REPORTS

Detection of Macrophage Migration Inhibitory Factor by Monoclonal Antibody in Sezary Syndrome

Christine Neumann, M.D., Renate Schlegel, M.D., Friedhelm Steckel, Ph.D., and Clemens Sorg, Ph.D. Department of Experimental Dermatology, University of Munster, Munster, F.R.G.

We have reported previously on the generation of a mono­clonal antibody against human macrophage migration in­hibitory factor (MIF), which is a mediator of cellular im­munity . Macrophage migration inhibitory factor activity in the migration assay was closely correlated with antibody reactivity. Using this antibody called lC5/B, we are now a ble to study the expression of MIF in situ. Here, we report on the detection of MIF in blood lymphocytes and skin of a patient with a leukemic cutaneous T-cell lymphoma with the characteristics of Sezary syndrome. Ninety percent of the patient's Ficoll Hypaque-isolated peripheral white blood cells were of the helper phenotype. By conventional im­munoperoxidase method, 94% reacted strongly positive

Macrophage migration inhibitory facto r (MIF) was the first Iymphokine to be described and was defined by its inhibitory effect on the migration

. of macro phages from capillary tubes [1). Its re­lease by cultured mononuclear cells upon antigen

stimulation has been considered to be an equivalent to the state of hypersensitivity in vivo. Relatively little information is avail­able on the production of MIF in vivo and its relation to the physiology and pathology of inflammatory reactions . An MIF­like activity was previously detected in the serum of animals which had been infected with bacillus Cal mette-Guerin and of patients with sarcoidosis, leprosy , and Sezary syndrome, a leu­kemic T -cell lymphoma of helper cell type [2-5) . However, in­formation on the distribution and function ofMIF in the organism is lacking. Recently this molecule has been characterized and has been shown to exert profound effects on macrophage differen­tiation [6,7]. From in vitro experiments it is generally concluded that MIF is produced by T cells when they are stimulated either

Manuscript received August 21, 1986; accepted for publication Decem­ber 23, 1986.

This work was supported by grant No. Ne 26011-1 from the Deutsche Forschungsgemeinschaft.

Reprint requests to: Dr. Christine Neumann , Universitats-Hautklinik, von Esma rch Strasse 56, 0-4400 Munster, F.R.G.

Abbreviations: 1CS/B : biotinylated antibody against the 14 kD molecular weight

species of human MIF CTCL: cutaneous T-cell lymphoma HTLV: human T lymphotropic virus IFN-y: gamma interferon IL-2: interleukin 2 MIF: macrophage migration inhibitory factor

with the antibody lC5/B. In contrast, using the immu­nofluorescence method only 25% reacted positive. This indicates that the majority of the tumor cells did not express the molecule on their membrane but only in the cyto­plasma. No other marker, such as interleukin 2 receptor, HLA-DR antigen, or interferon-gamma could be related to the expression of MIF. Also the cellular infiltrate in the skin was composed mainly of T helper cells and reacted positive with lC5/B. As less than 3% of normal blood lymphocytes reacted with lC5/B we suggest that the con­version to positivity may be a characteristic feature of the leukemic T-cell phenotype in Sezary syndrome. ] Invest D ermatol 88:670-674, 1987

by antigen or mitogen [1). Direct identification of the cells that contain MIF has now become possible by a monoclonal antibody, which we prepared against highly puri"fied MIF from mitogen­stimulated human peripheral blood mononuclear cells [8] . We show here that the malignant T cells of a patient with a leukemic cutaneous T-cell lymphoma (CTCL) of the helper type express MIF-related structures.

MATERIALS AND METHODS

Case History A 51-year-old woman with a 2-year history of erythroderma and grossly enJarged axillary and groin lymph nodes showed rising peripheral white blood cell counts some months before the investigation. At the time of investigation the total white cell count was 56,0001 ILl, a differential blood count showed 70% lymphocytes, 2% eosinophils, 2% monocytes, and 16% neutrophils . Most lymphocytes appeared atypical, being larger than normal with prominent nucleoli and approximately 20% had a highly indented nucleus. Lactate dehydrogenase in the serum was strongly elevated (900 units/ml). Anti-HTLV-I, -II antibod­ies, as evaluated by an enzyme-linked immunosorbent assay were negative. The examination of an enlarged lymph node of the groin (Prof. Dr. K . Lennert, Kiel, F.R.G.) revealed a T -zone lym­phoma, in agreement with a Sezary syndrome. By ultrasound investigation abdominal lymph nodes, spleen, and liver were found to be normal. A bone marrow smear showed no infiltration with malignant cells at the time of investigation .

Blood Cells Lymphoid cells were isolated from heparinized blood by Ficoll Hypaque centrifugation (Pharmacia , Uppsala, Sweden) and used for preparation of semi thin sections, cytocen­trifuge smears, and for flow cytofluorometry with an EPICS V (Coulter Electronics, Inc. , Hialeah , Florida). The percentage of cells that were labeled positive by indirect immunofluorescence technique was calculated by the Immuno Software supplied by

0022-202X/87/S03.S0 Copyright © 1987 by The Society for Investiga tive Dermatology, Inc.

670

VOL 88 , N O.6 JUNE 1987

Figure 1. Semithin section of Ficoll Hypaque-isolated peripheral blood cells. M a ny cells show highly convoluted nuclei and heterochromatin attached to their nuclear membrane. Bar = 10 p.m. Counterstain: Meyer's haema la un .

Coulter. For semi thin sections the cell button was fixed with glutaraldehyde (4%).

Antibodies M onoclonal antibodies diluted as indicated in the specification sheets of the suppliers were used: Leu 3a, Leu 4, anti-interleukin 2 (lL-2) receptor (Becton Dickinson, Heidelberg, F.R. G . ); anti-interferon-gamma (lFN-I') , (code 2067, Cell tech , GB), T4, TS, T6 (Coulter C lone, Krefeld , F.R.G .); Ki67 (pro­liferation marker) (Dakopatts, Hamburg, F.R.G.); OKM1, OKM5 (Ortho Diagnostics, H amburg, F.R.G.); and D-1-12 for detection ofHLA- DR antigen (kindly provided by Dr. S. Carrel, Lausanne, Switze rland). Production procedure and characteriza tion of the monoclonal antibody 1 C5 aga inst human MIF were described elsewhere [S]. Briefly, the antibody was generated against a 14 kD species of MIF from human peripheral blood mononuclea r cells a nd binds but does not neutralize MIF [8]. T he antibody was biotiny l ated according to Guesdon et al [9J and diluted 1: 400.

Skin Six-millimeter punch biopsies were taken from the fo re­arm a nd either fixed in bu ffe red formalin for routine hi stologic investigatio n or snap-frozen in liquid nitrogen for immunola­beling.

Inununoperoxidase Technique Six-micrometer frozen skin sections were air-dried with acetone (10 min) and preincubated with 0.1% hyd rogen peroxide and 1 % bovine serum albumin. Incubation with the relevant antibody was performed fo r 45 min at raoUl temperature. Secondary to the commercial antibodies peroxidase-conjugated goat antimouse IgG F(ab'h (Dianova,

Figure 2 . Cytoccntrifugc prcparations of Ficoll Hypaquc-isolatcd pe­ripheral blood ce lls from the patient. Bars = 30 p.m. Counters tain: Meyers haen~a l a un . A , Leukemic tumor cel ls stain positive fo r MIF with the ICS/B a ntibody. Monocytes are negative at the employed dilution of anti body. B, Leukemic tumor cells stain also positi ve with Leu 3a anti­body, characterizing thcm as T helper cells.

M IF IN C UTANEO US T-CELL LYMPHO MA 671

Table I. Percent Mononuclea r Peripheral Blood Cell s" From Cytocentri fuge Preparations T hat Stained Positive

by Immunoperoxidase

T4 T8 Leu 4 IC5 B Ki67 IL-2-rec IFN-1' D-I-12

90 2 93 94 12 8

' Prepared by Ficoll Hypaq ue, 200 cells were eva luated by the microscope.

Table II . Percent Mononuclear Peripheral Blood Cells T hat Stained Positive as D etected by EPIC S va Analysis

T4 Leu 4 lC5 B IL-2-rec

94 88 25 o ' Mononuclea r cells were prepared by Ficoll H ypaquc, 30,000 cell s were analyzed .

Hamburg, F.R.G.) was applied for 30 min at room temperature. Secondary to the biotinylated 1 C5/B antibody peroxidase-co n­jugated Streptaviclin (Amersham, Braunschweig, F. R. G.) was used. All antibod ies were dilu ted with phosphate-buffered saline, con­taining 1 % bovine serum albumin . Peroxidase activity was dem­onstrated w ith the 3-amino-9-ethylcarbazole reaction [10] . T he preparations were counterstained with Meyer's haem alaun (Merck, Darmstadt , F.RG.).

RESULTS

Blood Semithin sections of Ficoll H ypaque preparations from the peripheral blood of the patient revealed m any atypi cal lym­phocytes displaying characteristic features of CTCL (Fig 1).

Figure 2A depicts cells from cytocentrifu ge smears that stained strongly positive for MIF and Fig 2B shows that the cells display the T helper phenotype. Monocytes stained negative with 1 C5/B at the employed concentrations . Controls using biotinylated mOllse

Leu4 1C5

~

~ Q,) ", .. Q,) u u c c Q,)

Q,) u ., u en Q,) en ... Q,)

= 1 ... = = = 1.1.. -1.1..

Q,)

~' -... Q,) ...

Forward angle light scatter

Figure 3. Distribution of membrane-labeled cells according to their flu­orcscence intensity and size as measured by fo reward angle light scattcr. 3A shows thc resul ts with Leu 4 antibody, 3A 1 the corresponding control with mouse IgG. 3B shows the rcsults with l C51B antibody, 3B1 thc co rresponding control with biotinylated mouse IgG . A 'TOI I' S indicate ac­cumulation of cclls that stain posi tive above background. It can be secn that peaks of Leu 4-positive cells and 1 C51B-positive cells do not overlap. This indicates that large Lcu 4-positive cells do not express MIF on their mcmbranc.

672 NEUMANN ET AL THE JO URNAL OF IN VESTIGATIVE DERMATOLOGY

Figure 4. Skin sections. Counterstain: Mcycrs haemalaun. A, The dermal infil t rate stai ns st rongly posi tive with the pan T -cell marker Leu 4, (Bm' = 30 J,Lm) . B, T he same areas of the dermis sta in strongly positi ve for MIF w ith 1CS/B (Bar = 30 J,Lm). C , MIF-positi ve, morphologica ll y highly atypica l cel ls arC clearly visib le in the densely infiltrated upper dermis on higher magnifi cation (B(/r = 10 J,Lm). D, In the dermal infiltrate onl y very few cel ls stain positive with an an tibody to IFN-y (see ar/"Ow­heads) (Bar = 20 J,Lm ).

IgG and Streptavidin were constantl y negative. In cytocentrifu ge preparation from hea lth y cont rols onl y vcry few lymphocytes sta ined positive (:53%) .

Table I shows the percentage of mononuclear cells from cy­tocentrifuge preparations that stained positive w ith various o ther an tibod ies. Ninety percent of the cells were T cells w ith the helper phenotype (T4/Leu 3a), w hich is a typica l fi ndin g for the Sezary syndrome [11] . Ninety-fo ur percent stai ned positive for MIF w ith 1C5/B. In contrast, IFN-y as well as the IL-2 recepto rs and HLA­DR antigens D-1-1 2, which are activation markers, were not expressed. T he absence ofIL-2 receptors on th e circulating tumor cells together with the absence of antibodies to human T Iym­photropic virus (HTL V) I and HTL V II clearly distinguished this case from the Japanese form of ad ult T-cell leukemia [12]. Twelve

percent of the cells were in the cell cycle as revea led by posi tive staining with the Ki67 an tibody. Others have reported that Sezary cells in the blood are not cycl in g [13] . This discrepancy is probably explained by the aggressive course that the disease took at the time of investigat ion, since hepatosplenomegaly and bone mar­row infiltration occurred 3 months later.

An immunoflu o rescence test was don e to defin e more precisely the cellular loca lization of the M[F epitope. Table II shows the percentage of monon uclea r peripheral blood cells with posi tive membrane flu o rescence as revea led by EPICS V. Again , a high percentage (94%) were positive with the T helper cell marker (T4), indicating its localiza tion on the cell membrane. [n contrast, only 25% showed membrane fluorescence with lC5/B. Com­paring this result to the numbers of cells that stained positive by

VOL. 88, NO. (, JUN E 1987

imm un operoxidase, it beco mes apparent that MIF was mainl y loca ted in the cytopb sma of the cell s.

Figure 3 shows the distribution of the pati ent 's ce ll s accordin g to their size as measured by forward angle light sca tter and mem­bra n e fluorescence intensity usin g the 1 C5/B antibody and the pan T - cel l marker Leu 4. It can be seen that large Leu 4-positi ve cells did not express MIF on their membrane. From their size, those cells that did ex press MIF on their membranes resembled normal I ym phocytes.

Skin

Histo l ogy: Slides sta in ed w ith hematoxylin-eosin showed thick­ened epidermis with elonga ted rete ridges and a dense, band-like infi ltrate with nodu lar accentuation in the upper dermis. The in fi ltra te extended into the papill ary tips with so me exocytosis into the epidermis. The Iy m phocytes were larger than no rmal , many h ad a blast-like appearan ce, others showed highl y convo­luted nu clei, consis tent with the diagnosis of CTCL.

1I/IIIIuI1 operoxidase Srnillillg: The infiltrate was mainl y co mposed ofT lyrn.phocy tes (Fig 4A), w hich alm os t excl usively ca rried the T 4/Leu 3a marker for T helper cells co rrespondin g to the findings in the periphera l blood . The dermal T cells stained pos itive with anti-HLA-DR antibody, accordin g to what is known about ma­lignant T cells infiltrating the skin [1 4]. There was a moderate migration of these cell s into the epidermis. In the center of the densel y infiltrated areas so me dendritic cells stained positi ve with T6 and probably represented Langerhans cells. In the sa me area man y lynlphoid cells expressed the IL-2 receptor as detected by anti-Tac, indi cating that they had beco me activated in the dermis [IS]. M acrophages as detected by OKM1 and OKM5 were mod­era tely scattered throughout the derm is. Many cell s in the dermis sta ined positive w ith l C5/B even at high dilutions (Fig 48, C) . Quan tit y and pattern of distribution was similar to th e atypi ca l Leu 4-positive cells. Controls, including Streptavidin and bioti­ny lated mouse IgG, we re negati ve. Twenty to thirty percent of the infiltrating cells were cycling as they stain ed positive with Ki67. SOl"ne cells migratin g into the epidermis were positive with ICS/B. In co ntrast, the antibody aga inst IFN-I' detected only very fevv cells in the derma l infiltrates (Fig 4D).

D ISC U SSIO N

Usin g a monoclona l antibody to MIF we found th at circulating and skin-associated leukemic T cells in a patient w ith advanced Sezary syndrome reacted posit ive. This findin g is of interest be­cause so fa r MIF-like activity has been detected onl y in the serum of th ese patients and in the culture supern atants of peripheral bloo d n, o no nuclear ce lls [5]. We ca nnot decide from our res ults whether MIF is acti vely secreted by th e tumor cells o r re leased upon cell d ea th . The work on the characteriza tion of the antibody shows that the l C5 epitope most likely indicates the presence of MIF ~olecules . When m onito ring various separation procedures, the binding curves with 1 C5/B antibody overlap qualitatively and quantitativel y with those of MIF act ivity as measured by the migratio n assay [8]. Definite proof, however, for MIF production by the patient's tum or cells would be the detection of messenger RNA in the cy to plasma of the cell s.

Besides the high pro portion of circulating leukemic T cells that showed cytoplas mic stainin g, there was also a cohort of small T cells , vv h i e h in addition had M IF on their mem brane surface. As monoeytes clearly stained nega ti ve at the antibod y cO I~ centrat ion s used, passive adso rption of MIF to the cell membrane is unlikel y. Except for T-cell markers, other membrane antigens that may be found on activated T cells were negative. The absence of IL-2 receptors and HLA-DR antigens on circulating tum or cel ls dis­tinguishes the Sezary syndro m e from the acute ad ult T - cell leu­kem ia [1 2 ].

[n lyu1.phoid orga ns and periphera l blood of no rm al control persons on ly very fcw Iymphocytcs have been found to stain positive vvith 1 C5/B (G. Zwadlo et aI, unpublished; G. Gerhards

MIF IN CUTANEO US T - ELL LYMPHOMA 673

et aI, unpublished) . In contras t, in th e elicitation phase of contact eczema many of the lymphocytes in fi ltrating thc skin are MIF­positi ve (c. N eum ann et aI, unpublished). Thus, it may be that MIF is preferentially expressed by a distinct subpopulation that has a marked propensity to infiltrate the sk in. It may further be specul ated that it is this MIF-positive subpopulation that expands in the Sezary sy nd rome. This hypothesis secms in triguing, as it is in creasin gly recognized that the skin possesscs various impor­tant immune mechanisms [1 6,17]. In two further cases of eryth­rodermic CTCL but without leukemia we have also found a signifi cant propo rti on of MIF-positive cells in the dcrmal inftl­trate. In these nonleukcmic cases , however, the peripheral blood contained on ly a few MIF-positive cclls. Blastogenic leukemia in CTCL as seen in the patient presented here is an extremel y ra re, late event in the course of the disease. Further cases have to clarify w hether the appeara nce of MIF-positive tumor cells in the blood is typical for the leukemic stage of the disease.

The fun ctional signifi cance of MI F in CTCL is no t clear. As MIF has profound effects on the differentiation of m ononuclear phagocytes, it appears appropriate to investiga te this cell sys tem in paticnts w ith incrcased MIF expression. Others have sugges ted that MIF can abrogate cellular immune reactions 11 8]. Whether the immunosuppression that is seen in patients with CTCL can be related to MIF rem ains to be inves tiga ted.

We also investigated IFN-I' because it is another prod uct of activated T-cell subsets [1 9]. In contrast to MIF, thc blood cells of our patient did not stain positive w ith an antibody against IFN­I' and accordin gly the dermal infiltrate was almost ncgative. T his might be surprisin g since IFN-I' is sccreted upon antigenic stim­ulation and one of thc proposed etio logic facto rs of CTCL is chronic antigenic stimulati on of T cells in the sk in [20]. O n the o ther hand , the negativity for IFN-I' might have resulted from functional defects of the m alignant T cells, w hich undoubtedl y m ay occur in C T CL [21].

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21. Clemmensen OJ , Bendtzen K, Andersen V, Wulf HC , N iebur E, Thomsen K, Bendixen G: Lymphocyte fun ction and chromosome aberrations in patients with earl y mycosis fun goides and parap­sorias is en plaques. J In ves t Dermatol 81 :308~313, 1983