Feb 15, 1988 - Craniopharyngeomas. Meningeomas. I1. 111. (endothelio- matous). Other tumors. Chordomas. 3. Teratomas. 3. Chondroma. I. Vasc tumors. 2.
Inflammatory Infiltrates and Natural Killer Cell Presence in Human Brain Tumors ANDREAS STEVENS, MD, INES KLOTER, MD, AND WOLFGANG ROGGENDORF, MD
Immunohistochemical analysis of subpopulations of inflammatory cells in 8 1 primary and secondary human brain tumors was done. Natural killer (NK) cells, representing non-major histocompatibility complex-restricted, spontaneous cytotoxicity and monocytic cells are virtually absent in infiltrates of gliomas and account only for a minor percentage of inflammatory cells in brain metastases of carcinoma and in craniopharyngeomas. Infiltrates in gliomas consist almost exclusively of T-cells of the suppressor/cytotoxic type whereas infiltrates in carcinoma metastases and craniopharyngeomas contain considerable numbers of T-helper/inducer cells and B-cells. From this the authors conclude (1) that NK cells do not play a major role in tumor rejection, and (2) that the kind of inflammatory reaction does not depend upon the tumor site but more likely on the tumor type. No correlation between tumor differentiation and infiltrate composition is evident. Cancer 61:738-743,1988.
T
HE SlGNlFICANCE of inflammatory reactions in
tumor tissue had been of interest for clinicians, immunologists, and pathologists alike. The response may be humoral or cellular and does not correspond with tumor type, malignancy, or prognosis.’-’ I The blood-brain barrier and the lack of a lymphatic vessel system account for immunologic peculiarities of the central nervous system (CNS) which have led to many studies of immunologic responses in intracerebral neoplasms. These include analyses of T-cell populations in peripheral blood of tumor patient~,~.”-l~ humoral res p o n ~ e , ~ and ~ ’ ~of . ’ inflammatory ~ cells in tumor horn~genisates.’~,~~ Natural Killer (NK) cells as mediators of spontaneous, antibody-independent cytotoxicity not restricted by major histocompatibility complex (MHC) haplotypes”-19 are given a particular role in regulation of proliferation and tumor The capacity of these cells for spontaneous killing of tumor cells has prompted clinical trials using interferon activated NK cells in tumor patients, so far without any apparently positive
Dedicated to Professor Dr. J. Peiffer, Director of lnstitut fur Hirnforschung for his 65th birthday. From lnstitut fur Hirnforschung, Tubingen, Federal Republic of Germany. The authors thank B. Weidle, M. Graf. and G. Mundle for excellent technical assistance. Address for reprints: Andreas Stevens, MD, lnstitut fur Hirnforschung. Calwerstr.3, 7400 Tubingen, West Germany. Accepted for publication August 25. 1987.
Many subpopulations of NK cells have been defined showing varying combinations of myeloic, monocytic, and (T-)lymphocytic surface antigens. ’8~’9.25-29The subsets are thought to correspond with different stages of maturation and activation. The majority of cells capable of “natural” cytotoxicity reacts with the Leu7 monoclonal antibody28although highest levels of activity are ascribed to a Leu7-Leu 1 1b+ population.21930 To our knowledge there are only a few reports concerning NK cell presence in human neoplasms” and none about tumors of the CNS. Information about this however is essential for the forming of therapy concepts involving activation of NK cells or other cytotoxic effectors. In our study we examined 81 biopsy specimens of intracranial, intracerebral, and extracerebral human tumors with monoclonal antibodies (AB) against leucocytic surface antigens in order to determine the involvement of different subpopulations of immunocompetent cells, especially that of NK cells. We compared the cellular composition of inflammatory infiltrates in primary and metastatic tumors, showing differences in immune response.
Materials and Methods Selection of Biopsv Specimens Eighty-one surgical biopsy specimens of intracranial tumors were selected (Table I ) showing mononuclear infiltration. In 24 of these cases additional, deep-frozen specimens were available. 738
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TABLE I . Results
Histopathologic diagnosis
No.
Density of infiltration*
Percentage of specimens with NK cells
Dominant T-cell population
Frequency of NK cells
Monocytes
B-cells
I Gliomas (IV Acc to WHO classification) (1-11 Acc. to WHO classification)
(+)
(+I
I1
T-SuppfCytotox = T-helperfinducer T-helper/inducer
90%
++
86%
I1
+++ +++ ++
ND
45%
+++ +
15
++
T-Suppf Cytotox = T-helper/inducer
33%
(+I
Carcinoma metastases
20
Craniopharyngeomas
7
111
Meningeomas (endotheliomatous) Other tumors Chordomas Teratomas Chondroma Vasc tumors Pituitary adenoma Neurinomas Total
+ +
++ ++
-
ND
(+I
+
3 3 I 2 I 5 81
Ace: according; WHO: World Health Organization; Vasc: vascular: ND: not done; Supp: suppressor; Cytotox: cytotoxic; NK: natural killer. * Degree of infiltration: -: no identifiable infiltrate; +: scanty infiltrate, not more than one cell layer thick, surrounding a few, small vessels; or isolated lymphocytes lying scattered in the tumor paren-
chyma; ++: scanty infiltrate around numerous vessels. or more than one cell layer thick around a few vessels, or diffisely scattered lymphocytes in the tumor parenchyma; +++: multilayered (2-3 cell layers) infiltrates surrounding numerous vessels, or an intense cell cuffing of a number of vessels, or a diffuse dense infiltration of the tumor parenchyma.
Tissue Preparation
Evaluation
Frozen specimens: Cryostat sections were mounted on poly-L-lysine (Sigma, St. Louis, MO) coated slides, prefixed in aceton, air dried, wrapped in aluminium sheets, and stored at -70°C. Before immunohistochemical staining the slides were warmed up to room temperature. Parafin-embedded specimens: Standard 3 to 5-pm sections were cut, mounted on poly-L-lysine coated slides and deparaffinized. Monoclonal antibodies: Table 2 shows the antibodies used. The antibodies against leucocytic surface antigens are monoclonal IgG mouse antibodies, anti-1-light chain and anti-k-light chain antisera are polyclonal rabbit sera. All antibodies were applied to cryostate sections, the LC and Leu7 antibody also on paraffin section^.^'
The slides were evaluated by light microscopic study in a semiquantitative matter as described by Boker et aL6 The specimens were thoroughly viewed for intratumoral, perithmoral intraperitumoral, penvascular, and diffuse lymphocytic infiltrates; localization and relative density of reactive cells were noted.
Immunologic Staining Procedure The avidin-biotin method (ABC), modified after Hsu et aL3’ and the phosphatase antialkalic phosphatasemethod (modified after Stein et al.,33were used with equivalent results. Positive and negative controls were coprocessed.
TABLE 2. Antibodies Used:Respective Specifity and Source Specifity
Antibody
Leukocyte common antigen T-helper cells T-suppressor cells Monocytes NK cells B-cells B-cells B-cells
LC* OKT4,t Leu3a4 OKT8.t LeuZaS OKM 1 ,t LeuM5$ Leu74 Leu I 1bS Leu I2$ Anti-k-light chain4 Anti-I-light chain4
NK: natural killer. Dakopatts, Hamburg Germany. t Ortho Diagnostics, Neckargemiind, Germany. Becton Dickinson, Mechelen, Belgium. 8 Vector Laboratories, Heidelberg, Germany.
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FIG. 1. Glioblastorna multiforme: LC+ leucocytes gathered around pathologic vessel convolute (arrows) whereas normal vessels (stars) show no perivascular infiltrates (ABC method. original magnification X280).
Results Infiltrates of a composition similar to intratumoral infiltrates were found in brain tissue adjacent to neoplasms. Tumor-free brain biopsy specimens showed leukocytes and NK cells only intravasally. According to infiltrate composition (Table 1) the tumor specimens can be classified in three groups: (1) infiltrates in gliomas; (2) infiltrates in carcinoma metastases and craniopharyngeomas; and (3) other tumors.
Injiltrates in Gliomas We found scanty to midsize infiltrates in low-malignancy gliomas, and larger infiltrates in glioblastomas. The leukocytes were closely associated with small or, more often, pathologic vessels (Fig. 1). The density of infiltration vaned in different areas of the tumors without conceivable correlation to local tumor morphologic features. Leu 12+anti-k/anti- 1+ (B-)cells and OKM 1 + (monocytic) cells were rare, the infiltrates consisted almost exclusively of OKT8+Leu2+ (T-suppressor) lymphocytes. Single Leu7+ (NK) cells were found in five of 28 gliomas (four of them were glioblastomas with heavy lymphocytic infiltration), all lying within large lymphocytic penvascular cuffs. Leu 1 1+ (highly activated NK) cells were not noted.
Injiltrates in Carcinoma Metastases and Craniopliarvngeomas Infiltrate composition within carcinoma metastases and craniopharyngeomas were strikingly similar. Carcinoma metastases: Heavy lymphocytic infiltrates were found in perivascular spaces and along connective tissue. OKMl+ (monocytic) cells were present in the
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boundary area between infiltrate and tumor parenchyma, slightly more numerous than in gliomas. The ratio of OKT8+Leu2a+(T-~uppressor)to OKT4+Leu3a +(T-helper) cells vaned. OKT8+Leu2a+(T-~uppressor) cells showed slightly closer association with blood vessels than OKT4+Leu3a+(T-helper) cells. Significantly more specimens of this group (18 of 2 1 ) revealed Leu7+(NK) cells than specimens of group I (five of 28) or I1 ( 10 of 26). Also the percentage of Leu7+ (NK) cells within inflammatory infiltrates was higher, although never exceding an estimated 10%. Leu7+(NK) cells were closely associated with mononuclear infiltrates (Figs. 2A and 2B). In two cases Leu1 lb+ (highly activated NK) cells could be detected. No correlation of infiltrate composition and degree of dedifferentiation or primary tumor site (gastrointestinal, lung, urogenital, mamma) was evident. Craniopharyngeomas: In Craniopharyngeomas we saw pronounced infiltration within tumor cysts and nearby tumor parenchyma. Penvascular cuffing was not a prominent feature. Craniopharyngeomas showed the highest relative incidence of OKM 1 + (monocytic cells), most of them lined along the cyst walls. Most of the infiltrate cells were T-lymphocytes with a slight preponderance of OKT4+Leu3a+ (T-helper) cells. Six of seven specimens contained large numbers of Leu7+(NK) cells, seemingly without specific distribution.
Other Tumors Meningiomas showed widely scattered and generally scanty infiltration and occasional penvascular cuffing. The LC+ (lymphocytic) cells also were found inside the whorl-like formations typical of endotheliomatous meningioma (Fig. 3). In the same place single Leu7+ (NK) cells (five of 1 1 cases) could be found. The other tumors examined showed moderate infiltration with a slight dominance of OKT8+Leu2a+ (Tsuppressor) cells and moderate numbers of Leu-/+ (NK) cells in about one third of the cases. Reactivity of tumor cells with the Leu7 antibody was noted in some meningeoma, carcinoma, and glioblastoma specimens. Staining occurred in only a few cells and was either cytoplasmic or surface bound. Morphologic study of cell soma and nucleus allowed reliable identification of these cells as noninflammatory. Similar observations have been reported
Discussion Natural killer cells do not play a major role in cellular immune response to primary or secondary brain tumors. By numbers, T-cells of the cytotoxic/suppressor type dominate, but most of them are not specific for tumor antigens. In addition, cytotoxic effectivity may
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FIGS. 2A AND 2B. Metastasis of lung carcinoma: (A, top) LC+ inflammatory cells surrounded by tumor parenchyma; (B, bottom) the same infiltrate with demonstration of Leu7+ cells (APAAP method, X420).
not be judged by numerical presence. Cytotoxicity of NK cells can be quantitatively measured by a CrS’-releasing assay in tissue culture e ~ p e r i m e n t s ~in~ tissue .~~; sections there is still no effective means for assaying NK cell or T-cell cytotoxicity. Also, tissue culture experiments are done at effector-target rates of about 50/ 1, which is at least inverse to the ratio found in vital neoplasms. Low engagement of NK cells in tumor infiltrates is
not a phenomenon particular to brain tumors. Mori et aL3’and Levy et al.38report similar findings for analysis of cellular immune response to lymphomas. Natural killer cells are equally sparse in nontumor-related inflammatory infiltrates as in m y o ~ i t i s ~or ’ . ~abszess (unpublished results). Suppression of NK cell activity by neoplasms therefore cannot be assumed. The striking affinity of NK cells to carcinoma metastases and craniopharyngeomas as compared with
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Since prognostic rating of mononuclear infiltration in neoplasms is not unequi~ocaP-~.~' one may speculate infiltrate composition to be more important than the mere quantity of immunocompetent cells.
^.
I
.
.
FIG.3. Endotheliomatous meningeoma: LC+ cells are shown within typical whorl-like formation (APAAP method, X420).
gliomas (with equivalent lymphocytic infiltration) current can not be explained. It may be noted that a relative high NK cell number roughly correlated with the frequency of T-helper/inducer cells. No correlation could be established between infiltration type and tumor dedifferentiation and local tumor morphologic findings. Similar findings are reported by Mon et aL3' and Levy et al.38 Trenkwalder et ~ 1and. Maluish ~ ~ et ~ l . recently *~ reported about clinical trials evaluating the effects of alpha-interferon and cimetidine as stimulators of NK cells and macrophages in patients having various malignancies. The apparent lack of beneficiary effects may be explained partly by the paucity of NK cells (and macrophages) in vital tumor tissue. In tissue culture NK cells act spontaneously and independently from lymphocytes. In the tumor specimens we analyzed, NK cells always were closely associated with lymphocytic infiltrates, however, or proved to lie int ravasal. Our findings that infiltrate composition vanes in different tumors within the same organ indicates that not immunologic properties of the tumor site (the brain) but rather of the tumor tissue are responsible for the observed differences. Similar results were given by von Hahnwehr et ~ l . noting , ~ predominance of T-suppressor/cytotoxic cells in glioma infiltrates. Earlier ~tudies'*~-"*~' have shown that inflammatory infiltration is more frequent and more dense in metastases than in gliomas. It has been speculated that this is due to sialomucine coating of glioma cell^^.^' or to altered surface glycolipid composition and reduced antig e n i t ~We . ~ ~could confirm that truely extravasal lymphocytes are absent in normal brain and we were not able to demonstrate NK cells either.
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