J Clin Pathol 1987;40:1413-1417

Unusual immunophenotype displayed by histiocytes inhaemophagocytic lymphohistiocystosisT HERLIN,* G PALLESEN,t T KRISTENSEN,$ N CLAUSEN*

From the Departments of *Pediatrics, tLaboratory ofImmunohistology, University Institute ofPathology, and$Tissue Typing Laboratory, University Hospital, Aarhus, Denmark

SUMMARY Extensive immunophenotypic studies in a 2 '/2 month old girl with haemophagocyticlymphohistiocytosis were performed to characterise the proliferating histiocytes of the disease. Thecells strongly expressed conventional macrophage antigens, but unexpectedly, there was a dis-sociated expression of the CD la antigen (reacting with the monoclonal antibody NA 1/34 but notwith OKT6) and intracellular S-100 protein by the haemophagocytic lymphohistiocytosishistiocytes. These findings indicate that there is a "hybrid" phenotype between the two main armsof the mononuclear phagocyte system-namely, Langerhans' cells and phagocytic macrophages.

Haemophagocytic lymphohistiocytosis (HL) is a rare,usually familial, and fatal disease of early child-hood. 5̀ The onset of symptoms occurs before 3months of age in about two thirds of patients. Clinicalfeatures include recurrent fever, hepatosplenomegaly,anorexia, pallor, and cerebral irritability, but the clin-ical and pathological variability of this disease oftenresults in a delayed or post mortem diagnosis. In mostcases the disease is inherited as an autosomal recessivetrait,' 4 but 25% of cases have no known family his-tory.45 The pathognomonic cell in the tissueinfiltrates has a morphological resemblance to cells ofthe mononuclear phagocyte system. Defects in bothhumoral and cellular immunity have been describedby a few investigators.6 7We report a patient in whom a functional primary

B lymphocyte defect was detected before dissem-ination of HL. In an attempt to characterise theinfiltrating cell of HL and the underlying immunedeficiency in more detail we performed immu-nohistological investigations on tissue samples ofseveral organs frozen at necropsy using a wide panelof monoclonal antibodies.

Case report

A 2 /2 month old girl of previously normal health andgrowth was admitted with a severe acute illness, char-acterised by high fever, dehydration, and hepa-tomegaly. There was no clinically important familyhistory. Laboratory investigations showed anaemia(haemoglobin 5 6 mmol/l), hypoproteinaemia, raisedSGOT, thrombocytopenia (platelets 40 x 109/l),and hypertriglyceridaemia (serum triglycerides 3-7

Accepted for publication 25 June 1987

mmol/l, normal range 0-5-20 mmol/l). The bonemarrow was hyperplastic with normally differentiatedcells. A normal leucocyte count with relative lympho-cytosis was repeatedly observed. The child clinicallyreturned to normal within three weeks with conser-vative treatment, and the liver size decreased. Severalrelapses with high fever, diarrhoea, and hepa-tomegaly occurred at three to four week intervals.Final deterioration with generalised oedema, hepaticinsufficiency, a profuse haemorrhagic diathesis andseizures then developed and she died at the age of 10months.

IMMUNOLOGICAL STUDIESSerum IgA concentrations were low throughout theillness (0-03 to below 0-01 g/l, normal range 008-0-98g/l). Serum IgG fell from normal (3 0 g/l) at the age of4 months to below O-1 g/l at 6 months of age (normalrange 2-06-11 25 g/l). The concentrations of IgMwere within the normal range (0-28-0-71 g/l) andserum IgD and IgE were not increased. At 6 monthsof age immunological investigations were performedduring a quiescent period. Natural killer cell activitywas in the low to normal range, but, unlike controlleucocytes, enhanced killer cell activity after incu-bation with interferon was not observed. Lymphocyteantibody dependent, cell mediated cytotoxicity wasdepressed. The distribution of T and B lymphocytesin the peripheral blood was normal, the CD4:CD8ratio being 2-6. Lymphocyte responses to mitogenand antigen stimulation were normal. B lymphocytefunction, evaluated by an indirect plaque forming cellassay,8 showed no in vitro immunoglobulin G, A, orM synthesis, either in presence of the patient's own Tcells or with control T cells, indicating functional Blymphocyte defect. On the other hand, the patient's T

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cells, added to control B cells, completely suppressed showed jaundice, hepatosplenomegaly and cerebralimmunoglobulin synthesis. This further indicated a oedema, but no lymph node enlargement. Micro-high functional T suppressor cell associated activity. scopy showed a diffuse infiltration of lymphocytic

and histiocytic cells in the organs. The infiltrates werePathology particularly prominent in the spleen, liver, kidney,

lymph nodes, bone marrow and central nervous sys-Necropsy was performed 18 hours after death. It tem. In the brain the infiltrate was perivascular and

Table Immunophenotype ofproliferating histiocyte in a case of haemophagocytic lymphohistiocytosis

Antigen defined Haemophagcytic Phagocyticby antibody Antibody Source lymphohistiocytosis histiocytes Langerhans' cells

B Cel antigemCD9CD1OCD19CD20CD21CD22CD23CD24CD37CD38CD39CDw40

T cell antigensCDlaCDlaCDlbCD2CD3CD4CD5CD6CD7CD8CD27CD28CDw29p180

J2F 103.12B4BlHB5Tol5MHM6BAIG28-1OKT1OG28-10G28-5

OKT6NAI/34NU-T2DAKO-TI IUCHT1Anti-Leu-3aTu7lTu33Tu14DAKO-T8OKT18AKolt-24B4UCHLI

Myelomonocytic antigensCD11a CC5-07CD11b OKMICDI Ic F90.93CD13 MY7CD14 UCHMICD15 3C4CD31 SG134CDw32 2EICD33 L4F3CD34 MYIOCD35 To5CD36 5F1Macrophages Ber-MAC3Macrophages EMBI IFollicular dendritic

cells R4/23Follicular dendritic

cells Ki-M4

Miscefaueous antigemECD25CD30CD45Regulatory T cellsP1 10CD16HLA-DRTransferrin receptorS-100 proteinEpithelial membrane

antigen

Tu69Ki-l, Ber-H22DIAnti-Leu-8Anti-Leu-7Anti-Leu- I I bF100.28OKT9Anti-S-100

E29

WS*Own laboratorytWSWSWSWSWsWSWSOrtho DiagnosticWsWs

Ortho DiagnosticDakopattsNichirei CorporationDakopattsP BeverleyBecton DickinsonA ZieglerA ZieglerA ZieglerDakopattsWSNichirei CorporationWSP Beverley

WSOrtho DiagnosticsOwn laboratoryCoulterP BeverleyH SteinWsWSWSWSD MasonWsH SteinDakopatts

D Mason

A Feller

A ZieglerH SteinP BeverleyBecton DickinsonBecton DickinsonBecton DickinsonOwn laboratoryOrtho DiagnosticsDakopatts

D Mason

2+03+3+3+002+2+2+2+3+

3+2+1+I+2+002+1+03+3+

03+0003+03+002+3+1+3+

01+2+002+01+0002+(+)3+

Not assessed000000Not assessed1+3+I+1+

3+3+Not assessed001+00000000

01+0/+0/+2+01+001+00/+00001+0

0

002+0002+02±Not assessed

3+2+3+2+3+01+3+2+2+01+3+3+

0

0

3+2+3+1+2+03+3,-01+1+03+3+

0

0

03+3+003+1+3+

2+

0/+03+1+003+3+0

1+

Antibody obtained from the Third International Workshop on Human Leucocyte Differentiation Antigens, Oxford, September 1986. Degreeof staining: 0 = no staining; 0/+ = a fraction (minority) of cells stain; (+) = borderline staining; 1 + = weak staining; 2 + = moderatestaining intensity; 3 + = strong staining. tln collaboration with Dr T Plesner, the Leukaemia Typing Laboratory, Copenhagen.

1414 Herlin, Pallesen, Kristensen, Clausen

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Unusual immunophenotype in haemophagocytic lymphohistiocytosis

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S~~~~~~~~~~~~~~_,L4Sicm * .~~~~~~~~~ j s i w_to.wt *K<F Lr. ttk\YJ<, W M'" * c2 fFigure Frozen section of kidney. Glomerulus surrounded by an infiltrate ofHL histiocytes and some lymphocytes isshown. (a) Stainedfor CD6 antigen with Mab Tu33; (b) stainedfor CD38 antigen with Mab OKTJO; (c) stainedforCDla antigen with monoclonal antibody NAI/34; (d) with monoclonal antibody OKT6; (e) stained with polyclonal anti-body to S-100 protein; (f) stainedfor CD35 antigen (C3b-receptor) with monoclonal antibody To5. Strong labelling ofHLhistiocytes is seen in a, b, c, and e, and negative reactions in d andf. Inf strong labelling is obtained on the glomerularpodocytes. (a-f: immunoperoxidase.)

extended from the meninges into the deep cortex. Itwas less pronounced in the lungs and thymus. Eryth-rophagocytosis was a conspicuous feature of thehistiocytes, which seemed to be cytologically benign.In the liver a pronounced and diffuse fatty change wasseen. The' spleen was congested with severe depletionof the lymphoid zones.The lymph node structure was severely effaced.

Apart from the histiocytic infiltration expanding thesinuses, there was complete absence of follicles and a

general lymphocyte depletion of the residual T zones.The thymus was reduced in size, mainly due todepletion of cortical thymocytes (the so called "stressthymus"). There was, however, no evidence of con-genital thymic dysplasia.

IMMUNOPHENOTYPICAL STUDIESAs most leucocyte differentiation antigens have beenshown to be fairly resistant to disintegration afterdeath9 fresh tissue from lymph nodes, spleen, liver,

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1416lung and kidney was frozen in liquid nitrogen at nec-ropsy for immunohistological analysis. Frozen sec-tions (7 gm) were cut in a cryostat, fixed in acetoneand chloroform, and finally stained by a three stageimmunoperoxidase technique, as previouslydescribed.'0 The antibodies selected for this studywere all monoclonal except the anti-S-100 protein.Table 1 briefly describes their reactivity and source.Many of the monoclonal antibodies were obtained atthe Third International Workshop and Conferenceon Human Leucocyte Differentiation Antigens, heldin Oxford in September 1986, details of which are tobe published." Sections of kidney and lymph nodewere stained with all the antibodies, but other tissueswith a selected panel.The expression by HL histiocytes of the various

leucocyte antigens is given in table 1, and some exam-ples of the staining reactions are illustrated in thefigure. For comparison, the immunophenotypes ofdermal Langerhans' cells and conventional phago-cytic histiocytes (studied in sections from cystic breastdisease) were also examined, and the results areincluded in table 1.The nature of the lymphocytes in lymph nodes

and tissue infiltrates was also examined in thisimmunohistological analysis. The lymph nodes werevirtually depleted of B cells and plasma cells, and noantigen presenting follicular dendritic cells wereidentified using the relevant monoclonal antibodies.The number of T cells (CD2 +, CD3 +, CD5 +) wasreduced and comprised about equal numbers ofCD4+ and CD8 + cells. There was a remarkableincrease in the number of Leu-7 + cells (killer andnatural killer cells) compared with normal reactivelymph nodes.

Discussion

Langerhans' cells or interdigitating dendritic cells oflymph nodes express CD1 antigen and intracellularS-100 protein, and Birbeck granules are visible onelectron microscopy.'2 13 HL has been classifiedpreviously as one of the non-Langerhans' cellhistiocytoses as it lacks any of the above cellularmarkers.'4 In the present study it has been shown thatthe proliferating cells in HL may express a uniquephenotype sharing characteristics of both the Lan-gerhans' cell and the phagocytic macrophage lineage.

In HL the clinical presentation is non-specific andrather variable, and for this reason a diagnosis afterdeath is quite common.4 Although a positive familyhistory was not present in our patient, the clinicalfindings were consistent with those seen in HL andincluded hypertriglyceridaemia, hypofibrinogen-aemia, and immunological disturbances. This differsfrom what is seen in Langerhans' cell histio-cytosis,4 5 12 so called malignant histiocytosis (nowusually recognised as anaplastic large cell lymphoma

Herlin, Pallesen, Kristensen, Clausenof the Ki-l + type,'5 and virus associated hae-mophagocytic syndrome,16 which lacks central ner-vous system disease and is often associated withincreased immunoglobulin concentrations.The characteristic histiocytes of HL were first ob-

served at necropsy in our patient. Histologically, theybore no resemblance to the proliferating cells of thefirst two of the above mentioned disorders.

Extensive immunophenotypical analysis of freshfrozen tissue obtained at necropsy was performed tocharacterise the proliferating histiocytes of HL moreclosely. It disclosed that the cells strongly expressedthe conventional macrophage antigens defined bymonoclonal antibodies of CD groups" 4, 1la, lib,1 Ic, 13, 14, 31, w32, 34, and by the monoclonal anti-bodies Ber-MAC3 and EBMI1. The expression ofCD38, CD6, HLA-DR, S-100 protein and epithelialmembrane antigen may indicate a high degree of acti-vation in HL histiocytes. CD38 and HLA-DR antigenare non-lineage specific antigens that are associatedwith cell activation, and the CD6 antigen (expressedby normal macrophages at low density) is associatedwith activation in a functional subpopulation ofCD4+ T cells. A recent report stated that epithelialmembrane antigen is expressed in activated lympho-cytes,'7 and we have often seen poor expression of thisantigen in tissue macrophages. Thus the expression ofthis antigen in macrophages may well be associatedwith activation in this cell lineage as well.

Interesting results were obtained with monoclonalantibodies to the CDla antigen. Both antibodies usedreacted strongly with skin Langerhans' cells, butOKT6 gave repeatedly negative and NA1/34 intensepositive staining ofHL histiocytes (fig lc and d). Thisfinding may reflect a difference in epitope specificity ofthese antibodies.The expression of CDla antigen (as defined by

monoclonal antibodies NAI/34) and of S-100 proteinby the HL histiocytes was unexpected, and togetherwith the expression of conventional macrophage anti-gens, this indicates that there is a "hybrid" phenotypebetween the two main arms of the mononuclearphagocyte system-namely, Langerhans' cells andphagocytic macrophages. The former express CDlaantigen and S-100 protein in high density and littleor no "conventional" macrophage antigens; theopposite is true for phagocytic macrophages.The immunohistological localisation of S- 100 a and

13 subunits in the bone marrow derived mononuclearphagocyte system has recently been outlined by Taka-hashi et al'8 using monospecific antibodies to thesesubunits. They confirmed the presence of the (3 sub-unit in the Langerhans' cell arm of this system (Lan-gerhans' cell, indeterminate cells of the skin,interdigitating cells of the lymph nodes, histiocytosis-X cells). Moreover, they found the a subunit to bewidely distributed among cells of the other arm of thesystem-that is, monocytes, most tissue macrophages,

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Unusual immunophenotype in haemophagocytic lymphohistiocytosis 1417

lung alveolar macrophages, some Kupffer cells, butalso epitheloid cells and Langerhans' giant cells ingranulomatous reactions. Their results suggest thatcells of the macrophage system under normal condi-tions may be subdivided according to their immu-noreactivity solely with the S-100 oa subunit or solelywith the P subunit.The meaning of the aberrant S-100 ,B subunit ex-

pression in HL histiocytes in our case, which by mostother criteria were phagocytic histiocytes, is not un-derstood, but perhaps an abnormal activation of theHL histiocytes, as suggested by Goldberg andNezelof5 may be associated with an additional expres-sion of the S-100 Pi subunit in some cases.Few previous studies have been reported on the

immunological markers ofHL histiocytes. Wieczoreket al19 examined frozen tissue from one case with alimited panel of antibodies and reported the cells to benegative for S-100 protein, OKT6 (CD la), OKM1(CDllb), OKM5 and Leu-3a (CD4), but positivefor HLA-DR, Leu-M3, and Leu-M1. Goldberg andNezelof5 studied frozen tissue sections from two cases,and HL histiocytes were positive for OKM 1 (CD1 lb)and HLA-DR, but their results with several othermonoclonal antibodies were not considered to beconclusive, probably for technical reasons.5

Necropsy showed a total aplasia of B zones and apronounced depletion of T lymphocytes in lymphnodes and diffuse infiltrates of histiocytes. Althoughdefects of the immune system have been suspected inHL, few immunological studies have been done.6 7 20The distribution of peripheral blood T and B lympho-cytes investigated during a quiescent phase of the dis-ease was found to be normal. This agrees with thefindings of Ladisch et a16 who, in contrast to ourstudy, found defective lymphocyte mitogenesis. B cellfunction, as judged by immunoglobulin concen-trations, has usually been intact, although some ex-ceptions have been reported.46 Investigation of invitro immunoglobulin synthesis in our patient clearlyshowed a complete functional B cell defect, togetherwith enhanced suppressor T cell activity.A partial defect in natural killer cell activity with no

additional effect of in vitro interferon is a character-istic finding in HL.420 Repeated plasma exchangeshave led to improvement in natural killer cell activity,monocyte mediated antibody dependent cytotoxicity,and lymphocyte mitogenic responses. This indicatesthat the cellular defects are acquired rather than in-trinsic. It is still unclear, however, why the cellulardefects shown are not also reversible in vitro. Webelieve that the cellular immune defect is an importantcomponent of the HL syndrome, presumably con-tributing to the uncontrolled proliferation of the celllineage specific for HL.

This study was supported by the Danish CancerSociety. We are grateful to the research workers listed

in the table for generously providing monoclonalantibodies.References

I Farquhar JW, Claireaux AE. Familial haemophagocytic reticu-losis. Arch Dis Child 1952;27:519-26.

2 MacMahon HE, Bedizel M, Ellis CA. Familial erythrophagocyticlymphohistiocytosis. Peadiatrics 1963;32:868-79.

3 Perry MC, Harrison EG, Burgert EO, Gilchrist GS. Familialerythro-phagocytic lymphohistiocytosis. Report of two casesand clinico-pathologic review. Cancer 1976;38:209-18.

4 Janka GE. Familial erythrophagocytic lymphohistiocytosis. Eur JPediair 1983;140:221-30.

5 Goldberg J, Nezelof C. Lymphohistiocytosis. A multifactorialsyndrome of macrophagic activation. Clinicopathologicalstudy of 38 cases. Haematol Oncol 1986;4:275-90.

6 Ladisch S, Holiman B, Poplack DG, Blaese RM,Immunodeficiency in familial erythrophagocytic lympho-histiocytosis. Lancet 1978;i:58 1-3.

7 Ladisch S, Ho W, Matheson D, Pilkington R, Hartman G. Immu-nological and clinical effects of repeated blood exchange infamilial erythrophagocytic lymphohistiocytosis. Blood1982;60:814-21.

8 Tauris P. Plaque-forming cells in man. I. Basic technical resultsobtained with the protein A technique. Scand J Immunol1983;18:241-8.

9 Pallesen G, Knudsen LM. Leucocyte antigens in human postmortem tissues: their preservation and loss as demonstrated bymonoclonal antibody immunohistological staining. Histo-pathology 1985;9:791-804.

10 Pallesen G, Beverley PCL, Lane EB, Madsen M, Mason DY,Stein H. Nature of non-B, non-T lymphomas: an immu-nohistological study on frozen tissues using monoclonal anti-bodies. J Clin Pathol 1984;37:911-18.

1 1 McMichael AJ, Beverley PCL, Cobbold S, et al. Leucocyte typingIII. In: White cell differentiation antigens. Oxford: OxfordUniversity Press, 1987 (in press).

12 Nezelof C, Barbey S. Histiocytosis nosology and pathobiology.Pediatr Pathol 1985;2:441-80.

13 Beckstead J, Wood G, Turner R. Histiocytosis X cells andLangerhans' cell: enzyme histochemical and immunologicalsimilarities. Hum Pathol 1984;15:826-33.

14 Chu T, D'Angio GJ, Favara BE, Ladisch S, Nesbit M,Pritchard J. Histiocytosis syndromes in children. Lancet1987;i:208-9.

15 Stein H, Mason DY, Gerdes J, et al. The expression of theHodgkin's disease associated antigen Ki- 1 in reactive and neo-plastic lymphoid tissue. Evidence that Reed-Stemnberg cells andhistiocytic malignances are derived from activated lymphoidcells. Blood 1985;66:848-58.

16 Risdall RJ, McKenna RW, Nesbit ME, et al. Virus-associatedhemophagocytic syndrome. A benign histiocytic proliferationdistinct from malignant histiocytosis. Cancer 1979;44:993-1002.

17 Delsol G, Gatter KC, Stein H, et al. Human lymphoid cell expressepithelial membrane antigen. Implications for diagnosis of hu-man neoplasms. Lancet 1984;ii: 1124-9.

18 Takahashi K, Isobe T, Ohtsuki Y, Sonobe H, Takeda I, Akagi T.Immunohistochemical localization and distribution of S-100proteins in the human lymphoreticular system. Am J Pathol1984;1 16:497-503.

19 Wieczorek R, Greco MA, McCarthy K, Bonetti F, Knowles DM.Familial erythrophagocytic lymphohistiocytosis. Immu-nophenotypic, immunohistochemical and ultrastructuraldemonstration of the relation to sinus histiocytes. Hum Pathol1986;17:55-63.

20 Perez N, Virelizier J-L, Arenzana-Seisdedos F, Fischer A,Griscelli C. Impaired natural killer activity in lympho-histiocytosis syndrome. J Pediatr 1984:104:569-73.

Requests for reprints to: Dr Troels Herlin, Department ofPediatrics, University Hospital, DK-8000 Aarhus C,Denmark.

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