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Cytokine MicroenvironmentMature Dendritic Cells: Importance of the

ArePulmonary Langerhans Cell Histiocytosis Evidence That Langerhans Cells in Adult

Dominique, Allan J. Hance and Paul SolerAbdellatif Tazi, Joelle Moreau, Anne Bergeron, Stéphane

http://www.jimmunol.org/content/163/6/35111999; 163:3511-3515; ;J Immunol 

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Evidence That Langerhans Cells in Adult PulmonaryLangerhans Cell Histiocytosis Are Mature Dendritic Cells:Importance of the Cytokine Microenvironment1

Abdellatif Tazi, 2*† Joelle Moreau,* Anne Bergeron,* Stephane Dominique,‡ Allan J. Hance,*and Paul Soler2*

Because Langerhans cells (LC) in peripheral tissues are generally “immature” cells with poor lymphostimulatory activity, thecontribution of immune responses initiated by LC to the pathogenesis of pulmonary LC histiocytosis (LCH) has been uncertain.In this study we demonstrate that LC accumulating in LCH granulomas are phenotypically similar to mature lymphostimulatorydendritic cells present in lymphoid organs. LC in LCH granulomas intensely expressed B7-1 and B7-2 molecules, whereas normalpulmonary LC and LC accumulating in other pathologic lung disorders did not express these costimulatory molecules. Thepresence of B71 LC in LCH granulomas was associated with the expression in these lesions, but not at other sites in the lung, ofa unique profile of cytokines (presence of GM-CSF, TNF-a, and IL-1b and the absence of IL-10) that is known to promote thein vitro differentiation of LC into cells expressing a lymphostimulatory phenotype. Finally, LCH granulomas were the only sitewhere CD154-positive T cells could be identified in close contact with LC intensely expressing CD40 Ags. Taken together, theseresults strongly support the idea that an abnormal immune response initiated by LC may participate in the pathogenesis ofpulmonary LCH, and suggest that therapeutic strategies aimed at modifying the lymphostimulatory phenotype of LC may beuseful in the treatment of this disorder. The Journal of Immunology,1999, 163: 3511–3515.

Pulmonary Langerhans cell (LC)3 histiocytosis (LCH), alsoreferred to as histiocytosis X, is a rare disorder of un-known origin characterized by the presence in the lung of

destructive granulomas containing large numbers of LC (1, 2).Strikingly, the granulomatous lesions in LCH are centered onbronchioles, the only site in the normal human lung where LC,members of the dendritic cell (DC) lineage, are found (1–4).

The pathogenesis of LCH is not understood. Several arguments,however, support the hypothesis that this disorder is the result ofan abnormal immune reaction initiated by LC (2). First, the majorfunction of LC in the lung and other tissues is to sample Ags in thelocal milieu, process the proteins, and present the resulting anti-genic peptides in association with MHC molecules to T cells,thereby initiating immune responses (5). Second, LC and T lym-phocytes are the predominant cell types present in early lesions ofpulmonary LCH (2), and close contacts between the two cell types,identical to those observed in the course of hypersensitivity reac-tions in the skin are frequently observed in pulmonary LCH gran-ulomas (6, 7). Third, as expected for T cells responding to extrinsic

Ags presented by LC, T lymphocytes infiltrating early pulmonaryLCH lesions are predominantly CD41 T cells expressinga/bTCRs (7), and evidence for local activation of these lymphocyteshas been provided (8).

However, considerable evidence indicates that normal LC inperipheral tissues, including lung LC, have limited ability to acti-vate T cells in situ (5, 9, 10). Rather, tissue LC are thought to actas sentinels that avidly internalize and process Ags, but only sub-sequent to migration to regional lymph nodes and further differ-entiation do they acquire the capacity to stimulate T cells (5). Thistemporal and spatial separation in the functional capacities of LCin peripheral tissues and lymphoid organs has been suggested to bean important mechanism through which inappropriate pulmonaryimmune responses against innocuous inhaled Ags are avoided.Thus, the development of an immune response initiated by LC inpulmonary LCH would imply the presence of factors that not onlyincrease their number at the sites of the disease, but also enhancetheir ability to activate T cells locally.

In this regard, the interaction of B7 molecules (B7-1, B7-2)expressed by DC/LC with the CD28 receptor expressed on T cellsis of critical importance for the optimal T cell activation (reviewedin Ref. 11). The expression of B7 molecules by pulmonary LCfrom man has not been evaluated, but as would be expected forcells with weak lymphostimulatory activity, LC present in the nor-mal human skin and lung DC freshly isolated from rodents havebeen shown to be B7 negative (10, 12, 13). In contrast, whencultured in vitro, these cells up-regulate the expression of B7 mol-ecules at their surface and acquire strong lymphostimulatory ac-tivity (12–14). Several cytokines have been identified that modu-late the surface expression of B7 molecules by DC/LC in vitro andmay participate in the acquisition of lymphostimulatory activity byDC/LC in lymphoid tissues (11, 13, 15–17). In addition, cytokinescan also regulate the expression of CD40 on cells of DC/LC lin-eage, and ligation of CD40 by its ligand CD154 expressed by T

*Institut National de la Sante et de la Recherche Medicale U82, Unite de Formationet de Recherche Xavier Bichat, Paris, France;†Service de Pneumologie, Hopital Avi-cenne, Bobigny, France; and‡Service de Pneumologie, Hopital Charles Nicolle,Rouen, France

Received for publication April 21, 1999. Accepted for publication July 7, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by a grant from la Fondation Martine Midy.2 Address correspondence and reprint requests to Prof. Abdellatif Tazi or Dr. PaulSoler, Institut National de la Sante et de la Recherche Medicale U82, Unite de For-mation et de Recherche Xavier Bichat, BP 416, 75870 Paris cedex 18, France. E-mailaddress: abdellatif.tazi@avc.ap-hop-paris.fr or soler@bichat.inserm.fr3 Abbreviations used in this paper: LC, Langerhans cell; LCH, LC histiocytosis; DC,dendritic cell; KO, knock-out.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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cells can also induce B7 expression, thereby improving the lym-phostimulatory capacity of these cells (18–20).

If the LC present in large numbers in LCH lesions are respon-sible for initiating the abnormal granulomatous immune responsein situ, these cells, but not normal LC, would be expected to havean immunophenotype compatible with the expression of stronglymphostimulatory activity. In this study we demonstrate that thisis the case. LC in LCH granulomas intensely express both B7-1and B7-2, whereas normal pulmonary LC and lung LC accumu-lating in other pathologic situations are B7 negative. Further eval-uation permitted the identification of several factors unique toLCH granulomas that may promote the development of suchlymphostimulatory LC.

Materials and MethodsTissue specimens

Pulmonary LCH. Lung tissue was obtained from five patients with pul-monary LCH (three men and two women; 346 11 yr; all current smokers)at the time of open thoracotomy. Characteristic granulomas containing nu-merous CD1a1 LC were present in all samples. LC were identified un-equivocally at the electron microscopic level by the presence of numerousBirbeck granules.Normal lung tissue, peritumoral lung tissue, and lung cancers.Lungtissue was obtained from 10 patients at the time of thoracotomy for local-ized lung pathology. Five patients (four men and one woman, 606 13 yr;all nonsmokers) had benign pathology. Five patients had primary lungcarcinoma (four men and one woman; 606 14 yr; all smokers). The his-tologic types of cancer were as follows: adenocarcinoma (n 5 3) and squa-mous cell carcinoma (n5 2). Tumor tissue, tissue immediately adjacent tothe carcinoma, and grossly normal lung tissue (taken at a site distant fromthe lesion or from a different lobe) were evaluated separately. Lung tissuefrom nonsmoking patients was normal. Lung tissue from smokers and peri-tumoral tissue demonstrated mild diffuse or focal fibrotic changes in alve-olar walls, accumulation of pigment-laden macrophages, and alveolar ep-ithelial hyperplasia. All tumors and peritumoral tissues included in thisstudy were heavily infiltrated by CD1a1 LC as previously described (4).Murine tissues. C57BL/6 transgenic female mice in which the gene en-coding IL-10 was disrupted (IL-10 knock-out (KO) mice), and C57BL/6wild-type female mice were obtained from The Jackson Laboratory (BarHarbor, MA). After mice were sacrificed by sodium pentobarbital injec-tion, lungs, spleen, tongue, and ears were removed. The pancreas of NODmice, generously provided by Dr. C. Boitard (Institut National de la Santeet de la Recherche Medicale U342, Paris, France), which has previouslyshown to be infiltrated by DC-expressing B7 molecules, was also evaluatedas a positive control (21).

Immunohistochemical techniques

mAbs used with human tissues were as follows: anti-CD1a (BL6, Immu-notech, Marseille, France); anti-CD80 (anti-B7-1, MAB104, Immunotech);anti-CD86 (anti-B7-2, B70, PharMingen, San Diego, CA); anti-CD40(MAB89, Immunotech); anti-CD154 (Genzyme, Cambridge, MA); anti-CD3 (Leu-4, Becton Dickinson, San Jose, CA); anti-CD4 (Leu-3a1b Mul-ticlone, Becton Dickinson); anti-CD8 (Leu-2, Becton Dickinson); anti-CD28 (CD28.2, Becton Dickinson); anti-human IL-1b, (Genzyme); anti-human GM-CSF (Genzyme); anti-human TNF-a (Serotec, Oxford, U.K.);and anti-human IL-10 (JES3–12G8, PharMingen). Isotype-matched Abs(MOPC 21, anti-IgG1; MOPC 141, anti-IgG2, Sigma, Saint Louis, MO)were used as controls. Rat mAbs used for murine tissues were as follows:anti-CD80 (anti-B7-1, 1G10, PharMingen); anti-CD86 (anti-B7-2, GL1,Serotec); and anti-MHC class II (NIMR4, Southern Biotechnology Asso-ciates, Birmingham, AL). An isotype-matched Ab (R35-95, PharMingen)was used as a control.

For immunohistochemical staining, tissue fragments were immediatelyfrozen in liquid N2 and stored at280°C until use. Cryostats sections, 4- to6-mm thick, were fixed in acetone and reacted with appropriate dilutions ofAb. For human tissues, positive cells were revealed using a VectastainABC-alkaline phosphatase kit system (Vector Laboratories, Burlingame,CA) and the fast red substrate. For mouse tissues, samples were reactedsequentially with secondary biotinylated rabbit anti-rat IgG Abs that hadpreviously been adsorbed against mouse Igs (Vector Laboratories), avidin-alkaline phosphatase complexes (Vector Laboratories), and the fast redsubstrate. To test the specificity of immunostaining, Abs were omitted orreplaced by an isotype-matched control Ab. Under theses conditions no

positive cells were identified. The intensity of immunostaining was gradedfrom 0 (absent) to11 (strongly positive); complete agreement was ob-tained between two independent observers.

Results and DiscussionExpression of B7 by pulmonary LC and LC in LCH granulomas

LC strongly expressing CD1a Ags were observed within the bron-chiolar epithelium in all samples of normal lung obtained fromnonsmokers, and exhibited characteristic long cytoplasmic pro-cesses that intercalated between epithelial cells (Fig. 1A). On serialsections, no expression of B7-1 or B7-2 by these cells could bedetected (Fig. 1B). As previously described (4), no LC werepresent in normal alveolar parenchyma, but areas of alveolar epi-thelial hyperplasia in lung tissue from smokers were infiltrated byCD1a1 LC (data not shown). Although LC accumulated in largenumbers at these sites, no cells expressing B7 molecules could beidentified. Similarly, the lung carcinomas included in the studywere heavily infiltrated by CD1a1 cells with characteristic den-dritic shape and long cytoplasmic processes intercalated betweenmalignant cells (Fig. 1C). In all cases, immunostaining of serialsections for both B7-1 and B7-2 failed to demonstrate any positivecells (Fig. 1D).

As expected, LCH granulomas contained large numbers ofstrongly CD1a1 LC (Fig. 1E). Strikingly, essentially 100% ofthese cells were strongly positive for B7-1 and B7-2 molecules(Fig. 1,F andG). The alveolar walls and alveolar lumina adjacentto the lesions were frequently invaded by cellular infiltrates ex-tending from the granulomatous mass that contained large num-bers of CD1a1 LC. In both locations, LC also strongly expressedboth B7-1 and B7-2 molecules (data not shown). However, not allpulmonary LC from these patients expressed B7 molecules. Bothnormal bronchioles and areas of alveolar hyperplasia not involvedby the granulomatous reaction were present in the biopsies andcontained intraepithelial CD1a1 LC. In neither case were thesecells found to express B7-1 or B7-2 (data not shown).

Local cytokine profiles correlate with the expression of B7molecules by pulmonary LC

As indicated above, LC in LCH granulomas strongly expressedB7-1 and B7-2, but in the same biopsies, LC in uninvolved bron-chioles and those in areas of alveolar hyperplasia distant from thegranulomatous process did not express B7 molecules. Thus, theexpression of B7 was not an intrinsic property of LC from thesepatients and suggests that differences in the local milieu of thegranulomatous lesions resulted in the induction of expression ofthese costimulatory molecules. Therefore, we evaluated the pres-ence of cytokines known to modulate B7 expression in LC gran-ulomas and at sites where LC not expressing B7 molecules werefound.

As we have previously shown (4), GM-CSF was found to beexpressed at all sites where LC were present in the lung, regardlessof whether the LC expressed B7 molecules (data not shown). Sim-ilarly, TNF-a was abundantly expressed by normal bronchiolarepithelial cells, hyperplastic alveolar epithelium, and, as previ-ously described (22), by all of the lung carcinomas. Within theLCH granulomas, the LC themselves were the predominant celltype expressing both GM-CSF and TNF-a, although the smallnumber of macrophages infiltrating the lesions, like alveolar mac-rophages in the normal lung, also strongly expressed these twocytokines. Thus, although these cytokines may be important inrecruiting LC to lung epithelia and maintaining cell viability, theirpresence was not sufficient to induce the expression of B7 mole-cules on pulmonary LC in vivo.

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In contrast, the presence of IL-10, the principal cytokine capableof down-regulating the expression of B7 molecules by DC/LC invitro (13, 15–17) strongly correlated with the absence of expres-sion of these costimulatory molecules on LC in the specimensstudied. IL-10 was strongly expressed by normal bronchiolar ep-ithelium, hyperplastic alveolar epithelium and lung carcinomas(Fig. 2,A–C), sites where LC were found to be B7 negative. Con-versely, IL-10 was not detected in LCH granulomas (Fig. 2D).

The expression of IL-1b was the mirror image of that observedfor IL-10, i.e., this cytokine could not be detected in normal bron-chiolar epithelium, hyperplastic alveolar epithelium or lung carci-nomas (Figs. 2,E–G) but was strongly expressed by LC granulo-mas (Fig. 2H). The failure to detect IL-1b in lung cancers ornormal and hyperplastic epithelia was not a technical artifact be-cause alveolar macrophages and lung endothelial cells, cell popu-lations whose distribution is different from that of LC, were pos-itive for this cytokine in all biopsies (data not shown).

To evaluate whether the absence of IL-10 was sufficient to per-mit the expression of B7 molecules by LC in peripheral tissues, wecompared the expression of B7-1 and B7-2 by intraepithelial LC innormal and transgenic IL-10 K/O mice. DC expressing class IImolecules were easily identified both in the epidermis and in theairway epithelium of both wild-type and IL-10 K/O mice (Fig. 3A),but evaluation of serial sections demonstrated that these cells wereB7-1 and B7-2 negative (Fig. 3,B andC). These results could notbe attributed to an inability to identify B7 molecules because thespleen of wild-type and IL-10 K/O mice and the pancreas of NODmice were all infiltrated by numerous DC that were strongly pos-itive for B7-1 and B7-2 molecules (data not shown). Thus, theseresults demonstrate that the absence of IL-10, although perhapsnecessary, is not sufficient to permit the expression of B7 costimu-latory molecules by LC in peripheral tissues.

Taken together, these results demonstrate that the profile of cy-tokines expressed in LCH granulomas (presence of GM-CSF,TNF-a, and IL-1b; absence of IL-10) would be conducive to theexpression of B7 molecules by LC. The presence of IL-10 and the

absence of IL-1b would favor the maintenance of LC in an “im-mature” state at other sites where LC are found in the lung. In thisregard, it is noteworthy that although GM-CSF and TNF-a havebeen shown to up-regulate the expression of B7 molecules on LC(15), IL-1b, but not TNF-a, had a synergistic effect when com-bined with GM-CSF (23).

Expression of CD40 and CD40-ligand (CD154)

Because the interaction between CD154-positive T cells andDC/LC expressing CD40 Ags has been shown to up-regulate theexpression of B7 molecules by DC/LC (18–20), we also evaluatedthe expression of these molecules. All LC present in LCH lesionsintensely expressed CD40; both the intensity of staining and thedistribution of positive cells was very similar to that observed forB7-1 and B7-2 (Fig. 4A). T cells present within the granulomas didnot express CD40 (data not shown). The expression of CD40 byLC in normal bronchiolar epithelium and those infiltrating sites ofalveolar epithelial hyperplasia could not be evaluated, becauselung epithelial cells strongly express this Ag. Interestingly, how-ever, CD40 could not be detected on the numerous LC infiltratinglung carcinomas.

T lymphocytes were present in considerable numbers in LCHgranulomas, both interspersed between LC and surrounding thelesions. Most of these T lymphocytes were CD41 T cells, althoughCD81 T lymphocytes were also present, preferentially at the pe-riphery of the granulomas. In all patients, the majority of these Tcells were CD281. Strikingly, ;30% of lymphocytes associatedwith the LCH granulomas expressed CD154 (Fig. 4B). It is note-worthy that T cells present in the airways and parenchyma of nor-mal human lung were uniformly negative for CD154, and that Tcells present in the areas of the biopsies from patients with LCHthat were not involved by the granulomatous process were alsonegative for CD154. Similarly, although numerous CD31/CD281

T lymphocytes infiltrated lung carcinomas, sometimes in impres-sive numbers, no cell expressing CD154 were observed.

FIGURE 1. Expression of B7 molecules by pulmonary LC.A, CD1a1 LC in normal bronchiolar epithelium.B, Serial section from the same specimenshowing the absence of LC expressing B7-1 molecules.C, Numerous CD1a1 LC infiltrating a lung adenocarcinoma.D, The same tumor nodule wascompletely negative with anti-B7-2 immunostaining.E, Strongly CD1a1 LC in a pulmonary LCH granulomatous lesion. Serial sections of the samegranuloma showing that these cells were intensely stained with anti-B7-1 Abs (F). B7-1 negative lymphocytes (arrows) are present between the LC (inset).LC in LCH granulomas were also intensely stained with anti-B7-2 mAbs (G). Original magnifications:A, B, andE–G,3250;C andD, 3125; inset,3500.

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Lymphostimulatory LC and pulmonary LCH

Our results demonstrate that intraepithelial LC in the normal hu-man bronchi and LC infiltrating sites of alveolar hyperplasia andlung carcinomas do not express B7 costimulatory molecules insitu, findings similar to those observed for human epidermal LCand intraepithelial DC in rodent lungs (10, 12, 13). These findingsare compatible with the idea that pulmonary LC are immature cellsthat can capture and process exogenous Ags, but are unable togenerate an immune response locally. In striking contrast, LC inLCH granulomas strongly express B7-1, B7-2, and CD40 and thushave a phenotype typical of “mature” DC/LC found in lymphoidorgans, cells that express strong lymphostimulatory activity (5).

As discussed earlier, a variety of arguments support the idea thatLCH results from an uncontrolled immune response initiated byLC (2). Our findings add additional support to this hypothesis be-cause they explain how these LC, despite their location in a tissue

in which immature LC are normally present, would be able toprovide the costimulatory signals necessary for T cell activation.Our results also give insights into why LC in LCH granulomasmay develop their unusual lymphostimulatory phenotype. Wefound that the cytokine milieu at the site of LCH granulomas hasunique characteristics, in particular the presence IL-1b and theabsence of IL-10, that are propitious for the maturation of LC intoa population expressing B7 molecules and high levels of CD40.The presence in these lesions of large numbers of T cells express-ing CD154, a ligand expressed only transiently after activation,supports the idea that the LC are indeed activating T cells in situ.

These studies, evaluating established LCH granulomas, do notprovide insights into the mechanisms responsible for the initiationof the process. In this regard, it has recently been shown that LCin lesions from patients with both diffuse LCH and localized formsof the disease are of clonal origin (24, 25). However, several lines

FIGURE 3. Bronchiolar epithe-lium from an IL-10 K/O mouse. DCexpressing class II molecules wereidentified in the airway epithelium(A), but serial sections of the sametissue specimen were constantlyneg-ative for B7-1 (B) and B7-2 (C)immunostaining. Original magnifi-cations,3250.

FIGURE 2. Correlation between the expression of B7 molecules by pulmonary LC and the cytokines produced in the microenvironment of these cells.Bronchiolar epithelial cells (A), hyperplastic alveolar epithelial cells (B), and lung carcinomas (C), sites devoid of positive LC, produced IL-10, whereaspulmonary LC granulomas (D), which contained strongly B7 positive LC, were constantly negative for this cytokine. All sites positive for IL-10 werenegative for IL-1b (E, F, andG), whereas LC granulomas were intensely positive for this cytokine (H). Original magnifications,3250.

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of evidence strongly refute the idea that adult pulmonary LCH isa malignant disorder, including the high proportion of spontaneousremissions seen in pulmonary LCH, the very low proliferation rateof LC in pulmonary LCH granulomas, and the virtual absence ofLC in late lesions (2, 26). Nevertheless, such clonal LC could haveabnormalities that predispose to the initiation of the process, suchas increased sensitivity to maturation induced by cytokines or de-fects that interfere with the mobilization of LC toward regionallymphoid tissues. Pulmonary LCH is also strongly associated withcigarette smoking, and smoking-induced immune/inflammatoryresponses in peripheral airways may be required to initiate theabnormal differentiation of LC (27). Following these initial events,several pathways that would favor the persistence of granulomascontaining lymphostimulatory LC in LCH were identified in thesestudies. First, LC granulomas destroy the bronchiolar epithelium,thereby eliminating the only local source of IL-10. Second, LC inLCH granulomas themselves were shown to produce GM-CSF,TNF-a, and IL-1b, cytokines that could promote the maturation ofnewly arriving LC. Finally, the strong expression of CD40 by LCin LCH granulomas and the presence of T cells expressing CD154would promote CD40/CD154 interactions that increase the expres-sion of B7-1 and B7-2 on LC, induce cytokine production, andimprove the lymphostimulatory activity of LC.

In summary, this study further supports the idea that an immuneresponse initiated by LC is involved in the pathogenesis of adultpulmonary LCH and provides evidence that the unusual state ofactivation of LC within the lesions is influenced by cytokines pro-duced in the local milieu. These findings suggest that therapeuticstrategies based on the modulation of cytokine production may beuseful in this often disabling disorder for which no efficacioustreatment is currently available. Likewise, therapies designed toinduce the expression of costimulatory molecules by LC may behelpful in inducing immune responses against lung carcinomas.

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FIGURE 4. A, LC in pulmonary LCH granulomas intensely expressCD40 Ags.B, On a serial section of the same granuloma, many T cellsinfiltrating the granulomatous lesion were stained with the anti-CD154mAb. Original magnifications,3250.

3515The Journal of Immunology

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