator and B Cell Differentiationin Idiopathic Pulmonary Fibrosis Patients

Jianmin Xue,*,1 Daniel J. Kass,*,1 Jessica Bon,* Louis Vuga,* Jiangning Tan,*

Eva Csizmadia,† Leo Otterbein,† Makoto Soejima,* Marc C. Levesque,* Kevin F. Gibson,*

Naftali Kaminski,* Joseph M. Pilewski,* Michael Donahoe,* Frank C. Sciurba,* and

Steven R. Duncan*

We hypothesized B cells are involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a progressive, restrictive lung

disease that is refractory to glucocorticoids and other nonspecific therapies, and almost invariably lethal. Accordingly, we sought

to identify clinically associated B cell–related abnormalities in these patients. Phenotypes of circulating B cells were characterized

by flow cytometry. Intrapulmonary processes were evaluated by immunohistochemistry. Plasma B lymphocyte stimulating factor

(BLyS) was assayed by ELISA. Circulating B cells of IPF subjects were more Ag differentiated, with greater plasmablast

proportions (3.1 6 0.8%) than in normal controls (1.3 6 0.3%) (p < 0.03), and the extent of this differentiation correlated with

IPF patient lung volumes (r = 0.44, p < 0.03). CD20+ B cell aggregates, diffuse parenchymal and perivascular immune complexes,

and complement depositions were all prevalent in IPF lungs, but much less prominent or absent in normal lungs. Plasma

concentrations of BLyS, an obligate factor for B cell survival and differentiation, were significantly greater (p < 0.0001) in 110

IPF (2.05 6 0.05 ng/ml) than among 53 normal (1.40 6 0.04 ng/ml) and 90 chronic obstructive pulmonary disease subjects (1.59 6

0.05 ng/ml). BLyS levels were uniquely correlated among IPF patients with pulmonary artery pressures (r = 0.58, p < 0.0001). The

25% of IPF subjects with the greatest BLyS values also had diminished 1-y survival (46 6 11%), compared with those with lesser

BLyS concentrations (81 6 5%) (hazard ratio = 4.0, 95% confidence interval = 1.8–8.7, p = 0.0002). Abnormalities of B cells and

BLyS are common in IPF patients, and highly associated with disease manifestations and patient outcomes. These findings have

implications regarding IPF pathogenesis and illuminate the potential for novel treatment regimens that specifically target B cells

in patients with this lung disease. The Journal of Immunology, 2013, 191: 2089–2095.

Idiopathic pulmonary fibrosis (IPF) is a morbid, fibroproli-ferative disorder characterized by progressive lung restrictionand gas exchange abnormalities (1–3). The annual incidence

of IPF in the United States is ∼40,000, and afflicted patients havea median survival of ∼3 y (1–3).Although the pathogenesis of IPF is widely considered to be

enigmatic (2), abnormalities of adaptive immunity are commonamong patients with this disease (Refs. 4–22). HLA allele frequencyperturbations are a typifying feature of immunological disorders,and HLA-DRb1*15 is overrepresented among IPF subjects (20), aswell as being linked to dysregulated autoimmune responses in thispopulation (13). Infiltrates of activated T cells are present in IPF

lungs, and the magnitude of these abnormalities is proportionateto disease severity and patient mortality (19, 21, 22). CirculatingT cells among IPF patients are also Ag activated, clonally expanded,dysregulated, and have augmented productions of myriad proin-flammatory and profibrotic mediators, and many characteristics ofthese lymphocytes are associated with clinical manifestations (9, 18,19). One or more Ags in IPF lungs stimulate autologous CD4 T cells(9), including heat shock protein 70, which induces lymphocyteproliferation and IL-4 production (13).B cell studies in IPF are more limited, but examinations of dis-

eased lungs from these patients have shown the presence of highlyabnormal intrapulmonary B cell aggregates (4, 5) and overexpressionsof Ig genes (6). Potentially pathogenic immune complexes have beenfound in the sera, bronchoalveolar lavage, and pulmonary paren-chyma of IPF patients (7, 8, 13). Diverse circulating IgG autoanti-bodies are present in .80% of these subjects (8–16), and someparticular Ig specificities have been linked to disease severityand/or poor prognoses (10–13).We hypothesized that B cell characteristics and/or their asso-

ciated mediators may be correlated with clinical features of IPF. Ifso, these findings could have considerable importance. To begin,the course of IPF is unpredictable among afflicted individuals (2,3). Thus, facile, valid biomarkers could be very useful to identifypatients who are destined for poor near-term outcomes, and thusoptimize timings of lung transplantations, and/or aid in selectionof high-risk subjects for experimental treatments.Moreover, the absence of a definitive, mechanistic paradigm of

IPF pathogenesis has precluded the rational selection of therapies thatare directed specifically at the causal biological process(es) (2, 3).Medical treatments used for IPF to date, typically based on gluco-

*Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213; and†Department of Surgery, Beth Israel Deaconess Medical Center, Harvard MedicalSchool, Boston, MA 02215

1J.X. and D.J.K. contributed equally to this work.

Received for publication December 19, 2012. Accepted for publication June 20,2013.

This work was supported in part by National Institutes of Health Grants HL107172and HL084948.

Address correspondence and reprint requests to Dr. Steven R. Duncan, Pulmonary,Allergy, and Critical Care Medicine, Department of Medicine, University of Pitts-burgh, 628 NW MUH, 3459 Fifth Avenue, Pittsburgh, PA 15213. E-mail address:duncsr@upmc.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: BLyS, B lymphocyte stimulating factor; COPD,chronic obstructive pulmonary disease; FEV, forced expiratory volume; FVC, forcedvital capacity; IPF, idiopathic pulmonary fibrosis; PA, pulmonary artery; PAP, PApressure; SLE, systemic lupus erythematosus.

Copyright� 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

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corticoids and/or global antifibrotic drugs, are ineffectual, and thedisease continues to have a worse prognosis than several commonmalignancies (2, 3). Like IPF, many B cell–mediated lung diseasesare also refractory to nonspecific therapy with glucocorticoids.Conversely, however, these same syndromes often respond to focusedanti–B cell agents or other treatments that reduce concentrations ofpathogenic Abs (23–29). Hence, finding a compelling link betweenB cells and IPF progression might engender considerations for ex-perimental trials of recently developed agents that more specificallytarget these lymphocytes and/or their functions, and may providesome clinical benefit for these otherwise ill-fated patients.Accordingly, we conducted investigations to characterize B cells

and examine a critical B cell mediator in IPF patients that couldhave therapeutic implications.

Materials and MethodsSpecimens for autoantibody studies

Peripheral blood specimens were obtained from consecutive IPF patients (9,13, 19, 20), healthy volunteers, and subjects with cigarette smoking–at-tributable chronic obstructive pulmonary disease (COPD) and/or emphy-sema (30, 31), henceforth collectively denoted as COPD. Plasma wasobtained from these specimens by centrifugation, aliquoted, and storedat280˚C prior to use in the B lymphocyte stimulating factor (BLyS) assays.Diagnoses of lung disease were established by expert clinicians, who ana-lyzed all information and were blinded to the experimental laboratory tests.All IPF subjects fulfilled consensus diagnostic criteria (9, 19, 20) and hadnegative conventional autoimmune serological tests (13). COPD was diag-nosed by spirometry (30), and emphysema was detected by chest com-puterized tomography scans (31). Healthy controls were recruited fromvolunteers among hospital personnel or research registries. Those withtobacco smoking histories had normal spirometry and no radiographicevidence of emphysema.

Subpopulations of IPF and COPD subjects had pulmonary artery (PA)pressures (PAP) measured by right heart catheterizations during evaluationsfor possible lung transplantation, or other clinical indications. Thesecatheterizations were performed by cardiologists whowere independent andunaware of this study.

All subjects gave written informed consent. This study was approved bythe University of Pittsburgh Institutional Review Board.

B cell phenotypes

PBMCswere isolated from venous phlebotomy specimens by density gradientcentrifugation (9). B cells among the PBMCs were stained with panels ofmAb and characterized by flow cytometry, as fully detailed previously (32).B lymphocytes were identified as CD32/CD142/CD162/CD19+ cells, andfurther stratified for comparisons in this study as transitional (IgD+/CD38++),IgM+ IgA2 IgG2 switched memory (IgD+/CD27+), IgG+ switched memory(IgD2/CD27+/2), and plasmablasts (CD19low/+, CD202, CD38++, IgD2,CD27++) (Fig. 1A).

Lung specimens

Pulmonary explant specimen processing has been detailed elsewhere (9, 13).IPF and COPD explants were obtained during therapeutic transplantations.

Normal lungs not used for transplantations were procured from cadavericdonors during harvests of other organs (9, 13).

Immunohistochemistry

These studies were performed using methods that have been previously de-tailed (30). Primary Abs used on Zn-fixed lung sections were mouse anti-human IgG (Serotec, Raleigh, NC) and rabbit anti-human C4d (LSBio,Seattle, WA). Treatments with these Abs were followed by successive incu-bations with species and isotype-specific biotinylated secondary Abs andavidin-HRP (30). Mouse anti-human CD20 (Dako, Carpinteria, CA) and anti–Ki-67 (ThermoFisher, Kalamazoo, MI) were analogously used in formalin-fixed, paraffin-embedded lung specimens. Intrapulmonary B cell aggregateswere quantified by blinded observers who counted CD20+ cells in 30 suc-cessive, defined rectangular fields in each individual lung tissue specimen (atoriginal magnification 310). These data are expressed as numbers of CD20+

cells/mm2.

BLyS

Plasma was obtained by centrifugation of heparinized phlebotomy specimensand used in BLyS ELISA kits (R&D Systems), according to manufacturerinstructions. OD at 405 nm for replicate specimens were determined ina SpectraMax 190 plate reader (Molecular Devices, Sunnyvale, CA), blankedagainst untreated wells.

Statistical analyses

Two- and three-group comparisons of continuous variables were made byMann–Whitney or Kruskal-Wallis tests, respectively. Associations betweencontinuous variables were established by linear regression. Logistic re-gression was used to perform multivariate analyses. Survival analyses wereperformed using product-limit estimation, with intergroup comparisons bylog rank. Hazard ratios and 95% confidence interval were established byproportional hazard regression. The p values ,0.05 were considered sig-nificant. Unless otherwise denoted, data are depicted as means 6 SE.

ResultsSubjects and plasma specimens

Plasma specimens for BLyS assays were collected between De-cember 28, 2005 and December 16, 2011. Characteristics of theaggregate lung disease populations used in these studies are de-tailed in Table I. Normal controls (n = 53) were 636 1 y old, 64%male, and 57% were former or current smokers.

B cell phenotypes

To conduct an initial evaluation for B cell abnormalities in therespective cohorts, the phenotypes of their circulating lymphocyteswere prospectively determined by flow cytometry among recentlyrecruited consecutive subjects. Characteristics of the subjects whoprovided these specimens are detailed in Supplemental Table I.B cell phenotype distributions among the IPF patients were

abnormal compared with healthy subjects, and similar to those ofthe subjects with COPD (Fig. 1B), a clinically distinct lung diseasein which pathogenic autoantibodies have been implicated (30).

Table I. Demographic and clinical characteristics of lung disease subjects in whom BLyS was quantified

IPF COPD

n 110 90Age (y) 69 6 1 (71, 51–87) 64 6 1 (65, 47–83)a

Gender (% male) 73 44a

FVC % predicted 62 6 2 (59, 25–113) 81 6 2 (82, 35–136)a

FEV1 % predicted 75 6 2 (73, 31–130) 53 6 3 (54, 12–121)a

FEV1/FVC 0.85 6 0.01 (0.85, 0.71–1.22) 0.48 6 0.02 (0.46, 0.15–0.81)a

DLCO % predicted 47 6 2 (47, 14–110) 51 6 2 (48, 12–122)Smoking history (%) 55 100a

Data are depicted as means6 SE, and in parentheses: (median, minimum-to-maximum values). FVC % predicted = FVC, asa percentage of predicted values; FEV1 % predicted = FEV in the first second of expiration, as a percentage of predicted values;FEV1/FVC = the ratio of FEV in the first second to FVC; DLCO % predicted = diffusing capacity for carbon monoxide, asa percentage of predicted values. Smoking history denotes subjects with $5 pack years of cigarette smoking.

ap , 0.0001 for nonparametric comparisons between the COPD and IPF cohorts.

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The magnitude of plasmablast differentiation among the IPF subjectswas inversely correlated with forced vital capacities (FVC), a mea-sure of lung restriction (Fig. 1C). There was a trend in the COPDcohort for an analogous inverse correlation between the proportion ofplasmablasts among B cells and the ratio of forced expiratory volume(FEV) in the first second of expiration to FVC (FEV1/FVC), a de-fining criterion of expiratory airflow obstruction (r = 0.32, p = 0.09).Plasmablast differentiation among the COPD subjects was sig-nificantly correlated with diffusing capacities for carbon monoxide,a correlate of intrapulmonary gas exchange (Fig. 1D).

In situ B cells

Having found evidence of circulating B cell abnormalities inIPF patients, we performed additional studies to confirm theselymphocytes were also present within patient lungs. Focal aggregatesof CD20+ B cells were present in all IPF lungs examined by im-munohistochemistry (n = 11), typically proximate to small airways,and to a somewhat lesser extent near small blood vessels (Fig. 2A).Fewer and typically more scattered CD20+ cells were evident innormal lungs (n = 9) (Fig. 2B). Ki-67 was only infrequently seenamong the IPF B cells (Fig. 2A).Analogous B cell aggregates were also evident in COPD

lung sections (data not shown), as previously described and illus-trated (33).

BLyS

BLyS plasma concentrations were significantly greater among IPFsubjects than in both normal and COPD controls (Fig. 3A). BLySconcentrations were highest among those IPF subjects who had PA

hypertension, defined as PA mean pressure .25 mmHg with PAwedge pressure ,15 mmHg, and also among those who diedwithin 1 y of the specimen acquisitions (Fig. 3B). The BLyS

FIGURE 1. Phenotypes of circulating B cells. (A) Cells were stained with panels of mAb and gated on CD19+, CD20–, CD3–, and IgD– B cells.

Plasmablasts among these (CD2hi, CD38hi ) are denoted in the upper right quadrant of this example. (B) Selected phenotypic characteristics of circulating

B cells among COPD (n = 29), IPF (n = 25), and normal controls (n = 20). The lowest, second lowest, middle, second highest, and highest box points

represent 10th, 25th, median, 75th, and 90th percentiles, respectively. Means are represented by squares. Trans refers to transitional (recent bone marrow

emigrant B cell) and successively differentiated developmental stages: non-isotype switched (IgA–, IgG–) memory (nsMem), isotype (IgG) switched

memory (sMEM), and efficient Ab-producing plasmablasts (PB). Absolute sMEM values are 103 those depicted in this figure. The p values denote

Kruskal–Wallis comparisons of the three cohorts: *denotes the group that is significantly different from the other two. Note increased B cell differentiation

among the disease cohorts (COPD and IPF) relative to healthy controls, and the general similarities between those two lung disease subpopulations. (C) The

percentage of plasmablasts (PB) among circulating B cells was inversely correlated with FVC, as a percentage of predicted values (FVC%p) in IPF

subjects. (D) The percentage of plasmablasts (PB) among circulating B cells was inversely correlated with DLCO%p in COPD subjects.

FIGURE 2. Intrapulmonary CD20+ B cells. (A) Representative images

show sections from normal lungs (left panel) and IPF lungs (middle panel)

immunostained for CD20. These B cells are only infrequently positive for Ki-

67 (right panel) (original magnification 3100). (B) Direct counting confirmed

CD20+ cells are more numerous in IPF compared to normal lung specimens.

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values were significantly correlated with PAP among individualswith IPF (Fig. 3C). Pulmonary function tests were obtained 6.1 60.3 mo in 64 of the IPF subjects (those still alive, not transplanted,or not too ill to perform these tests). BLyS concentrations in thesepatients tended to be inversely associated with subsequent changesin FVC (r = 0.26, p = 0.04).To examine for possible associations of BLyS with subsequent

outcomes, the IPF patients were stratified into the quartile withhighest circulating concentrations of this mediator versus the 75%of subjects with lower BLyS levels. Actuarial analyses confirmedthat subjects with the greatest concentrations of BLyS had worse1-y outcomes (Fig. 3D). Post hoc actuarial analyses limited totabulations of deaths, with omission of those subjects who hadtransplantations during the year after their specimen acquisitions,also showed greater 1-y absolute survival among the IPF subjectswith lower BLyS concentrations (Fig. 3E).There were no significant gender differences of BLyS levels

among the IPF patients (2.1 6 0.1 versus 1.9 6 0.1 ng/ml, formales and females, respectively, p = 0.66), but there was a trend forgreater proportions of males within the quartile of subjects withhighest BLyS concentrations (Table II). Otherwise, there were no

appreciable associations of the clinical/demographic parametersin Table II with levels of this mediator. None of these clinical/demographic characteristics were associated with survival inde-pendently of BLyS. PAP was also not an independent correlate ofoutcome in this study cohort.To date, very few of the COPD cohort have died (precluding

meaningful analyses of survival correlates). However, BLyS levelsin COPD subjects did not significantly correlate with PAP (r = 0.20,p = 0.21) or measures of pulmonary function (data not shown).There were no differences of circulating BLyS concentrationsbetween males (1.56 6 0.08 ng/ml) and females (1.62 6 0.06 ng/ml) among the COPD cohort. Proportions of males and females inthe highest quartile of BLyS concentrations among COPD subjectswere 25 and 24%, respectively.

Intrapulmonary immune complexes and complement

Given the association between BLyS and PAP, we examined lungsections for evidence of Ab-mediated processes involving pul-monary blood vessels (Fig. 4). Diffuse parenchymal immunecomplexes and complement depositions were much more exten-sive in IPF lungs compared with normals, as has been previously

FIGURE 3. BLyS levels. (A) Plasma BLyS

concentrations were significantly greater in IPF

subjects (n = 110) than in either COPD (n = 90)

or normals (n = 53). (B) Among the IPF cohort,

BLyS levels were highest among those who

died during the next year (n = 25), and in those

who had pulmonary artery hypertension (PAH)

(n = 17), compared to those with lesser pul-

monary artery (PA) mean pressures (Nl) (n =

28). PAH was defined as PA mean pressures

.25 mmHg with PA wedge (PAW) pressures

,15 mmHg. Subjects with PAW .15 were not

tabulated here (n = 5). These five excluded

subjects had PA mean pressures .25, and their

BLyS concentrations were 1.92 6 0.32 ng/ml).

(C) BLyS concentrations were correlated with

pulmonary artery pressures among IPF subjects.

(D) The quartile (25%) of IPF subjects with

greatest BLyS levels (High) had worse out-

comes than those subjects with lesser BLyS levels

(Low). Cross-hatches and numbers in parentheses

denote censored events (end of observation). (E)

Absolute mortality was also greater among IPF

patients with the highest quartile plasma BLyS

concentrations (High) after omission of the sub-

population that had lung transplantations during

the observation interval.

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described (13). Also, in contrast to normal lung specimens, theIPF sections were characterized by thickened blood vessel wallsand adventitia that are associated with wider circumferential IgGand C4d staining. These findings were particularly conspicuous inareas proximate to extensive fibrotic abnormalities of lung pa-renchyma. The presences of intrapulmonary immune complexesand complement depositions in COPD have already been detailed(30).

DiscussionAg-stimulated B cells undergo incremental maturations that resultin highly differentiated lymphocytes with increased efficacy for theelaboration of avid, isotype-switched IgG Abs (34). In comparisonswith healthy controls, B cell phenotypes among patients withrecognized autoantibody diseases, such as rheumatoid arthritis andsystemic lupus erythematosus (SLE), are more differentiated, withlesser proportions of early B cells and greater relative proportionsof mature lymphocytes, including autoantibody-producing plas-mablasts (35–37). Moreover, the magnitude of this B cell differ-entiation is a biomarker for the clinical activities of these diseases(35–37). The present data show the extent of B lymphocyte dif-ferentiation among IPF subjects is similarly abnormal (Fig. 1B)

and is also analogously correlated with the pulmonary function ofthese lung disease patients (Fig. 1C).Other findings in this study confirm previous reports of abnormal

B cell aggregates within IPF lungs (4, 5) (Fig. 2). The B cellaccumulations in these specimens are predominately Ki-67 neg-ative, and thus are almost certainly attributable to trafficking fromextrapulmonary compartments rather than local proliferation. Thisparticular finding is also congruent with a previous report (5).BLyS, also known as B cell–activating factor, is a TNF ligand

family cytokine that is produced by a variety of leukocytes, andprovides essential, nonredundant signals necessary for B lymphocytesurvival, maturation, and Ab production (29, 38). Circulating BLySis increased in patients with many autoantibody-mediated diseases,including SLE and rheumatoid arthritis, and levels of this mediatoramong individuals with these diseases are associated with theirclinical manifestations (29, 38, 39). The present data show thatplasma concentrations of BLyS are similarly increased in IPF sub-jects, as well as also being correlated with important clinical featuresand prognoses of these patients (Fig. 3). PAP were measured in thisstudy among patient subpopulations that had these determinationsduring evaluations for lung transplantations and/or other clinicalindications (e.g., for disproportionate dyspnea). Accordingly, it ispossible the cohorts with these measures were biased, which couldhave included an enrichment of patients with pulmonary arteryhypertension. Thus, the association of BLyS with PAP may not beas rigorous in cross-sectional, unselected IPF populations. None-theless, the IPF subpopulation analyzed in this study also includedmany patients with normal PAP in whom the BLyS-PAP associ-ation was still evident (Fig. 3C). Most importantly, circulatingBLyS concentrations were also highly associated with patientoutcomes (Fig. 3D, 3E), and the latter analyses are not subject toselection bias.The findings in this study are further evidence that B cell ab-

normalities are common in IPF subjects (4–16) and linked toclinical manifestations and mortality (10–13). Studies of B cells inIPF are ongoing, but there are several plausible mechanisms bywhich these lymphocytes could have pathogenic effects.Tissue-bound Abs produced by B cells can cause cytotoxicities

and promote neutrophil recruitment by the formation of Ab-Ag(immune) complexes and complement fixation (Fig. 4), and/orby activation of NK cells (17, 34, 40, 41). Observations in thisstudy confirm previous findings of diffuse immune complex andcomplement depositions in IPF lungs (13), and additionally showthese processes are proximally associated with intrapulmonaryblood vessel pathology (Fig. 4).Autoantibodies can also deleteriously alter target cell functions

(13, 16, 42, 43) by cross-linking cell surface autoantigen-receptorcomplexes that transduce and enhance proinflammatory responses,

Table II. Demographic and clinical characteristics of IPF subjects stratified by plasma BLyS levels

Lowest BLyS Highest BLyS

n 82 28Age (y) 70 6 1 (71, 53–87) 67 6 2 (69, 51–82)Gender (% male) 68 86Lung biopsy (%) 51 61FVC % predicted 63 6 2 (61, 31–113) 59 6 4 (57, 25–102)DLCO % predicted 49 6 2 (48, 14–109) 44 6 4 (40, 14–91)Smoking history (%) 59 44Immunosuppressants (%) 19 18

Highest BLyS denotes those IPF subjects with mediator levels in the greatest quartile of values, whereas lowest BlySdenotes the 75% of subjects with lower values. Data are depicted as means 6 SE, and in parentheses: (median, minimum-to-maximum values). Immunosuppressants denote subjects taking any single agent or various permutations of prednisone (5–20mg/day), azathioprine, IFN-g, mycophenolate, or tacrolimus. See Table I legend for other explanations. None of thesecharacteristics differed significantly between the subpopulations (p = 0.07 for the intergroup gender comparisons).

FIGURE 4. Intrapulmonary IgG and C4d. In contrast to normal lungs,

the IPF sections are characterized by more intense IgG and C4d staining

throughout the parenchyma, and particularly intense staining around blood

vessels (black arrows). Staining was also present in lymphoid aggregates

(red arrows) and intraluminal cells and debris (green arrows). Inset images

show the lack of staining in nonimmune controls (original magnification

3100).

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or after gaining access to intracellular autoantigens (44). Previousstudies have shown that autoantibodies of IPF patients increase theproduction of profibrotic TGF-b by alveolar epithelia (16). Anti–heat shock protein 70 IgG isolated from these patients activatesmonocytes and increases their elaborations of IL-8 (13), a che-mokine biomarker of IPF that has been implicated in the patho-genesis of this disease (45).Although perhaps less widely appreciated, activated B cells per

se also directly elaborate numerous cytokines and other mediatorsthat have vasoactive, proinflammatory, and profibrotic effects (34).Intrapulmonary lymphoid aggregates are an abundant source ofdiverse, highly active mediators, essentially always an abnormalfinding, and most likely have pathogenic consequences (5, 34, 46,47). Lymphoid aggregates in proximity to pulmonary blood ves-sels are associated with anatomic and functional vascular abnor-malities among IPF and other disease populations (5, 47).Activated B cells are also efficient APCs for T lymphocytes (34).

In turn, Ag (or autoantigen)-stimulated T cells produce myriadpathogenic mediators (48), and have been singularly implicated asthe initiators of many disease-associated inflammatory cascades,including those that result in pathological fibrosis (41, 48, 49).Numerous T cell abnormalities have been described in IPF patients,including reactivity to autologous lung proteins and intrapulmonaryautoantigens, and several characteristics and functions of theselymphocytes are correlated with clinical manifestations and/orpatient outcomes (9, 13, 18, 19, 21, 22).The present data, in conjunction with other independent evidences

in IPF patients (4–22), illuminate several parallels between this lungdisease and recognized autoimmune syndromes (34–39, 41, 42, 46,49). Thus, these collective findings may be an impetus to considerinnovative experimental trials for this inexorable and highly lethalsyndrome. Severe autoantibody-mediated lung diseases in particular(e.g., Goodpasture’s disease, anti–Jo-1–associated interstitial lungdisease, Wegener’s granulomatosis, anti-alloantigen–mediated re-jection, etc.) are usually inadequately responsive or even refractoryto corticosteroid-based therapy. However, these disorders are oftenmore responsive to treatments with modalities that remove auto-antibodies [e.g., plasma exchange (23)] and/or B cell–depletingagents, including rituximab (24–27), and a preliminary reportindicates this approach may also possibly benefit IPF patients(50). Alternatively, or in addition, critical B cell functions canbe targeted by interfering with the maturation and survival ofthese lymphocytes (29, 38). A highly specific anti-BLyS agent(belimumab) was recently approved by the Food and Drug Admin-istration for treatment of Ab-mediated SLE, and this drug appears tohave efficacy and minimal toxicity in this indication (28).If humoral autoimmunity plays a pathogenic role in IPF causality

or progression, as suggested by the current findings and severalother reports (4–17), mechanistic therapies that specifically targetB cells could perhaps benefit patients afflicted with this morbidand heretofore untreatable disease (2, 3, 50).

DisclosuresThe authors have no financial conflicts of interest.

References1. Fernandez Perez, E. R., C. E. Daniels, D. R. Schroeder, J. St. Sauver,

T. E. Hartman, B. J. Bartholmai, E. S. Yi, and J. H. Ryu. 2010. Incidence,

prevalence, and clinical course of idiopathic pulmonary fibrosis: a population-

based study. Chest 137: 129–137.2. Borchers, A. T., C. Chang, C. L. Keen, and M. E. Gershwin. 2011. Idiopathic

pulmonary fibrosis: an epidemiological and pathological review. Clin. Rev. Al-

lergy Immunol. 40: 117–134.3. O’Connell, O. J., M. P. Kennedy, and M. T. Henry. 2011. Idiopathic pulmonary

fibrosis: treatment update. Adv. Ther. 28: 986–999.

4. Campbell, D. A., L. W. Poulter, G. Janossy, and R. M. du Bois. 1985. Immu-nohistological analysis of lung tissue from patients with cryptogenic fibrosingalveolitis suggesting local expression of immune hypersensitivity. Thorax 40:405–411.

5. Marchal-Somme, J., Y. Uzunhan, S. Marchand-Adam, D. Valeyre, V. Soumelis,B. Crestani, and P. Soler. 2006. Cutting edge: nonproliferating mature immunecells form a novel type of organized lymphoid structure in idiopathic pulmonaryfibrosis. J. Immunol. 176: 5735–5739.

6. Zuo, F., N. Kaminski, E. Eugui, J. Allard, Z. Yakhini, A. Ben-Dor, L. Lollini,D. Morris, Y. Kim, B. DeLustro, et al. 2002. Gene expression analysis revealsmatrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc.Natl. Acad. Sci. USA 99: 6292–6297.

7. Dall’Aglio, P. P., A. Pesci, G. Bertorelli, E. Brianti, and S. Scarpa. 1988. Studyof immune complexes in bronchoalveolar lavage fluids. Respiration 54(Suppl.1): 36–41.

8. Dobashi, N., J. Fujita, M. Murota, Y. Ohtsuki, I. Yamadori, T. Yoshinouchi,R. Ueda, S. Bandoh, T. Kamei, M. Nishioka, et al. 2000. Elevation of anti-cytokeratin 18 antibody and circulating cytokeratin 18: anti-cytokeratin 18 an-tibody immune complexes in sera of patients with idiopathic pulmonary fibrosis.Lung 178: 171–179.

9. Feghali-Bostwick, C. A., C. G. Tsai, V. G. Valentine, S. Kantrow, M. W. Stoner,J. M. Pilewski, A. Gadgil, M. P. George, K. F. Gibson, A. M. Choi, et al. 2007.Cellular and humoral autoreactivity in idiopathic pulmonary fibrosis. J. Immunol.179: 2592–2599.

10. Ogushi, F., K. Tani, T. Endo, H. Tada, T. Kawano, T. Asano, L. Huang,Y. Ohmoto, M. Muraguchi, H. Moriguchi, and S. Sone. 2001. Autoantibodies toIL-1 a in sera from rapidly progressive idiopathic pulmonary fibrosis. J. Med.Invest. 48: 181–189.

11. Kurosu, K., Y. Takiguchi, O. Okada, N. Yumoto, S. Sakao, Y. Tada, Y. Kasahara,N. Tanabe, K. Tatsumi, M. Weiden, et al. 2008. Identification of annexin 1 asa novel autoantigen in acute exacerbation of idiopathic pulmonary fibrosis. J.Immunol. 181: 756–767.

12. Taille, C., S. Grootenboer-Mignot, C. Boursier, L. Michel, M. P. Debray,J. Fagart, L. Barrientos, A. Mailleux, N. Cigna, F. Tubach, et al. 2011. Identi-fication of periplakin as a new target for autoreactivity in idiopathic pulmonaryfibrosis. Am. J. Respir. Crit. Care Med. 183: 759–766.

13. Kahloon, R. A., J. Xue, A. Bhargava, E. Csizmadia, L. Otterbein, D. J. Kass,J. Bon, M. Soejima, M. C. Levesque, K. O. Lindell, et al. 2013. Patients withidiopathic pulmonary fibrosis with antibodies to heat shock protein 70 have poorprognoses. Am. J. Respir. Crit. Care Med. 187: 768–775.

14. Grigolo, B., I. Mazzetti, R. M. Borzı, I. D. Hickson, M. Fabbri, L. Fasano,R. Meliconi, and A. Facchini. 1998. Mapping of topoisomerase II alpha epitopesrecognized by autoantibodies in idiopathic pulmonary fibrosis. Clin. Exp.Immunol. 114: 339–346.

15. Yang, Y., J. Fujita, S. Bandoh, Y. Ohtsuki, I. Yamadori, T. Yoshinouchi, andT. Ishida. 2002. Detection of antivimentin antibody in sera of patients with id-iopathic pulmonary fibrosis and non-specific interstitial pneumonia. Clin. Exp.Immunol. 128: 169–174.

16. Wallace, W. A., and S. E. Howie. 2001. Upregulation of tenascin and TGFbetaproduction in a type II alveolar epithelial cell line by antibody against a pul-monary auto-antigen. J. Pathol. 195: 251–256.

17. Magro, C. M., W. J. Waldman, D. A. Knight, J. N. Allen, T. Nadasdy,G. E. Frambach, P. Ross, and C. B. Marsh. 2006. Idiopathic pulmonary fibrosisrelated to endothelial injury and antiendothelial cell antibodies. Hum. Immunol.67: 284–297.

18. Kotsianidis, I., E. Nakou, I. Bouchliou, A. Tzouvelekis, E. Spanoudakis,P. Steiropoulos, I. Sotiriou, V. Aidinis, D. Margaritis, C. Tsatalas, and D. Bouros.2009. Global impairment of CD4+CD25+FOXP3+ regulatory T cells in idio-pathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 179: 1121–1130.

19. Gilani, S. R., L. J. Vuga, K. O. Lindell, K. F. Gibson, J. Xue, N. Kaminski,V. G. Valentine, E. K. Lindsay, M. P. George, C. Steele, and S. R. Duncan. 2010.CD28 down-regulation on circulating CD4 T-cells is associated with poorprognoses of patients with idiopathic pulmonary fibrosis. PLoS One 5: e8959.

20. Xue, J., B. R. Gochuico, A. S. Alawad, C. A. Feghali-Bostwick, I. Noth,S. D. Nathan, G. D. Rosen, I. O. Rosas, S. Dacic, I. Ocak, et al. 2011. The HLAclass II allele DRB1*1501 is over-represented in patients with idiopathic pul-monary fibrosis. PLoS One 6: e14715.

21. Parra, E. R., R. A. Kairalla, C. R. Ribeiro de Carvalho, E. Eher, and V. L. Capelozzi.2007. Inflammatory cell phenotyping of the pulmonary interstitium in idiopathicinterstitial pneumonia. Respiration 74: 159–169.

22. Daniil, Z., P. Kitsanta, G. Kapotsis, M. Mathioudaki, A. Kollintza, M. Karatza,J. Milic-Emili, C. Roussos, and S. A. Papiris. 2005. CD8+ T lymphocytes in lungtissue from patients with idiopathic pulmonary fibrosis. Respir. Res. 6: 81.

23. Erickson, S. B., S. B. Kurtz, J. V. Donadio, Jr., K. E. Holley, C. B. Wilson, andA. A. Pineda. 1979. Use of combined plasmapheresis and immunosuppression inthe treatment of Goodpasture’s syndrome. Mayo Clin. Proc. 54: 714–720.

24. Sem, M., O. Molberg, M. B. Lund, and J. T. Gran. 2009. Rituximab treatment ofthe anti-synthetase syndrome: a retrospective case series. Rheumatology 48:968–971.

25. Martinu, T., D. N. Howell, and S. M. Palmer. 2010. Acute cellular rejection andhumoral sensitization in lung transplant recipients. Semin. Respir. Crit. CareMed. 31: 179–188.

26. Borie, R., M. P. Debray, C. Laine, M. Aubier, and B. Crestani. 2009. Rituximabtherapy in autoimmune pulmonary alveolar proteinosis. Eur. Respir. J. 33: 1503–1506.

27. Keir, G. J., T. M. Maher, D. M. Hansell, C. P. Denton, V. H. Ong, S. Singh,A. U. Wells, and E. A. Renzoni. 2012. Severe interstitial lung disease in con-nective tissue disease: rituximab as rescue therapy. Eur. Respir. J. 40: 641–648.

2094 BLyS IN IPF

at Univ of Pittsburgh, H

SLS on Septem

ber 30, 2014http://w

ww

.jimm

unol.org/D

ownloaded from

28. Furie, R., M. Petri, O. Zamani, R. Cervera, D. J. Wallace, D. Tegzova,J. Sanchez-Guerrero, A. Schwarting, J. T. Merrill, W. W. Chatham, et al.; BLISS-76 Study Group. 2011. A phase III, randomized, placebo-controlled study ofbelimumab, a monoclonal antibody that inhibits B lymphocyte stimulator, inpatients with systemic lupus erythematosus. Arthritis Rheum. 63: 3918–3930.

29. Cancro, M. P., D. P. D’Cruz, and M. A. Khamashta. 2009. The role ofB lymphocyte stimulator (BLyS) in systemic lupus erythematosus. J. Clin. In-vest. 119: 1066–1073.

30. Feghali-Bostwick, C. A., A. S. Gadgil, L. E. Otterbein, J. M. Pilewski,M. W. Stoner, E. Csizmadia, Y. Zhang, F. C. Sciurba, and S. R. Duncan. 2008.Autoantibodies in patients with chronic obstructive pulmonary disease. Am. J.Respir. Crit. Care Med. 177: 156–163.

31. Bon, J., C. R. Fuhrman, J. L. Weissfeld, S. R. Duncan, R. A. Branch,C. C. Chang, Y. Zhang, J. K. Leader, D. Gur, S. L. Greenspan, and F. C. Sciurba.2011. Radiographic emphysema predicts low bone mineral density in a tobacco-exposed cohort. Am. J. Respir. Crit. Care Med. 183: 885–890.

32. Levesque, M. C., M. A. Moody, K. K. Hwang, D. J. Marshall, J. F. Whitesides,J. D. Amos, T. C. Gurley, S. Allgood, B. B. Haynes, N. A. Vandergrift, et al.2009. Transmitted/Founder HIV-1 induces B cell polyclonal differentiation andapoptosis with massive gastrointestinal tract germinal center loss in the earlieststages of infection. PLoS Med. 6: e1000107.

33. Hogg, J. C., F. Chu, S. Utokaparch, R. Woods, W. M. Elliott, L. Buzatu,R. M. Cherniack, R. M. Rogers, F. C. Sciurba, H. O. Coxson, and P. D. Pare.2004. The nature of small-airway obstruction in chronic obstructive pulmonarydisease. N. Engl. J. Med. 350: 2645–2653.

34. Browning, J. L. 2006. B cells move to center stage: novel opportunities forautoimmune disease treatment. Nat. Rev. Immunol. 5: 564–575.

35. Jacobi, A. M., K. Reiter, M. Mackay, C. Aranow, F. Hiepe, A. Radbruch,A. Hansen, G. R. Burmester, B. Diamond, P. E. Lipsky, and T. Dorner. 2008.Activated memory B cell subsets correlate with disease activity in systemic lupuserythematosus: delineation by expression of CD27, IgD, and CD95. ArthritisRheum. 58: 1762–1773.

36. Souto-Carneiro, M. M., V. Mahadevan, K. Takada, R. Fritsch-Stork, T. Nanki,M. Brown, T. A. Fleisher, M. Wilson, R. Goldbach-Mansky, and P. E. Lipsky.2009. Alterations in peripheral blood memory B cells in patients with activerheumatoid arthritis are dependent on the action of tumour necrosis factor. Ar-thritis Res. Ther. 11: R84.

37. Anolik, J. H., J. Barnard, A. Cappione, A. E. Pugh-Bernard, R. E. Felgar,R. J. Looney, and I. Sanz. 2004. Rituximab improves peripheral B cell abnor-malities in human systemic lupus erythematosus. Arthritis Rheum. 50: 3580–3590.

38. Bossen, C., and P. Schneider. 2006. BAFF, APRIL and their receptors: structure,function and signaling. Semin. Immunol. 18: 263–275.

39. Dillon, S. R., B. Harder, K. B. Lewis, M. D. Moore, H. Liu, T. R. Bukowski,N. B. Hamacher, M. M. Lantry, M. Maurer, C. M. Krejsa, et al. 2010. B-

lymphocyte stimulator/a proliferation-inducing ligand heterotrimers are ele-vated in the sera of patients with autoimmune disease and are neutralized byatacicept and B-cell maturation antigen-immunoglobulin. Arthritis Res. Ther. 12:R48.

40. Mayadas, T. N., G. C. Tsokos, and N. Tsuboi. 2009. Mechanisms of immunecomplex-mediated neutrophil recruitment and tissue injury. Circulation 120:2012–2024.

41. Fu, S. M., U. S. Deshmukh, and F. Gaskin. 2011. Pathogenesis of systemic lupuserythematosus revisited 2011: end organ resistance to damage, autoantibodyinitiation and diversification, and HLA-DR. J. Autoimmun. 37: 104–112.

42. Yokota, S.-I., S. Chiba, H. Furuyama, and N. Fujii. 2010. Cerebrospinal fluidscontaining anti-HSP70 autoantibodies from multiple sclerosis patients augmentHSP70-induced proinflammatory cytokine production in monocytic cells. J.Neuroimmunol. 218: 129–133.

43. Lu, M. C., N. S. Lai, H. C. Yu, H. B. Huang, S. C. Hsieh, and C. L. Yu. 2010.Anti-citrullinated protein antibodies bind surface-expressed citrullinated Grp78on monocyte/macrophages and stimulate tumor necrosis factor alpha production.Arthritis Rheum. 62: 1213–1223.

44. Jang, J. Y., J. G. Jeong, H. R. Jun, S. C. Lee, J. S. Kim, Y. S. Kim, andM. H. Kwon. 2009. A nucleic acid-hydrolyzing antibody penetrates into cells viacaveolae-mediated endocytosis, localizes in the cytosol and exhibits cytotoxicity.Cell. Mol. Life Sci. 66: 1985–1997.

45. Richards, T. J., N. Kaminski, F. Baribaud, S. Flavin, C. Brodmerkel,D. Horowitz, K. Li, J. Choi, L. J. Vuga, K. O. Lindell, et al. 2012. Peripheralblood proteins predict mortality in idiopathic pulmonary fibrosis. Am. J. Respir.Crit. Care Med. 185: 67–76.

46. Aloisi, F., and R. Pujol-Borrell. 2006. Lymphoid neogenesis in chronic inflam-matory disease. Nat. Rev. Immunol. 6: 205–217.

47. Perros, F., P. Dorfmuller, D. Montani, H. Hammad, W. Waelput, B. Girerd,N. Raymond, O. Mercier, S. Mussot, S. Cohen-Kaminsky, et al. 2012. Pulmonarylymphoid neogenesis in idiopathic pulmonary arterial hypertension. Am. J.Respir. Crit. Care Med. 185: 311–321.

48. Monaco, C., E. Andreakos, S. Kiriakidis, M. Feldmann, and E. Paleolog. 2004.T-cell-mediated signalling in immune, inflammatory and angiogenic processes:the cascade of events leading to inflammatory diseases. Curr. Drug TargetsInflamm. Allergy 3: 35–42.

49. Wynn, T. A. 2004. Fibrotic disease and the TH1/TH2 paradigm. Nat. Rev.Immunol. 4: 583–594.

50. Donahoe, M., N. Chien, J. S. Raval, K. F. Gibson, M. Saul, Y. Zhang, andS. R. Duncan. 2013. Autoantibody-targeted treatments for acute exacerbations ofidiopathic pulmonary fibrosis. Presented at the Am Thorac Soc International Meet-ing, May 2013. Am. J. Resp. Crit. Care Med. 187: A5712. Available at: http://cms.psav.com/cPaper2013/ats2013/myitinerary. Accessed: June 4, 2013

The Journal of Immunology 2095

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