Canine Cutaneous Histiocytoma Is an

American Journal ofPathology, Vol. 148, No. 5, May 1996Copyright © American Societyfor Investigative Pathology

Canine Cutaneous Histiocytoma Is anEpidermotropic Langerhans Cell HistiocytosisThat Expresses CD1 and Specific f32-lntegrinMolecules

Peter F. Moore,* Mark D. Schrenzel,*Verena K. Affolter,*Thierry Olivry,t and Diane NaydantFrom the *Department ofPathology, Microbiology, andImmunology and Veterinary Medical Teaching Hospital,tSchool of Veterinary Medicine, University of California,Davis, Davis, California, and the Department ofCompanion Animal Medicine,t College of VeterinaryMedicine, North Carolina State University, Raleigh,North Carolina

Canine cutaneous histiocytoma (CCH) is a com-mon, benign neoplasm ofthe dog. Histiocytomasmost commonly occur as solitary lesions thatundergo spontaneous regression. The age-spe-cific incidence ratefor histiocytomas drops pre-cipitously after 3 years, although histiocytomasoccur in dogs ofaU ages. Langerhans ceUs (LCs)in humans and dogs express abundant majorhistocompatibility complex class II moleculesand a variety of leukocyte antigens characteris-tic of dendritic ceU differentiation includingCDla, CDib, CD1c, and CDIJc. The immunophe-notype of CCH resembled that of cutaneous LCsby virtue of the expression of CD] molecules(CDJa, -b, and -c), CD11c, and major histocom-patibility complex class I. Furthermore, histio-cytoma ceUls had a tropismfor epidermis, whichwas also consistent with an epidermal LC lin-eage. The expression ofadhesion molecules sucbas CDiib (variable), CD44, CD54 (ICAM-)), andCD49d (VLA-4) in CCH indicated that the infiltrat-ing cells had some ofthe characteristics of acti-vated LCs, as these molecules are not expressedby normal, resting canine epidermal LCs. CCHdid not express Thy-i or CD4. Thy-i expressionis a characteristic of human and canine dermaldendrocytes, which are perivascular dendriticantigen-presenting ceUs closely related to epider-

mal LCs. CD4 expression is prevalent in humanLC histiocytosis, and in this respect CCH differedfrom human LC histiocytosis. Here we demon-strate that CCH is a localizedform of self-limit-ing LC histiocytosis, which predominantly ex-presses an epidermal LCphenotype. CCH occursas solitary or, less commonly, as multiple cuta-neous nodules orplaques, which rarely may ex-tend beyond the skin to local lymph nodes. Re-gression ofCCH occurs spontaneously in the vastmajority of cases in primary and secondarysites, and is mediated by CD8+ a18 T ceUs. Thehighfrequency ofCCH within the general caninepopulation offers the potential that the dog mayprovide an interesting model system to furtherthe understanding ofLCproliferative disorders,particularly the self-limiting, cutaneousform ofhuman LC histiocytosis. (Am J Pathol 1996,14&-1699-1 708)

Canine cutaneous histiocytoma (CCH) is a common,benign, cutaneous neoplasm of the dog.1-7 Histio-cytomas usually occur as solitary lesions that un-dergo spontaneous regression. The age-specific in-cidence rate for histiocytomas drops precipitouslyafter 3 years, although histiocytomas do occur indogs of all ages.2 Reports of recurrence of histiocy-tomas at the same or other sites are rare, and theoccurrence of multiple tumors is considered un-usual. Epidermal invasion by cells of histiocytomafrequently occurs, and intra-epidermal nests of his-tiocytes resemble Pautrier's aggregates, character-istically found in epidermotropic lymphoma (mycosis

Supported in part by the Laboratory for Companion Animal Health,School of Veterinary Medicine.

Accepted for publication January 15, 1996.

Address reprint requests to Dr. Peter F. Moore, VM Pathology,Microbiology, and Immunology, University of California, Davis, CA95616.

1699

1700 Moore et alAJP May 1996, Vol. 148, No. 5

Table 1. Clinical Summary of Cutaneous Histiocytoma Cases

Number Age range (years; Epitheliotropism Lesion recurrence LymphadenopathyPresentation of cases mean ± SD) (n cases) (n cases) (n cases)

Solitary lesions 16 0.3-12.0 (4.3 ± 3.5) 10/13 1 1Multiple lesions 7 0.2-9.0 (2.5 ± 3.0) 6/7 1 3

fungoides). Epidermal invasion in histiocytoma or thepresence of simultaneous multiple histiocytomas es-

pecially in aged dogs can present a diagnostic di-lemma, and distinction from mycosis fungoides andnon-epitheliotropic cutaneous lymphoma is difficulton purely morphological grounds.

Immunophenotypic analysis of histiocytic popula-tions in humans has enabled differentiation of a va-

riety of macrophages and dendritic/antigen-present-ing cells (APCs).8 The dendritic cells of human skininclude epidermal Langerhans cells (LCs) and der-mal dendrocytes, which are distinguished by theirrespective locations and antigen expression pro-files.8-12 LCs in humans and dogs express abundantmajor histocompatibility complex (MHC) class 11 mol-ecules and a variety of leukocyte antigens charac-teristic of dendritic cell differentiation includingCDla, CD1c, and CD1 1 c.1 1 1318 Dermal dendro-cytes in humans and canines are perivascular den-dritic cells located most prominently in the superficialdermis. They are identified by their expression ofMHC class 11, Thy-1, and factor Xlila, which is an

enzyme of the coagulation cascade; initial reportssuggest that they did not express CD1a.9'10 Subse-quently, a detailed analysis of MHC class II+ dermalcell suspensions revealed heterogeneous popula-tions of cells of both the monocyte/macrophage lin-eage and dendritic/APC lineage. The dendritic/APCsexpressed the phenotype CDl a'0,CD1 b,CD c,

CD1 1 b,CD1 1 chi; these cells also expressed intracy-toplasmic factor Xllla and were potent in alloantigenpresentation to T cells.12 This has recently beenconfirmed independently.19 Hence, dermal dendro-cytes are more closely related phenotypically to LCsthan was originally thought, and expression of CD1molecules by skin histiocytes is a marker of dendrit-ic/APC lineage and serves to differentiate these cellsfrom macrophages. Within the epidermis, CDla ex-

pression is the most reliable marker of LC differenti-ation. LCs can up-regulate the expression of addi-tional molecules, which are not evident or minimallyexpressed in resident epidermal LCs in unstimulatedskin; this is indicative of activation or further differ-entiation. These molecules include CD4, very lateactivation antigen-4 (VLA-4 or CD49d), CD44, andintercellular adhesion molecule-1 (ICAM-1 ).20,21

We hypothesized that CCH was a localized LChistiocytosis (LCH) based on the cutaneous localiza-tion of the tumors, the morphological characteristicsof the cells, and the epidermotropic tendency ob-served in many histiocytomas. In this paper, we de-scribe the immunophenotypic characteristics of sol-itary and multiple histiocytomas and the lymphoidinfiltrate that coincides with spontaneous regression.Furthermore, we present evidence that histiocyto-mas represent a proliferative disorder of epidermalLCs, which are of the dendritic/APC lineage.

Materials and Methods

Clinical SummaryExcised tumor tissues from 23 cases of CCH wereobtained from regional veterinary practices and theVeterinary Medical Teaching Hospital at University ofCalifornia, Davis, for evaluation (Table 1). The diag-nosis of histiocytoma was based on characteristicmorphological and clinical criteria.2'5 The affecteddogs were from 2 months to 12 years of age (mean,3.7 ± 3.4 years). Two dogs had an original diagnosisof mycosis fungoides based on epidermal invasionby the infiltrates and the advanced age of the dogs.Their lesions regressed spontaneously shortly aftersurgical biopsy. In the majority of dogs, surgicalexcision was curative, although in two dogs, thetumors recurred at the original site several monthsafter surgical excision and were again excised with-out further complication. Seven dogs had tumors inmultiple sites. In three of these dogs, lesions wereexcised or regressed spontaneously. In the otherfour dogs, new lesions appeared over a 4- to 16-month period. The lesions eventually regressedspontaneously in two dogs, the third dog died frombacterial septicemia secondary to colonization of theulcerated skin lesions, and the fourth dog was eu-thanized because of lymphadenopathy of the pres-capular lymph nodes. In a related case, a 3-month-old Boxer presented with obliteration of the cervicallymph node chain, which occurred 2 weeks afterexcision of a solitary histiocytoma on the cheek. Twoof these lymph nodes were excised and submittedfor immunophenotyping. Lymphadenopathy in the

CD1 Expression in Canine Histiocytoma 1701AJPMay 1996, Vol. 148, No. 5

Table 2. Monoclonal Antibodies Specific for Canine Leukocyte Antigens

Antigen MAb Isotype Reference Source

CDia CA9.AG5 IgGl Submitted P. MooreCD1b CC20 IgG2a 22 C. Howard (Compton, UK)CDic CA13.9H11 IgGl Submitted P. MooreCD3s Polyclonal 24 Dako (Carpinteria, CA)CD4 CA13.1E4 IgGl 48 P. MooreCD5 YKIX.322 IgG2a 49 S. Cobbold (Oxford, UK)CD8a CA9.JD3 IgG2a 48 P. MooreCD11a CA11.4D3 IgGl 16 P. MooreCD11b CA16.3E10 IgGl 16 P. MooreCD11c CA11.6A1 IgGl 16 P. MooreaD CA11.8H2 IgGl 29 P. Moore

CA18.3C6 IgGi 29 P. MooreCD18 CA1.4E9 IgGl 50 P. Moore

CA16.3C10 IgGl Unpublished P. MooreCD21-like CA2.1D6 IgGl 48, 49 P. MooreCD44 S5 IgGl 51 B. Sandmaier (Seattle, WA)CD45 CA12.10C12 IgGl 48, 49 P. MooreCD45RA CA4.1D3 IgG1 48, 49 P. MooreCD49d-like (VLA-4) CA4.5B3 IgGl 48, 49 P. MooreCD54 (ICAM-1) CL18.1D8 IgGi 52 C. Smith (Houston, TX)CD79a (MB-1) HM57 26 M. Jones (Oxford, UK)CD90 (Thy-1) CA1.4G8 IgGi 49 P. MooreMHC class 11 CA2.1C12 IgGi 49 P. MooreTCRaf3 CA15.8G7 IgGl 53 P. MooreTCR-y CA20.8H1 IgG2a 54 P. Moore

remaining lymph nodesweeks without treatment.

resolved over the next 4 toplasmic peptide sequence derived from humancDNA, this sequence is also conserved in a variety of

26species.

AntibodiesMonoclonal antibodies (MAbs) specific for canineleukocyte antigens were produced in the author'slaboratory (CA prefix) or were obtained from listedsources (Table 2). CD1b was detected by a cross-reactive MAb (CC20) specific for bovine CDib.22CC20 also reacts with COS cells transfected withhuman CDi B.22 Specificity of CC20 for canine CDi bwas confirmed by comparison of immunoprecipi-tates of biotin-labeled thymocytes with CA9.AG5(CD la, 49 and 12 kd), CA13.9H1 1 (CD1 c, 43 and 12kd), and CC20 (CDib, 45 and 12 kd). The 12-kdsubunit likely represented 32-microglobulin, whichassociates with CD1 and MHC class molecules.23Hence, canine CDla, -b, and -c molecules werebroadly similar to their human homologues with re-spect to antigen molecular weight and also with re-spect to cell and tissue distribution by flow cytometryand immunohistology23 (manuscript submitted). Ca-nine CD3E was detected by a polyclonal rabbit anti-serum (A452, Dako Corp., Carpinteria, CA) that isspecific for a 13-mer peptide sequence from thecytoplasmic domain of human CD3E.24 This se-quence is highly conserved in several mammalianspecies including canine.2526 CD79a (MB-i) wasdetected by a MAb specific for a conserved intracy-

Tissue Handling and ImmunohistochemistryTissues from histiocytomas or normal canine controltissues (skin, tongue, thymus, spleen, and tonsil)were fixed in 10% neutral buffered formalin; addi-tional blocks were snap frozen in dichlorodiflu-oromethane (Freon 22) or isopentane cooled to theirfreezing points in liquid nitrogen. Formalin-fixed tis-sue was embedded in paraffin, and 4- to 6-,um sec-tions were stained with hematoxylin and eosin (H&E).Frozen sections were stained by a streptavidin-horseradish-peroxidase method according to manu-facturer's instructions (Zymed, South San Francisco,CA) and previously described methods.16 MAbsspecific for canine leukocyte antigens (Table 2) wereapplied to sections as diluted tissue culture super-natants based on previous titration on frozen sec-tions of normal canine spleen and thymus. Negativecontrols consisted of substitution of specific MAbwith isotype-matched nonspecific MAb or omissionof primary antibody. MAbs were also applied to fro-zen sections of normal spleen to ensure that specificand characteristic staining of leukocyte populationswas achieved in each run. Normal epidermal LCs inskin adjacent to the histiocytoma served as a posi-tive control for CDia expression. This latter control


Figure 1. Normal canine skin illustrating CD1a+ dendritic cells withinthe epidermis. Immunoperoxidase; magnification, X340.

was particularly important, as MAb CA9.AG5 detectsan allotypic variant of CDia, which is expressed byapproximately 80% of dogs (manuscript submitted);a negative staining result in the tumor had to beinterpreted with caution. It should also be noted thatthe epitopes recognized by MAbs used in this studyare destroyed by formalin fixation, with the exceptionof epitopes recognized by polyclonal anti-CD3E,anti-MB-1, anti-aD (CA18.3C6), and anti-CD18(CA16.3C10). These latter reagents were useful inaccurately differentiating cutaneous T cell lymphomafrom histiocytoma in formalin-fixed, paraffin-embed-ded sections (data not shown).

Results

Immunophenotypic Characteristics ofCanine Langerhans CellsCanine LCs were identified in frozen sections of skinand mucosal sites, such as tongue and tonsillar ep-ithelium, as MHC class 11+, CDla+, CDlb+ (vari-able), CDlc+ intra-epithelial dendritic cells, consis-tent with previous observations in humans (Figure1).12,17 Canine LCs had consistent, preferential ex-pression of individual f32-integrins (CDi 1/CD1 8) andisoforms of the leukocyte common antigen family(CD45) similar to that described for human epider-mal LCs.12'27'28 In particular, normal canine LCs ex-pressed CD18 and the associated CD1 1 moleculesCD1ia and CD1 l1c but did not express CD1 lib or aD,the novel /32-integrin expressed by splenic red pulpmacrophages.29 Canine LCs also expressed CD45but did not express CD45RA. Thy-1 was not ex-pressed by epidermal LCs, although Thy-1+, MHCclass II+, CDla, -b, and -c+, and CDiic+ dendritic

Figure 2. Epidermal invasion by nests of tumor histiocytes in cutane-ous histiocytoma. Tumor histiocytes possess abundant cytoplasm andirregular nuclear profiles, which are either oval, indented, orfolded.H&E; magnification, x 485.

cells were observed in the canine dermis. Thesewere likely dermal dendritic/APCs, which also havebeen reported to express factor Xllla.9'10123031.However, Thy-1 was broadly expressed; vascularendothelium and dermal fibroblasts also expressedThy-1. Additionally, canine epidermal LCs did notexpress CD4, CD44, CD54 (ICAM-1), or CD49d.These molecules have been demonstrated, althoughsometimes inconsistently, on normal epidermal LCsor freshly isolated epidermal LCs from humans ormice. 14'15'20'21'2832,33 Hence, expression of CD1molecules, particularly CDia and CDic, were thebest criteria of LC differentiation in canine epidermis.However, expression of CD1 a by epidermal LCs wasdetectable in only approximately 80% of dogs withMAb CA9.AG5, as this MAb recognized an allotypicvariant of CDia. The presence of epidermal LCs inCD1 a-allotype-negative dogs was confirmed by theirexpression of MHC class 11 and CDic in serial sec-tions of the respective tissues.

Morphological and ImmunophenotypicCharacteristics of CCHHistiocytomas occurred as nodular lesions that infil-trated the superficial and deep dermis and subcutis.Epidermal invasion by histiocytes was observed in16 of 20 cases, and in 11 cases this feature wasprominent (Figure 2). Epidermal invasion was notassessable in 3 cases; in 2 of these, complete epi-

CDM Expression in Canine Histiocytoma 1703AJP May 1996, Vol. 148, No. 5

;~~~~~~~~~~~~~~~~~~~~N ; 4'AK. >R:S ,

Figure 3. CDla expression by tumor histiocytes in the dermis in cuta-neous histiocytoma. Normal CD1a+ LCs are present within the epider-mis. Immunoperoxidase; magnification, X340.

dermal ulceration prevented evaluation. Histiocyteswithin tumors possessed abundant eosinophilic cy-toplasm and either round, indented, or folded nuclei(Figure 2). In the 2 cases of lymph node involvement,tumor histiocytes obliterated the paracortex and fol-licular domains and the cortical and medullary si-nuses.The tumor cells within CCH consistently and dif-

fusely expressed CD1 b, CD1 c, CD1 1 a, CD1 1 c, andMHC class 11 in all instances. Tumor cells expressedCDia in 19 of 23 cases (Figure 3). Normal LCs inskin adjacent to the histiocytomas did not expressCDia in the 4 CDia-negative tumors, which indi-cated that these dogs did not express the CDiaallotype recognized by CA9.AG5. In tumors that ex-hibited prominent epidermal invasion, CD1a expres-sion was prominent in both the dermal and epider-mal histiocytes (Figure 4). Tumor cells did notexpress aD, a novel f32-integrin that is highly ex-pressed by splenic red pulp macrophages.29CD1 1 b was expressed by histiocytes either diffuselyor regionally in 63% of cases; a similar distribution ofthe ,31-integrin (VLA-4) and ICAM-1 was observedwithin histiocytomas in 91 and 94% of cases, respec-tively. CD44 was intensely expressed by tumor his-tiocytes in all instances. Prominent high endothelialvenules were visible in the superficial and deep der-mis of histiocytomas stained with anti-Thy-1 MAb.Intense endothelial expression of Thy-1 was ob-served in these high endothelial venules, althoughtumor histiocytes lacked Thy-1 expression (Figure 5).Likewise, tumor histiocyte expression of CD4 was notobserved.

Marked lymphoid infiltration, often accompaniedby necrosis of histiocytoma cells, was observed in

Figure 4. Epidennal invasion by nests of CDla+ tumor histiocytes incutaneous histiocytoma. Immunoperoxidase; magnification, X 250.

52% of CCH lesions. The lymphoid infiltrates wereinitially heaviest in perivascular locations near thejunction of the subcutis and the deep dermis in sometumors (Figure 6). In other tumors, the lymphoid in-filtrates were diffuse and radiated from perivascularlocations at all levels of the lesion. The infiltratinglymphocytes expressed T cell receptor-af3 and CD3and were almost exclusively of the CD8 subset (Fig-ure 6). CD4 lymphocytes and B lymphocytes(CD21+ and CD79a+) were encountered less fre-quently.

In formalin-fixed, paraffin-embedded sections,histiocytomas could be readily distinguished fromcutaneous lymphomas by demonstration of intenseCD18 expression by tumor histiocytes (using

~~~~Ai~~~~~~~~~~r

Figure 5. Thy-1 expression kV endothelial cells in dermal high endo-thelial venules in cutaneous histiocytoma, Several reactive Thyl:dermal dendritic cells are interspersed among Thyv 1 tumor histio-cytes. Immunoperoxidase; magnification, X340.

.71=


Figure 6. CD8e lymphoid infiltrate in regressing cutaneous histiocy-toma. Immunoperoxidase; magnification, X340.

CAl 6.3C10) and lack of CD3 and CD79a expressionto rule out T cell and B cell proliferative disorders,respectively (data not shown). These three reagentshave much utility in routine diagnostic pathologywhen fresh, unfixed tissue is routinely unavailable.

DiscussionThis investigation has demonstrated that tumor his-tiocytes in CCH are phenotypically related to LCs byvirtue of their expression of a spectrum of CD1 mol-ecules. Tumor histiocyte expression of CDla, whichis normally restricted in its expression to corticalthymocytes and dendritic/APCs in the thymic me-dulla and cutaneous or mucosal epithelia (LCs), wasmost supportive of this conclusion. Furthermore, his-tiocytoma cells have a tropism for skin, particularlyfor epidermis. This behavior is also consistent withan epidermal LC lineage. In previous investigationsof CCH, it was concluded that tumor cells were likelyof mononuclear phagocytic lineage but were unlikelyto be related to LCs because they lacked Birbeck'sgranules.3 4 However, it was later shown that normalcanine epidermal LCs lacked Birbeck's granulesand did not express broadly distributed antigenicand enzymatic markers commonly associated withLCs in other species, such as S100 and ATPase.13However, as we have shown here, canine epidermalLCs resemble their counterparts in humans in termsof their leukocyte antigen expression profile, partic-ularly their abundant expression of CDla, CDlc,CD11c, and MHC class 11 molecules.

LCs are members of the dendritic/APC lineageand are located within squamous epithelia such asepidermis and certain mucosal epithelia.34 Dendrit-

ic/APCs originate from bone marrow and occur atlow frequency in blood but are widely distributed inlymphoid and nonlymphoid tissues.8 Mature dendrit-ic/APCs are morphologically, immunophenotypi-cally, and functionally distinct from cells of mono-cyte/macrophage lineage.8 However, dendritic/APCs and monocytes may originate from a commonbone marrow precursor, and diversion of cells com-mitted to a monocytic lineage to dendritic/APCs hasnot been excluded.3536 The conditions that directdifferentiation and growth of dendritic/APCs arepoorly understood, although recent evidence impli-cates a major role for granulocyte/macrophage col-ony-stimulating factor (GM-CSF) and tumor necrosisfactor (TNF)-a in the differentiation of LCs fromCD34+ hemopoietic precursors.37 The infrequencyof dendritic/APC precursors among bone marrowcells implies that dendritic/APCs could potentiallymature outside of bone marrow from stimulation ofcirculating CD34+ hemopoietic precursors by locallyderived GM-CSF and TNF-a.37 In support of thishypothesis, intradermal injection of GM-CSF leads tothe selective accumulation of CD1a' LCs in the der-mis of human subjects.38 Additionally, keratinocytesand skin-seeking lymphocytes can produce GM-CSF and TNF-a, and dermal mast cells have TNF-ain cytoplasmic granules.39'40 Hence, current opinionfavors the derivation of dendritic/APCs from a com-mon hemopoietic precursor and suggests a role forlocally derived cytokines in directing the localizationand differentiation of specific subsets of dendritic/APCs. The possibility of inter-relationships betweensub-lineages of cutaneous dendritic/APCs is appar-ent. The observed morphological and immunophe-notypic differences between dendritic/APCs in thedermis and epidermis (LCs) may be more reflectiveof local environmental factors than major lineagedifferences. 12The ,2-integrins and CD1 molecules are impor-

tant markers for the identification of specific macro-phage and dendritic/APC populations. The ,B2-inte-grins CD11b, CD11c, and aD are differentiallyexpressed by histiocytic populations in normal ca-nine tissues.16'29 For instance, hepatic Kupffer cellspredominantly express CD11b. In spleen, CD11c isabundantly expressed by dendritic/APCs, which in-clude interdigitating dendritic cells in T cell domainsand marginal zone dendritic cells. In contrast,splenic red pulp macrophages predominantly ex-press aD. CD1 1ic is also preferentially expressed byepidermal LCs and perivascular dendritic/APCs(dermal dendrocytes). Hence, expression of CD1i cby resident histiocytic populations best correlateswith dendritic/APC lineage. Likewise, the expression

CD1 Expression in Canine Histiocytoma 1705AJP May 1996, Vol. 148, No. 5

of CD1 molecules is also highly regulated in thethymus and peripheral lymphoid organs.23 CD1 ex-pression is down-regulated as thymocytes becomecommitted to either CD4 or CD8 expression. Hence,CD1 expression maps to the thymic cortex; CD1expression in the thymic medulla is limited to resi-dent dendritic/APCs. Beyond the thymus, CD1 ex-pression is restricted and correlates with cells in-volved in antigen presentation. CDlc is expressedby dendritic/APCs and by subpopulations of B cellsand monocytes. CDla in particular is expressedmainly by epidermal and mucosal LCs and is incon-sistently expressed by dendritic/APCs within lym-phoid tissue. Collectively, the expression of CDla,CD1c and CD11c reliably identifies epidermal LC incanine skin. In normal canine dermis, the exact lin-eage relationships of dendritic/APC which expressCD1a, CD1b, CD1c, Thy-1 or CD11c are still unset-tled, as is the case in humans.12Tumor histiocytes in CCH expressed MHC class 11,

CD1 b, CD1 c, and CD1 1 c in all cases. CD1 a expres-sion was detected on infiltrating histiocytes in allcases in which the CDla allotype, detected by MAbCA9.AG5, was present (19 of 23 cases). Therefore,the phenotype of infiltrating histiocytes in cutaneoushistiocytoma was identical to the phenotype of nor-mal epidermal LCs with respect to the expression ofCD1 and )32-integrins, the most definitive markers forthe identification of canine macrophages and den-dritic cells.

Up-regulation of the expression of a ,B1-integrin(VLA-4), CID1 b, CD44, and ICAM-1 was observedin a variable proportion of histiocytomas. The expres-sion of CD44, VLA-4, and ICAM-1 were particularlyprevalent (90 to 100% of cases). These three mole-cules are not expressed by canine epidermal LCs.Up-regulation of VLA-4 in CCH may be of signifi-cance in determining the migration of cells to thelesion or from the site of the lesion to draining lymphnodes. For instance, up-regulation of CD44, VLA-4,ICAM-1, and MHC class 11 has been observed oncultured murine LCs and is thought to simulate thefunctional differentiation of LCs to mature dendritic/APCs that occurs during LC migration to lymphnodes after antigen challenge.20 The migration ofthese activated LCs is believed to depend on up-regulation of appropriate cell adhesion molecules.Under these circumstances, up-regulation of VLA-4is also observed and may play an important role inLC migration across activated endothelium that ex-presses VCAM-1, one of the ligands of VLA-4, orthrough connective tissue that contains fibronectin,another ligand for VLA-4.41

Investigation of leukocyte cell adhesion moleculeexpression in human LCH highlighted differencesbetween LCH cells and epidermal LCs, which weresuspected to be involved in abnormal homing anddifferentiation.18 Specifically, these authors notedconsistent, intense expression of CD1 1c and incon-sistent, variable CD1 lb expression. Intense CD44,CD54 (ICAM-1), and CD58 expression was consis-tently observed. They concluded that LCH cells ex-press markers expected of epidermal LCs when theyare activated and migrate to draining lymph nodes.LCH is currently believed to represent a disorder ofimmune regulation,42 although evidence for clonalLC proliferation in LCH has also been reported.33The phenotype of the LCH cell resembles that ofactivated epidermal LCs, which are capable of mi-gratory behavior. This activated phenotype may con-tribute to the aberrant migration, persistence, andproliferation of abnormal cells in LCH.The adhesion molecule expression pattern in CCH

is quite similar to human LCH. Similar pathogeneticmechanisms, which dictate abnormal differentiation,homing, and migration may be operative in cutane-ous histiocytoma, although the lesions in the majorityof cases of cutaneous histiocytoma are usually morelimited in extent and remain localized to the skin.However, we are aware of four instances of con-firmed migration of tumor histiocytes from the skin todraining lymph nodes. Two of these cases werepresented here, and two cases were communicatedto us by Liz Wilson. An additional two cases devel-oped lymphadenopathy in association with cutane-ous histiocytoma, although migration of tumor histio-cytes was not confirmed by histopathology. Draininglymph nodes are not routinely sampled in cases ofCCH, because a favorable prognosis is anticipated.Therefore, the true incidence of migration of CCH tolymph nodes may in fact be higher. In most in-stances, spontaneous regression occurred, evendespite obliteration of draining lymph nodes. Theonly exception was a dog that had multiple skintumors and developed massive lymphadenopathy.Euthanasia was performed at the request of theowner. No evidence of spread beyond the involvedlymph node was found at necropsy. Hence, CCH isa self-limited form of LCH that remains largely local-ized to the skin, despite the up-regulated expressionof adhesion molecules capable of conferring aber-rant migratory capability. Localized histiocytomasand migratory histiocytomas did not differ with re-spect to the expression of these molecules. Otherfactors must also influence the migratory potential oftumor histiocytes in this disease.


The factors that determine the onset of regressionin CCH are unknown. Evidence of regression is usu-ally observed in lesions that have been present foronly a few weeks, although regression can be de-layed for many months in problem cases. Regard-less, regression is mediated by CD8+ a3 T cells;only scant numbers of CD4+ T cells are observed inCCH lesions. By implication, the stringent co-stimu-latory requirements of CD8+ cytotoxic effector Tcells must have been met for them to mediate re-gression.43 This could occur in several ways. Migra-tion of tumor histiocytes to draining lymph nodescould activate resident dendritic/APCs and CD4+ Tcells, which would assist in CD8+ cytotoxic T cellactivation via exogenous interleukin-2. Alternatively,tumor histiocytes may undergo local differentiationand up-regulate co-stimulatory molecules of the B7family and thereby directly activate CD8+ cytotoxic Tcells and mediate their own destruction.43 Studiesare currently underway to test the latter hypothesis.A major point of departure between human LCH

and CCH is the lack of expression of CD4 by thecells of histiocytoma. Up-regulation of CD4 expres-sion is consistently observed in human LCH anddistinguishes LCH cells from resident epidermalLCs, which do not express significant amounts ofCD4.28'33 Preliminary observations indicate that his-tiocytic expression of CDla, -b, and -c and CD4 isobserved in other, more generalized histiocytic dis-eases of canine skin (Affolter and Moore, unpub-lished observations), such as systemic histiocytosis,which mostly afflicts Bernese Mountain dogs,44 andcutaneous histiocytosis.45 These diseases are LCHsthat occur in the context of disordered immune reg-ulation and are responsive to immunosuppressivetherapy with steroids (cutaneous histiocytosis) orwith cyclosporin A (systemic histiocytosis; Affolterand Moore, unpublished observations). It is possiblethat CD4 expression in this context may be a keyfeature that indicates an activated cell phenotypeassociated with more aggressive behavior.

The availability of MAbs specific for molecules thatdetermine canine leukocyte differentiation, migra-tion, and adhesion will enable further investigation ofcell lineage and pathogenetic mechanisms in cuta-neous lymphoid and histiocytic neoplasia in dogs.The distinction of epidermally invasive CCH fromepidermotropic lymphoma (mycosis fungoides)46 isreadily achievable by the application of these mark-ers. Also, the stage is set for a re-evaluation of theclassification and pathogenesis of the spectrum ofcanine histiocytic proliferative disorders.44,45,47Here, we have provided evidence that CCH is alocalized form of LCH in which regression is ex-

pected to occur. Other forms of canine LCH, whichencompass more aggressive clinical and patholog-ical disorders, exemplified by systemic histiocytosisand malignant histiocytosis,44,47 have been more re-cently identified (Affolter and Moore, unpublishedobservations). The high frequency of these diseaseswithin the general canine population (histiocytoma)or in genetically defined segments of the caninepopulation (malignant and systemic histiocytosis) of-fers the potential that the dog may contribute inter-esting model systems to further the understanding ofLC proliferative disorders of diverse etiology.

AcknowledgmentsThe authors thank the veterinarians at Midtown Ani-mal Hospital in Davis for their perseverance in iden-tifying case material. We also thank Dr. Liz Wilson forcommunicating her experience of two cases of mi-gratory histiocytoma to us.

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Canine Cutaneous Histiocytoma Is an