7 Clin Pathol 1997;50:559-562

Distribution of endogenous tumour necrosisfactor a in gliomas

M Maruno, J S Kovach, P J Kelly, T Yanagihara

AbstractAims-To determine the distribution andcellular origin of endogenous tumournecrosis factor a (TNFa) in the cellularcomponents ofhuman gliomas.Methods-Frozen sections of 26 gliomas(four astrocytomas (As); two oligo-astrocytomas (OA); one anaplastic astro-cytoma (AA); one anaplastic oligo-astrocytoma (AOA); 18 glioblastomas(GB)) were examined immunohisto-chemically using antihuman TNFa andanti-Leu-M5 (CD1lc) antibodies. Addi-tional studies with double immunohisto-chemical procedures were performed withanti-glial fibrillary acidic protein andanti-neurofilament antibodies.Results-Eighty per cent of the AA, AOA,and GB (16 of 20) had a positive reactionfor TNFa, but only 17% ofAs and OA (oneof six) were positive. Positive cells were

seen in both the tumour tissue andadjacent brain tissues. TNFa protein was

detected not only in the tumour cells butalso in the endothelium of tumour vesselsas well as reactive astrocytes and neurons.

Conclusions-Endogenous TNFa ispresent in cells of various origins in glialtumours including tumour vessels; how-ever, the role of TNFa may be different indifferent types of cells or altered microen-vironment.( Clin Pathol 1997;50:559-562)

Keywords: endogenous tumour necrosis factor a;gliomas; glioma cells; endothelial cells

Department ofNeurology, MayoClinic, Rochester,Minnesota, USAM MarunoT Yanagihara

Department ofNeurologic SurgeryP J Kelly

Mayo ComprehensiveCancer CenterJ S Kovach

Correspondence to:Dr Maruno, Department ofNeurosurgery, OsakaUniversity Medical School,2-2 Yamadaoka, Suita, Osaka565, Japan.

Accepted for publication8 April 1997

Cytokines are essential mediators of cell to cellinteractions in various pathophysiologicalprocesses. Tumour necrosis factor a (TNFa) isone of the major macrophage derived cytokinesthat was originally found to mediate cytostaticand cytotoxic effects against malignant cells invitro, and to cause haemorrhagic necrosis incertain types of tumours in vivo.' TNFapossesses multiple biological and pleiotropicfunctions such as cytotoxity, and promotion ofcell differentiation and growth, as well as play-ing many immunomodulatory roles within thecytokine network.2A

It is well known that TNFa exerts itsbiological activity by binding to specific cellsurface receptors.4 Two distinct types of TNFreceptors weighing 55 kDa (type I) and 75 kDa(type II) have been identified.5 Although, thedistribution ofTNF receptors in glioma tissuesis obscure, there are few reports in theliterature suggesting the distribution of TNFa

and endogenous TNFa possession by variousglioma cell types.6 Hence, we examined immu-nohistochemically the distribution of endog-enous TNFa in human glioma tissues

Materials and methodsTwenty six glioma samples were investigated(four astrocytomas (As); two oligo-astrocytomas (OA); one anaplastic astrocy-toma (AA); one anaplastic oligo-astrocytoma(AOA); 18 glioblastomas (GB)). Histologicaldiagnosis was performed according to theWHO classification.7 Immediately after surgi-cal removal, the samples were frozen in isopro-pyl alcohol cooled with dry ice and stored at-80°C. Each sample was sectioned to 8 pmthickness in a cryostat microtonle at -20°C,mounted on gelatin coated glass, dried at roomtemperature, and kept at -80°C until furtherprocessing.

For immunohistochemical procedures, thesections were fixed in 4% paraformaldehydefor five minutes, washed in 0.01 M phosphatebuffered saline (PBS) and then immersed in60% and 100% methanol. After washing inPBS, non-specific protein binding was blockedby incubation in 1.5% normal goat serum in

Table I Immunohistochemical study ofhuman gliomas forTNFa and monocytelmacrophage staining

TNFaHistology Monocytel

Specinmen ofglioma Tumour Brain macrophage

1 AS - NA ++2 As - NA ++3 As + NA ++4 As - +++5 OA - NA +6 OA - NA +++7 AA - NA ++8 AOA + ++ +++9 GB + + +++10 GB + NA +++11 GB + NA ++12 GB - + +++13 GB ++ ++ +++14 GB + NA +++15 GB + NA +++16 GB + NA +++17 GB +++ NA ++18 GB + NA ++19 GB - NA ++20 GB ++ NA ++21 GB ++ NA ++22 GB - + ++23 GB + NA ++24 GB + NA ++25 GB + NA ++26 GB + ++ +++

Immunoreaction was arbitrarily categorised as: very frequentstaining (+++), ->20% of the background cells; moderately fre-quent (++), <20% of the background cells; only a few (+), < 5%of the background cells; and none (-), 0% cells.As, astrocytoma; AA, anaplastic astrocytoma; GB, glioblastoma;OA, oligo-astrocytoma; AOA, anaplastic oligo-astrocytoma;NA, surrounding brain tissue not included in the specimen.

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Figure 1 (A) Immunostainingfor TNFa in glioblastoma. Cytoplasm of most of the glioblastoma cells was positive for TNFa. Several cells show a strongpositive reaction (original magnification x400). (B) Immunostainingfor TNFa in surrounding brain tissue. Positive reaction for TNFa was present in thecytoplasm of numerous cells in the brain tissue adjacent to a glioblastoma (original magnification x200). (C) Positive reaction was completely abolished byusing the antibody for TNFa after absorption with recombinant TNFa (original magnification x200). (D) Immunostaining for CDI Ic in glioblastoma.The monocytes/macrophages were seen moderately frequently (original magnification x200).

1% bovine serum albumin containing 0.2%Triton X100 for 20 minutes. The sections werethen incubated overnight at 4°C with theprimary antibody, rabbit polyclonal antihumanTNFa (1/1000) (a generous gift from Genen-tech, California, USA). Normal rabbit serumwas used in lieu of anti-TNFa at the samedilution as negative control. Further incuba-tion for 30 minutes with biotinylated antirabbitIgG and 30 minutes with avidin-biotin com-plex conjugated with alkaline phosphatase(Vectastain; Vector, California, USA) was per-formed followed by a final incubation for 30minutes with Vector Red (Vector). The cellnuclei were visualised by counterstaining withhaematoxylin. For identifying macrophages,mouse monoclonal anti-Leu-M5 (CDllc,3.3 gg/ml) (Becton Dickinson, California,USA), specific for human monocyte/macrophage antigen, was used. This wasfollowed by incubation in antimouse IgG,avidin-biotin peroxidase complex (ABC-PO)(Vectastain; Vector), and 3,3'-diamino-benzidine tetrahydrochloride (DAB). As nega-tive control, normal mouse IgG was used inlieu of anti-Leu-M5 (CD 1 ic) at the same pro-tein concentration.To investigate the cellular origin of TNFa,

we performed double immunohistochemical

procedures to visualise TNFa and a cellmarker in the same cell. After the chromogenicreaction with Vector Red for TNFa, thesections were washed in 0.1 M glycine HCIbuffer (pH 2.2) for 60 minutes, in Tris HCIbuffer for 30 minutes, and then incubated in0.15% hydrogen peroxide in Tris HCI bufferfor 10 minutes. After washing, the sectionswere incubated in 1.5% normal horse serumfor 20 minutes and then separately incubatedovernight at 4°C with mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody(Boehringer Mannheim, Indiana, USA), ormouse monoclonal anti-neurofilament (NF)antibody against 68 kDa monomer (Boe-hringer Mannheim). After washing, the sec-tions were incubated in biotinylated horse anti-mouse IgG for 30 minutes, and then withABC-PO for 30 minutes, and finally incubatedwith DAB and hydrogen peroxide, followed bycounterstaining with haematoxylin.

ResultsENDOGENOUS TNFaEighty per cent ofthe AA, AOA, and GB (16 of20) were positive for TNFa, but only 17% ofAs and OA (one of six) were positive (table 1).Cells with positive reaction for TNFa werepresent not only in the tumour tissues (fig 1A)

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Endogenous tumour necrosis factor a in gliomas

ticularly numerous within the tumour paren-

chyma and comprised over 20% of the cellularelements (table 1). However, no correlationwas seen between the expression of endog-enous TNFa and the level of monocyte/macrophage infiltration.

DOUBLE IMMUNOHISTOCHEMICAL REACTIONS

Six GB were examined with double immuno-histochemical reactions for TNFa and GFAP.While some cells in tumour tissues had positivereactions for both (fig 2A), the majority showeda positive reaction for TNFa alone. Four GBwere examined with double immunohisto-chemical reactions for TNFct and NF.Whereas, some cells with large cytoplasm,especially in the adjacent brain tissues, showedpositive reactions for TNFa and NF simulta-neously (fig 2B), most of them showed a posi-tive reaction for TNFa only.

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Figure 2 (A) Double immunohistochemical study for TNFa and GFAR Three cpositive reaction for both (redfor TNFa and brown for GFAP) are shown in the cThese cells were, thus, identified to be astrocytic in origin (original magnification >(B) Double immunohistochemical study for TNFa and NE:A cell in the centre wipositive reaction for both (redfor TNFa and brown for NF) was identified to be a(original magnification x400).

but also in the adjacent brain tissuesThe reaction was recognised predominthe cytoplasm and was evident in varioiof cells including multinucleated cellpleomorphic cells, and small cells. Cellpositive reaction were most numerouw

boundary of the tumour tissues. Furthsome endothelial cells of the vesselstumours or adjacent brain were alsopositive for TNFa. No reaction was fcontrol sections. In addition, theantibody for TNFa before incubaticrecombinant TNFa (Boehringer Marresulted in disappearance of thereaction (fig 1 C).

REACTION FOR MONOCYTES/MACROPHAGE

A variable number of cells had a positition with anti-Leu-M5 (CD1 c) instudy samples (fig ID, table 1). Sonmonocytes/macrophages were found to

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X It is well known that TNFa acts as a potentimmunomodulatory molecule affecting thefunction of many cells involved in the immuneresponse, such as T cells, B cells, neutrophils,monocytes, and macrophages." In the centralnervous system (CNS), astrocytes can producea variety of immunoregulatory molecules uponstimulation. These include interleukin (IL)-1,'0IL-3," prostaglandin E,'° interferon a and ,12

IL-6," and TNFa.'4"6 Another CNS cell, themicroglia, can also be stimulated to secreteIL-I,'7 TNFa,'5 16 and IL-6.'3 However, thetumour cells and endothelial cells in the

t territory oftumour were shown to be the majortargets of exogenous TNFa in humangliomas. 18

The present study demonstrated that cellscontaining TNFa protein were more abundantin malignant gliomas (AA, AOA, GB) than inbenign ones (As, OA). Cells containing TNFa

ells with protein have been detected in the brain tissuesentre. of patients with multiple sclerosis"9 and sub-<400). acute sclerosing panencephalitis,20 but not innth the normal brain.'9 In Guillain-Barre syn-neuron

drome, the serum concentration of TNFa israised, with a good correlation between TNFa

fig iB). and severity of the disease.2' Although TNFaantly in was shown to be produced and secreted byus types reactive astrocytes located mainly in areas withIs, large lymphocytic infiltration,6 we could not find anys with a correlation between the presence of TNFas at the protein positive cells and the number ofermore, infiltrating monocytes/macrophages in glio-within mas.

weakly Macrophages were believed to be the princi-

ound in pal source of TNFa, however, recently it wasuse of discovered that TNFa is secreted by a variety:n with of cells during the course of microbial infec-inheim) tions, neoplastic diseases, and autoimmunepositive disorders.6 In addition, microglial cells are

known to be the major producers of TNFa inthe brain.'6 22

-s TNFa is also known to induce astrocyteyve reac- proliferation in cultured cells,23 24 suggestingall the that it plays the role of growth factor producingaetimes, prominent gliosis such as that found in a vari-be par- ety of CNS disease. The presence of cells con-

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Maruno, Kovach, Kelly, Yanagihara

taining TNFa protein predominantly in themalignant gliomas substantiates the possibilitythat TNFa may be a growth factor for humangliomas.The present study further demonstrated that

vascular endothelium within gliomas and adja-cent brain tissues were also positive for TNFa,although weakly. Vascular endothelium hasbeen shown to be a direct target for the actionof TNFa,'8 25 modulating many endothelial cellfunctions such as the coagulant activity andimmunological properties.""'8 In addition tothese effects of TNFa, several other activitieshave been observed, such as alteration of theproperties of endothelial cells, upregulation ofmajor histocompatibility complex class I anti-gens, intercellular adhesion molecule-i, andother surface molecules that promote mono-cyte and neutrophil adherence to vascularendothelium. TNFa has also been shown toincrease tissue factor procoagulant activity,enhance plasminogen activator inhibitor- 1, andsuppress expression of thrombomodulin, re-sulting in a net change of haemostatic proper-ties of endothelium from an anticoagulant to aprocoagulant state." AsTNFa may have a

selective effect on the tumour vessels ingliomas,'"25 this cytokine could possibly be anadjuvant to the treatment of malignant glio-mas.While our findings from double immunohis-

tochemical procedures revealed that endog-enous TNFa recognised in tumour tissuesmight have originated from the glial cells orneurons, the precise origin of TNFa in thesecells could not be ascertained. A possibilityexists that macrophages may produce TNFa,and the astrocytes and neurons may be the tar-get cells for its binding.'9 Based on the resultsof our double immunohistochemical proce-dures, their is a strong possibility that malig-nant glioma cells can produce TNFa. Al-though TNFa production on a per cell basis islower compared with that by microglia or mac-rophages, a small but localised TNFa sourcehas been recognised in astrocytes.'6 It is antici-pated that our findings of immunohistochemis-try regarding the cellular origin of endogenousTNFa could be clarified by other techniquessuch as in situ hybridisation.

The present investigation was supported by the grant CA 42724andCA 50905 from the National Cancer Institute.

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