141
Ausf. N.Z.J. Surg. 1991.61. 141-146
SURGICAL RESEARCH TUMOUR NECROSIS FACTOR-ALPHA IMMUNODETECTION IN BLOOD MONOCYTES AND SERUM: PRELIMINARY FINDINGS IN WEIGHT- LOSING CANCER PATIENTS J. L. MCCALL,I*
s. FUNAMOTO, ** K. y U N 3 + AND B. R. PARR?*
Departments of *Surgery and 'Pathology, Otago University Medical School. Dunedin, New Zealand The peptide tumour necrosis factor-alpha (TNF-a) is a central mediator of the host response. Identifying where and when TNF-a is produced may give insights into its potential role in various pathophysiologicalstates. This paper describes a quantitative analysis of TNF-a expression in peripheral blood monocytes (PBM) at the single cell level. A pilot study has been undertaken, using this method to assess TNF-a expression in PBM from healthy volunteers and cancer patients. We also report mildly elevated serum TNF-a levels in the cancer patients. using an immunoradiometric assay (IRMA) sensitive to I p@mL of recombinant TNF-a. The results of this preliminary investigation suggest that TNF-a production may be altered in cancer patients. Key words: cachectin, cancer, immunocytochemistry, monacytes, tumour necrosis factor-alpha.
Introduction The monokine tumour necrosis factor-alpha (TNF-a, also known as cachectin) is a host-derived peptide involved in the pathophysiology of inflammatory and metabolic responses to infection and possibly malignancy. I*' TNF-a plays a major mediating role in septic shock.3 It also initiates energy substrate mobilization which, in the long term, can cause depletion of host tissues.2q4But considerabledebate continues over what role, if any, TNF-a might have in patients with weight-losing cancer. In part this debate stems from the unresolved question of whether TNF-a production is increased in cancer patients. Mononuclear phagocytes (blood monocytes and tissue macrophages) are the principal cellular source of TNF-a. I These cells have been the focus of studies attempting to determine the propensity for TNF-a production in different pathophysiological states. A standard approach has been to culture the cells in vitro and measure spontaneousor stimulated TNF-a production in the More recently, TNF immunostaining techniques have been used in histological and cytological preparations, to study cytokine expression at a cellular level. "-I5
' MB. ChB. 'MD.
MD. WD. 'MD. FRACS.
Correspondence: Dr 1. L. McCall. Department of Surgery. Box
913. Dunedin. New Zealand. Accepted for publication 8 August 1990.
This paper describes a method of TNF immunostaining in peripheral blood monocytes (PBM), and its application in a pilot study of healthy volunteers and cancer patients. The results of PBM immunostaining are compared with those of serum and mononuclear cell (MNC) supernatant TNF-a levels in the two groups. Methods SU EJ ECTS
Ten healthy volunteers and 14 cancer patients were studied after obtaining signed informed consent. Patient details are given in Table 1. Percentage weight loss was assessed by subtracting measured weight from recall well weight, and expressing the deficit as a percentage of well weight. No patient had sepsis, but one was receiving corticosteroids (dexamethasone 4mg 6-hourly) at the time of study. Serum for TNF-a assay was obtained from all subjects and heparinized blood for MNC separation was obtained from the ten healthy subjects and ten of the 14 cancer patients. MNC ISOLATION A N D CULTURE
The MNC were separated from heparinized whole blood by centrifugation through Ficoll-Hypaque (Histopaque-1077; Sigma, St Louis, MO) and washed twice in RPMI 1640 supplemented with 10% fetal calf serum (RPMVFCS; both Sigma). Cells were then resuspended in RPMVFCS and added to 35mm petri dishes, each containing a
142
MCCALL ET A L
clean sterile cover slip. The cell density of the suspension was determined and subsequent results adjusted to a standard density of 107cells/mL. Suspended cells were cultured for 2 h at 37'C in a humidified chamber with 5% COz, with or without 10pglmL of lipopolysaccharide (LPS; E. Coli serotype 0127:BS, Sigma). After culture the medium was collected and centrifuged to obtain the cell free supernatant and the cover slips were removed for immunocytochemistry of the adherent PBM monolayer. Supernatants from cultures with no LPS added had less than 0.5 endotoxin unitslmL as determined by Limulus amoebocyte lysate assay (E-Toxate. Sigma). All supernatants and serum samples were stored at -70°C. awaiting TNF-a assay.
IMMUNOASSAY FOR TNF-a
Table 1. Patient details Diagnosis Colorectal adenocarcinoma
Stage
No.
Weight loss
Dukes' C Dukes' 'D
3
< 10%
2
Nil
3
< IOY" > 10%
Liver mets
I
< IOO/v
Node+ve
I
Nil
Node +ve
I
< loo/"
3
Jejunal adenocarcinoma Breast carcinoma (infiltrating ductal) Lung adenocarcinoma
Each experiment was accompanied by a control in which normal rabbit immunoglobulin (Dakopatts, Glostrup, Denmark) was substituted for the primary antibody at an equivalent concentration. Antibody specificity was also tested by pre-incubating the primary antibody solution with IOpg/mL of recombinant human TNF-a (rHTNF-a; provided by Genentech Inc., South San Francisco, CA) for I h at room temperature prior to use. All slides were assessed by two independent observers, who counted at least I000 cells from the central part of each cover slip, scoring the percentage of positive staining for TNF-a. The results were recorded from one 'blind' observer (JLM), then compared with a second (SF) to assess interobserver variability.
Both serum and MNC culture supernatants were assayed for TNF-a using a highly specific immunoradiometric assay (TNF-IRMA; Medgenix, Fleurus, Belgium) sensitive to I pg/mL of rHTNF-a. The assay does not detect the related lymphokine, TNF-B (lymphotoxin). Briefly, standards and samples were added, along with "'I-labeled anti-TNF-a monoclonal antibody, to tubes pre-coated with oligoclonal anti-TNF-a antibodies. The solutions were incubated for 20h at room temperature, decanted and tubes washed prior to reading in a gamma counter. Values for each were determined by reference to the standard curve. STATISTICAL METHODS
MONOCYTE IMMUNOCYTOCHEMISTRY
The PBM monolayers were fixed in 4% phosphate buffered paraformaldehyde for 5 min, washed in phosphate buffered saline (PBS) and treated with 3% H202for IOmin at room temperature to inhibit endogenous peroxidase activity. After two further PBS rinses, cells were incubated in 5% normal goat serum for IOmin at 37"C, followed by rabbit anti-TNF-a (a gift from Prof. A. Scuderi, University of Arizona) diluted I :500 in 3% normal human serunV0. I '/O Triton-X 100 (Sigma) in PBS for 60min at 37°C. The specificity of the antibody for TNF-a has been demonstrated previously. I '.I6 Cover slips were rinsed again in three changes of PBS. incubated with biotinylated goat anti-rabbit antibody (Vector, Burlington, CA) diluted I : 100, followed after rinsing with streptavidin-horseradish peroxidase (Bethesda Research Laboratories, Gaithersburg, MD) diluted I :400,both for IOmin at 37°C. Finally, the cells were overlaid with freshly prepared 3-amino-9-ethylcarbazolesolution for IOmin, washed, counterstained with haematoxylin and mounted for light microscopy.
Linear regression and limits of agreement at the 95% confidence interval were used to compare data
obtained by two observers," and Spearman's signed rank correlation coefficient ( r , ) was used to test other associations. The modified unpaired 1-test was used to test for differences between unpaired group data.
Results SERUM TNF-II
Serum TNF-a levels were modestly elevated in cancer patients at 9.5 f 5.3 pg/mL (mean f s.d.) compared with healthy subjects (3.7 f 2. I pg/mL), and this difference was statistically significant ( P C0.01; Table 2). The highest serum levels occurred in patients with a history of weight loss (Fig. I ) and there appeared to be a possible association between serum TNF-a levels and the degree of weight loss ( r , = 0.6,P = 0.025).
I43
TNF-a IN CANCER PATIENTS
Table 2. Results of serum and supernatant TNF-a assay and monocyte immunostaining Normals Range
Test
Mean
Serum TNF-a (pg/mL) s u p LPS- (pg/mL) Sup LPS+ (pg/mL) MO LPS- ( Y o ) MO LPS+ (Yo) Sup:Mo(LPS-) Sup: Mo (LPS+)
3.7 671 1628 17 55
40 30
(1-8) (34-1606) (652-3023) (5-26) (43-68) (7-87) (12-56)
Mean
Cancer pts Range
9.5 1222 2105 19
38 158 82
(3-20) ( 144-3786)
(508-3582) (0.5-72) (6-72) (22-471) (25-208)
P*
< 0.01 NS NS NS 0.05 lo% Cancer patients
Fig. 1. Serum TNF-a levels in healthy subjects and cancer patients according to the degree of weight loss. Arrow indicates patient on dexamethasone. PBM IMMUNOSTAINING
The staining in TNF-a positive monocytes (Fig. 2a) occurred predominantly in a cytoplasmic perinuclear location. Very occasional cells showed a more diffuse granular type of staining. No staining was seen in control slides where anti-TNF-a antiserum was substituted with normal rabbit immunoglobulin (Fig. 2b), and staining could be completely prevented by pre-incubating the antiserum with rHTNF-a prior to immunostaining. There was a good correlation between data obtained by two independent observers, with respect to the percentage of PBM stained positively for TNF-a ( r = 0.97. P < 0.001). By limits of agreement analysis. independently assessed scores lay within 11 YO of each other at the 95% confidence interval. In contrast to the higher serum levels, mean percentage of PBM staining positively for TNF-a with LPS stimulation was less, overall, for cancer patients than for healthy volunteers (Table 2). This mean, however, was comprised of a wide range of values. Several weight-losing patients showed
Fig. 2. (a) PBM immunostained for TNF-a (arrow) and (b) control slide treated with normal rabbit immunoglobulin. Magnified lo00 times.
McCALL ETAL.
144
staining (MNC supernatant TNF-a :YO +ve staining PBM) was significantly elevated in cancer patients with and without LPS (Table 2). Interestingly, this ratio also correlated positively with the degree of weight loss in the cancer patients (rs= 0.58; P < 0.01; Fig. 5). Supernatant TNF-a levels in cancer patients with and without LPS did not differ significantly from healthy subjects, although their range of values was wider (Table 2). No significant correlation was found between PBM staining and serum TNF-a levels, or between MNC supernatant and serum TNF-a levels.
u)
E" $
40
U
2
I-
# " LPS LPs+ Cancer patients
LPSLPs+ Healthy subjects
O
Fig. 3. Percentage of PBM staining positively for TNF-a with and without LPS stimulation in healthy subjects, in (0) non weight-losing cancer patients, (0) cancer patients with < 10% weight loss, and (m) cancer patients with > 10%weight loss.
i"
i! -1
? 3ooo
E
Em
c
E
P
I4 -1
(*) 0
_i
0
0
20
0
0
0
0
0
40
%+vestaining PBM
80
60
0
0 0
0
0 0
,t.. 0
0
,
20
. .
, 40
Fig. 5. Ratio of MNC supernatant TNF-a to PBM staining (MNC supernatant levels: % +ve staining PBM), for healthy subjects and for cancer patients according to the degree of weight loss. (0)+LPS: ( 0 )-LPS.
Discussion This paper describes an immunocytochemical method of analysing TNF-a production in monocytes. Verification of the staining specificity has been described in greater detail elsewhere and is further supported by the absence of staining with TNF-a pre-absorbed antisera. The simple method of analysis, similar in principle to that described by Skidmore er al. has been validated by showing an acceptable concordance between independent observers' scores.I9 A pilot study, using this method of determining TNF-a expression by PBM from healthy volunteers and cancer patients, has been presented. Comparisons have been made with conventional methods of gauging TNF-a production, namely measurement of serum and MNC supernatants levels. The findings of this study need to be tempered by the small sample size. In addition, weight loss is only a crude and relatively insensitive indicator of metabolic status.2o Nevertheless it is significant that serum levels of TNF-a were found in this study to be modestly elevated in cancer patients. more so in those with a history of weight loss. These levels
''
0
0
0
YI
0
0
0
0
0
0
lo00
O.
.
.
%ve staining PBM
, 60
.
.
I
60
Fig. 4. Percentage of PBM staining positively for TNF-a against MNC supernatant TNF-a levels for (a) healthy subjects and (b) cancer patients ( 0 ) +LPS; (0)-LPS. PBM STAINING VS MNC SUPERNATANT TNF-(8
A positive correlation between PBM staining and
MNC supernatant TNF-a levels was observed overall (rs= 0.62, P < 0.001). When considered separately, the correlation was similar for both healthy subjects (rs= 0.73, P < 0.001 ; Fig. 4a) and cancer patients (rs= 0.7 I ; P < 0.001; Fig. 4b). The ratio of supernatant TNF-a levels to PBM
TNF-u IN CANCER PATIENTS
I45
are still low compared with those found in sepsis.” They are below the detection limits of most assays employed previously, which may ex lain the negative findings of these studies.8*’6*22*2 Balkwill et al. found TNF-a-like activity in the serum of 50% of cancer patients tested, but re rted no correlation with a history of weight loss.epThe substance they detected was biologically inactive and probably represented a subunit of TNF-aZSAlthough we did not test for bioactivity, Grau et al. have previously reported that the immunoassay employed in this study does detect biologically active TNF-a.Z6 High dose corticosteroids have been shown in vivo to inhibit TNF-a prod~ction.’~ It is interesting to note therefore that the one weight-losing patient on dexamethasone had a relatively low serum TNFa level compared with his weight-losing peers (Fig. 1). Although lymphocytes are capable of TNF-a production, PBM are held to be the principal source among MNC in the blood. This is also true in vitro following short incubation times of less than 6 h.13 The positive correlation between PBM staining for TNF-a and MNC supematant TNF-a levels was therefore not unexpected (Fig. 4a, 4b). However, monokine synthesis and release is a dynamic process, about which these two measurements can provide complementary information. High supernatant levels in the face of low PBM staining may reflect depletion of cellular sources after a secretory ‘burst’. The relative contributions of each are reflected in the ratio of MNC supernatant levels to PBM staining, with a higher ratio indicating relative depletion of monocytic TNF-a. These ratios were significantly higher in cancer patients (Table 2), as illustrated in Fig. 5 . Since MNC supernatant TNF-a levels were not elevated in these patients, depletion of monocytic TNF-a may reflect prior activation and release of TNF-a in vivo. This is compatible with the mildly elevated serum levels. Down regulation of TNF-a synthesis by PBM is another possibility, although not supported by previous studies.’-” These studies report a greater range of TNF-a responses among cancer patients with advanced disease, suggesting the possibility of temporal fluxes in the state of PBM activation in v ~ v o . ~ In * ~contrast, -~ production of interleukins 1 and 2 have been found to be deficient in advanced maIignancy.8*10,28 Extra-circulatory sources of TNF-a also occur in vivo but are much more difficult to quantify. In colorectal cancer, for example, loco-regional TNFa expression is seen in tumour-infiltrating macrophage^'^.^' and some regional lymph nodes (unpubl. obs.). Whether these sources could generate putative systemic effects in addition to their paracrine role is speculative, but one study has demonstrated attenuation of cachexia in tumour-
P
bearing rats with anti-TNF-a antibodies, despite an absence of detectable TNF-a in the blood.29 In summary, an immunocytochemicalanalysis of TNF-a expression in monocytes has been developed and applied to healthy volunteers and a small group of weight-stable and weight-losing cancer patients. Whereas serum TNF-a levels were mildly elevated, monocyte immunostaining tended to be lower, particularly in relation to MNC supernatant levels, in some cancer patients with weight loss. These results suggest an altered state of TNF-a regulation in some cancer patients, possibly resulting in enhanced TNF-a production. A larger study, looking simultaneously at gene and protein expression, is required to evaluate these possibilities further.
Acknowledgements This work was supported by the Cancer Society of New Zealand Inc. JLM was recipient of the Royal Australasian College of Surgeons Foundation New Zealand Fellowship (1989). We thank Professor A. M. van Rij for critical review and advice.
References 1. BEUTLERB. & CERAMI A. (1989) The biology of cachectin/TNF a primary mediator of the host response. Ann. Rev. Immunol. 7,625-55. 2. TRACEY K.J., WEIH., MANOGUE K. R. et al. (1988)
Cachectidtumor necrosis factor induces cachexia, anaemia, and inflammation. J. Exp. Med. 167, 1211-27. H. R., GUILLOU P. J. & WILMORED. W. (1989) Tumour necrosis factor and bacterial sepsis. Brit. J. Surg. 76.670-1. 4. EVANS R. D.,ARGILE~ J. M. & WILLIAMSON D. H. (1989) Metabolic effects of tumour necrosis factor-a (cachectin)and interleukin-I. Clin. Sci. 77, 357-64. 5 . ADERKA D..FISHER S.,LEVOY..HOLTMANN H., HAHN T. & WALLACHD. (1985) Cachectin/tumournecrosis-factor production by cancer patients. Lancer ii, 1190. 6. ZEMBALA M., MYTARB.. WOLOSZYNM., POPIELA T., URACZW. & CZUPRYNA A. (1988) Monocyte TNF production in gastrointestinal cancer. Lancet ii, 3.
MlCHIE
1262. 7. NARAK., ODAGW H., FUJUM.
er al. (1987) Increased production of tumor necrosis factor and prostaglandin & by monocytes in cancer patients and its unique modulation by their plasma. Cancer Immunol. Inmunother. 25, 126-32. 8. MOLDAWER L. L., D R m C. & LUNDHOLM K. (1988) Monocytic production and plasma bioactivities of interleukin-I and tumour necrosis factor in human cancer. Eur. J. Clin. Invest. 18.486-92. 9. IKEMOTO S., TAKC~OSHI K.. NISHIOS., WADAS. & MAEKAWA M. (1989) Correlation of tumor necrosis factor and prostaglandin & production of monocytes in bladder cancer patients. Cancer 64,2076-80. 10. ECONOMOU J. S.,COLQUHOUN S. D.,ANDERSON T.M.
I46
et a/. (1988) Interleukin-1 and tumor necrosis factor production by tumor-associated mononuclear leukocytes and peripheral mononuclear leukocytes in cancer patients. Int. J. Cancer 42, 7212-4. S. & PARRY B. R. 1I . MCCALL J. L., YUNK.. FUNAMOTO (1989)I n vivo immunohistochemicalidentificationof tumor necrosis factorkachectin in human lymphoid tissue. Amer. J . Pathol. 135,421-5. 12. BEISSERT S.. BERGHOU M., WAASE I., LEFSIENG., SCHAUER A., h Z E N M A l E R K. & KRONKM. (1989) Regulation of tumor necrosis factor gene expression in colorectal adenocarcinoma:I n vivo analysis by in situ hybridization. Proc. Narl Acad. Sci. USA 86, 5064-8. 13. ANDERSSON U., ADOLFG., DOHUTEN M., MOLLER G. & SJGGREN H-0.(1989) Characterization of individual tumor necrosis factor alpha and beta-producing cells after polyclonal T cell activation. J. Immunol. Methods 123,233-40. 14. CHENSUE s. w., REMlCK D. G . , SHMYR-FORSCH c., BEAU T. F. & KUNKELS. L. (1988) Immunohistochemical demonstration of cytoplasmic and membrane-associated tumor necrosis factor in murine macrophages.Amer. J . Pathol. 133,564-72. 15. BARATH P., FISHBEIN M. C., CAOJ., BERENSON J., HELFANT R. H. & FORRFSTER J. S. (1990) Detection and localization of tumor necrosis factor in human atheroma. Amer. J. Cardiol. 65, 297-302. 16. SCUDERI P., LAMK. S., RYANK. J. et a / . (1986) Raised serum levels of tumour necrosis factor in parasitic infections. Lancer ii, 1364-5. J. M. & ALTMAND. G. A. (1986) Statistical 17. BLAND methods for assessing agreement between two methods of clinical measurement. Lancer i, 307-10. 18. HUNTSBERGER D. V. & LEAVERTON P.E. (1970)Sfatisrical Inference in rhe Biomedical Sciences. Allyn and Bacon Inc., Boston. 19. SKIDMORE B. J., STAMNES S. A., TOWNSEND K. er a / . (1989) Enumeration of cytokine secreting cells at the
MCCALL ET AL.
single-cell level. Eur. J. Immunol.19, 1709-14. G . P.,LEINSTER S. J., DAVIS J. C. & HIPKIN 20. COPELAND L. J. (1987) Insulin resistance in patients with colorectal cancer. Brit. J. Surg. 74, 1031-6. 21. MARANO M. A., FONGY., MOLDAWER L. .L. et a / . (1990) Serum cachectirdtumor necrosis factor in critically ill patients with bums correlates with infection and mortality. Surg. Gynecol. Obstet. 170. 32-8. 22. SOCHER S. H., MARTINEZ D., CRAIG J. B.. KUHNG. & OLIFFA. (1988) Tumor necrosis factor not detectable in patients with clinical cancer cachexia. J. Null Cancer Insr. 80,595-8. P., HOEESS., VINERC. et a / . (1987) Tumour 23. SELEY necrosis factor in man: Clinical and biological observations. Brit. J. Cancer 56. 803-8. 24. BALKWILL F., BURKE F., TALBOT D. el a / . (1987) Evidence for tumour necrosis factorkachectin production in cancer. Loncet ii, 1229-30. P. J . , HOEESs., VINERc., JACKSON E., SMITH 25. SELEY 1. E. & McELwAlN T. J. (1988) Endogenous tumour necrosis factor in cancer patients. Lancet i, 483. T. E., MOLYNEUX M. E. et al. 26. GRAUG. E., TAYLOR (1989) Tumor necrosis factor and disease severity in children with falciparum malaria. N . Engl. J . Med. 320, 1586-91. 27. GOLDMAN M., AERAMOWICZ D., DE PAUW L., WIDERA I., VEREERSTRAETEN P. & KINNAERT P. (1989) OKT3induced cytokine release attenuation by high-dose methylprednisolone. Lancet ii, 802-3. C. & GUILLOU P. J . 28. MONSON J. R. T., RAMSDEN (1986) Decreased interleukin-2 production in patients with gastrointestinal cancer. Brit. J. Surg. 73, 483-6. 29. SHERRY B. A., GELIN1.. FONO Y. et a / . (1989) Anticachectirdtumor necrosis factor-a antibodies attenuate development of cachexia in tumor models. FASEB J. 3, 1956-62.
Ausf. N.Z.J. Surg. 1991.61. 141-146
SURGICAL RESEARCH TUMOUR NECROSIS FACTOR-ALPHA IMMUNODETECTION IN BLOOD MONOCYTES AND SERUM: PRELIMINARY FINDINGS IN WEIGHT- LOSING CANCER PATIENTS J. L. MCCALL,I*
s. FUNAMOTO, ** K. y U N 3 + AND B. R. PARR?*
Departments of *Surgery and 'Pathology, Otago University Medical School. Dunedin, New Zealand The peptide tumour necrosis factor-alpha (TNF-a) is a central mediator of the host response. Identifying where and when TNF-a is produced may give insights into its potential role in various pathophysiologicalstates. This paper describes a quantitative analysis of TNF-a expression in peripheral blood monocytes (PBM) at the single cell level. A pilot study has been undertaken, using this method to assess TNF-a expression in PBM from healthy volunteers and cancer patients. We also report mildly elevated serum TNF-a levels in the cancer patients. using an immunoradiometric assay (IRMA) sensitive to I p@mL of recombinant TNF-a. The results of this preliminary investigation suggest that TNF-a production may be altered in cancer patients. Key words: cachectin, cancer, immunocytochemistry, monacytes, tumour necrosis factor-alpha.
Introduction The monokine tumour necrosis factor-alpha (TNF-a, also known as cachectin) is a host-derived peptide involved in the pathophysiology of inflammatory and metabolic responses to infection and possibly malignancy. I*' TNF-a plays a major mediating role in septic shock.3 It also initiates energy substrate mobilization which, in the long term, can cause depletion of host tissues.2q4But considerabledebate continues over what role, if any, TNF-a might have in patients with weight-losing cancer. In part this debate stems from the unresolved question of whether TNF-a production is increased in cancer patients. Mononuclear phagocytes (blood monocytes and tissue macrophages) are the principal cellular source of TNF-a. I These cells have been the focus of studies attempting to determine the propensity for TNF-a production in different pathophysiological states. A standard approach has been to culture the cells in vitro and measure spontaneousor stimulated TNF-a production in the More recently, TNF immunostaining techniques have been used in histological and cytological preparations, to study cytokine expression at a cellular level. "-I5
' MB. ChB. 'MD.
MD. WD. 'MD. FRACS.
Correspondence: Dr 1. L. McCall. Department of Surgery. Box
913. Dunedin. New Zealand. Accepted for publication 8 August 1990.
This paper describes a method of TNF immunostaining in peripheral blood monocytes (PBM), and its application in a pilot study of healthy volunteers and cancer patients. The results of PBM immunostaining are compared with those of serum and mononuclear cell (MNC) supernatant TNF-a levels in the two groups. Methods SU EJ ECTS
Ten healthy volunteers and 14 cancer patients were studied after obtaining signed informed consent. Patient details are given in Table 1. Percentage weight loss was assessed by subtracting measured weight from recall well weight, and expressing the deficit as a percentage of well weight. No patient had sepsis, but one was receiving corticosteroids (dexamethasone 4mg 6-hourly) at the time of study. Serum for TNF-a assay was obtained from all subjects and heparinized blood for MNC separation was obtained from the ten healthy subjects and ten of the 14 cancer patients. MNC ISOLATION A N D CULTURE
The MNC were separated from heparinized whole blood by centrifugation through Ficoll-Hypaque (Histopaque-1077; Sigma, St Louis, MO) and washed twice in RPMI 1640 supplemented with 10% fetal calf serum (RPMVFCS; both Sigma). Cells were then resuspended in RPMVFCS and added to 35mm petri dishes, each containing a
142
MCCALL ET A L
clean sterile cover slip. The cell density of the suspension was determined and subsequent results adjusted to a standard density of 107cells/mL. Suspended cells were cultured for 2 h at 37'C in a humidified chamber with 5% COz, with or without 10pglmL of lipopolysaccharide (LPS; E. Coli serotype 0127:BS, Sigma). After culture the medium was collected and centrifuged to obtain the cell free supernatant and the cover slips were removed for immunocytochemistry of the adherent PBM monolayer. Supernatants from cultures with no LPS added had less than 0.5 endotoxin unitslmL as determined by Limulus amoebocyte lysate assay (E-Toxate. Sigma). All supernatants and serum samples were stored at -70°C. awaiting TNF-a assay.
IMMUNOASSAY FOR TNF-a
Table 1. Patient details Diagnosis Colorectal adenocarcinoma
Stage
No.
Weight loss
Dukes' C Dukes' 'D
3
< 10%
2
Nil
3
< IOY" > 10%
Liver mets
I
< IOO/v
Node+ve
I
Nil
Node +ve
I
< loo/"
3
Jejunal adenocarcinoma Breast carcinoma (infiltrating ductal) Lung adenocarcinoma
Each experiment was accompanied by a control in which normal rabbit immunoglobulin (Dakopatts, Glostrup, Denmark) was substituted for the primary antibody at an equivalent concentration. Antibody specificity was also tested by pre-incubating the primary antibody solution with IOpg/mL of recombinant human TNF-a (rHTNF-a; provided by Genentech Inc., South San Francisco, CA) for I h at room temperature prior to use. All slides were assessed by two independent observers, who counted at least I000 cells from the central part of each cover slip, scoring the percentage of positive staining for TNF-a. The results were recorded from one 'blind' observer (JLM), then compared with a second (SF) to assess interobserver variability.
Both serum and MNC culture supernatants were assayed for TNF-a using a highly specific immunoradiometric assay (TNF-IRMA; Medgenix, Fleurus, Belgium) sensitive to I pg/mL of rHTNF-a. The assay does not detect the related lymphokine, TNF-B (lymphotoxin). Briefly, standards and samples were added, along with "'I-labeled anti-TNF-a monoclonal antibody, to tubes pre-coated with oligoclonal anti-TNF-a antibodies. The solutions were incubated for 20h at room temperature, decanted and tubes washed prior to reading in a gamma counter. Values for each were determined by reference to the standard curve. STATISTICAL METHODS
MONOCYTE IMMUNOCYTOCHEMISTRY
The PBM monolayers were fixed in 4% phosphate buffered paraformaldehyde for 5 min, washed in phosphate buffered saline (PBS) and treated with 3% H202for IOmin at room temperature to inhibit endogenous peroxidase activity. After two further PBS rinses, cells were incubated in 5% normal goat serum for IOmin at 37"C, followed by rabbit anti-TNF-a (a gift from Prof. A. Scuderi, University of Arizona) diluted I :500 in 3% normal human serunV0. I '/O Triton-X 100 (Sigma) in PBS for 60min at 37°C. The specificity of the antibody for TNF-a has been demonstrated previously. I '.I6 Cover slips were rinsed again in three changes of PBS. incubated with biotinylated goat anti-rabbit antibody (Vector, Burlington, CA) diluted I : 100, followed after rinsing with streptavidin-horseradish peroxidase (Bethesda Research Laboratories, Gaithersburg, MD) diluted I :400,both for IOmin at 37°C. Finally, the cells were overlaid with freshly prepared 3-amino-9-ethylcarbazolesolution for IOmin, washed, counterstained with haematoxylin and mounted for light microscopy.
Linear regression and limits of agreement at the 95% confidence interval were used to compare data
obtained by two observers," and Spearman's signed rank correlation coefficient ( r , ) was used to test other associations. The modified unpaired 1-test was used to test for differences between unpaired group data.
Results SERUM TNF-II
Serum TNF-a levels were modestly elevated in cancer patients at 9.5 f 5.3 pg/mL (mean f s.d.) compared with healthy subjects (3.7 f 2. I pg/mL), and this difference was statistically significant ( P C0.01; Table 2). The highest serum levels occurred in patients with a history of weight loss (Fig. I ) and there appeared to be a possible association between serum TNF-a levels and the degree of weight loss ( r , = 0.6,P = 0.025).
I43
TNF-a IN CANCER PATIENTS
Table 2. Results of serum and supernatant TNF-a assay and monocyte immunostaining Normals Range
Test
Mean
Serum TNF-a (pg/mL) s u p LPS- (pg/mL) Sup LPS+ (pg/mL) MO LPS- ( Y o ) MO LPS+ (Yo) Sup:Mo(LPS-) Sup: Mo (LPS+)
3.7 671 1628 17 55
40 30
(1-8) (34-1606) (652-3023) (5-26) (43-68) (7-87) (12-56)
Mean
Cancer pts Range
9.5 1222 2105 19
38 158 82
(3-20) ( 144-3786)
(508-3582) (0.5-72) (6-72) (22-471) (25-208)
P*
< 0.01 NS NS NS 0.05 lo% Cancer patients
Fig. 1. Serum TNF-a levels in healthy subjects and cancer patients according to the degree of weight loss. Arrow indicates patient on dexamethasone. PBM IMMUNOSTAINING
The staining in TNF-a positive monocytes (Fig. 2a) occurred predominantly in a cytoplasmic perinuclear location. Very occasional cells showed a more diffuse granular type of staining. No staining was seen in control slides where anti-TNF-a antiserum was substituted with normal rabbit immunoglobulin (Fig. 2b), and staining could be completely prevented by pre-incubating the antiserum with rHTNF-a prior to immunostaining. There was a good correlation between data obtained by two independent observers, with respect to the percentage of PBM stained positively for TNF-a ( r = 0.97. P < 0.001). By limits of agreement analysis. independently assessed scores lay within 11 YO of each other at the 95% confidence interval. In contrast to the higher serum levels, mean percentage of PBM staining positively for TNF-a with LPS stimulation was less, overall, for cancer patients than for healthy volunteers (Table 2). This mean, however, was comprised of a wide range of values. Several weight-losing patients showed
Fig. 2. (a) PBM immunostained for TNF-a (arrow) and (b) control slide treated with normal rabbit immunoglobulin. Magnified lo00 times.
McCALL ETAL.
144
staining (MNC supernatant TNF-a :YO +ve staining PBM) was significantly elevated in cancer patients with and without LPS (Table 2). Interestingly, this ratio also correlated positively with the degree of weight loss in the cancer patients (rs= 0.58; P < 0.01; Fig. 5). Supernatant TNF-a levels in cancer patients with and without LPS did not differ significantly from healthy subjects, although their range of values was wider (Table 2). No significant correlation was found between PBM staining and serum TNF-a levels, or between MNC supernatant and serum TNF-a levels.
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Fig. 3. Percentage of PBM staining positively for TNF-a with and without LPS stimulation in healthy subjects, in (0) non weight-losing cancer patients, (0) cancer patients with < 10% weight loss, and (m) cancer patients with > 10%weight loss.
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Fig. 5. Ratio of MNC supernatant TNF-a to PBM staining (MNC supernatant levels: % +ve staining PBM), for healthy subjects and for cancer patients according to the degree of weight loss. (0)+LPS: ( 0 )-LPS.
Discussion This paper describes an immunocytochemical method of analysing TNF-a production in monocytes. Verification of the staining specificity has been described in greater detail elsewhere and is further supported by the absence of staining with TNF-a pre-absorbed antisera. The simple method of analysis, similar in principle to that described by Skidmore er al. has been validated by showing an acceptable concordance between independent observers' scores.I9 A pilot study, using this method of determining TNF-a expression by PBM from healthy volunteers and cancer patients, has been presented. Comparisons have been made with conventional methods of gauging TNF-a production, namely measurement of serum and MNC supernatants levels. The findings of this study need to be tempered by the small sample size. In addition, weight loss is only a crude and relatively insensitive indicator of metabolic status.2o Nevertheless it is significant that serum levels of TNF-a were found in this study to be modestly elevated in cancer patients. more so in those with a history of weight loss. These levels
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Fig. 4. Percentage of PBM staining positively for TNF-a against MNC supernatant TNF-a levels for (a) healthy subjects and (b) cancer patients ( 0 ) +LPS; (0)-LPS. PBM STAINING VS MNC SUPERNATANT TNF-(8
A positive correlation between PBM staining and
MNC supernatant TNF-a levels was observed overall (rs= 0.62, P < 0.001). When considered separately, the correlation was similar for both healthy subjects (rs= 0.73, P < 0.001 ; Fig. 4a) and cancer patients (rs= 0.7 I ; P < 0.001; Fig. 4b). The ratio of supernatant TNF-a levels to PBM
TNF-u IN CANCER PATIENTS
I45
are still low compared with those found in sepsis.” They are below the detection limits of most assays employed previously, which may ex lain the negative findings of these studies.8*’6*22*2 Balkwill et al. found TNF-a-like activity in the serum of 50% of cancer patients tested, but re rted no correlation with a history of weight loss.epThe substance they detected was biologically inactive and probably represented a subunit of TNF-aZSAlthough we did not test for bioactivity, Grau et al. have previously reported that the immunoassay employed in this study does detect biologically active TNF-a.Z6 High dose corticosteroids have been shown in vivo to inhibit TNF-a prod~ction.’~ It is interesting to note therefore that the one weight-losing patient on dexamethasone had a relatively low serum TNFa level compared with his weight-losing peers (Fig. 1). Although lymphocytes are capable of TNF-a production, PBM are held to be the principal source among MNC in the blood. This is also true in vitro following short incubation times of less than 6 h.13 The positive correlation between PBM staining for TNF-a and MNC supematant TNF-a levels was therefore not unexpected (Fig. 4a, 4b). However, monokine synthesis and release is a dynamic process, about which these two measurements can provide complementary information. High supernatant levels in the face of low PBM staining may reflect depletion of cellular sources after a secretory ‘burst’. The relative contributions of each are reflected in the ratio of MNC supernatant levels to PBM staining, with a higher ratio indicating relative depletion of monocytic TNF-a. These ratios were significantly higher in cancer patients (Table 2), as illustrated in Fig. 5 . Since MNC supernatant TNF-a levels were not elevated in these patients, depletion of monocytic TNF-a may reflect prior activation and release of TNF-a in vivo. This is compatible with the mildly elevated serum levels. Down regulation of TNF-a synthesis by PBM is another possibility, although not supported by previous studies.’-” These studies report a greater range of TNF-a responses among cancer patients with advanced disease, suggesting the possibility of temporal fluxes in the state of PBM activation in v ~ v o . ~ In * ~contrast, -~ production of interleukins 1 and 2 have been found to be deficient in advanced maIignancy.8*10,28 Extra-circulatory sources of TNF-a also occur in vivo but are much more difficult to quantify. In colorectal cancer, for example, loco-regional TNFa expression is seen in tumour-infiltrating macrophage^'^.^' and some regional lymph nodes (unpubl. obs.). Whether these sources could generate putative systemic effects in addition to their paracrine role is speculative, but one study has demonstrated attenuation of cachexia in tumour-
P
bearing rats with anti-TNF-a antibodies, despite an absence of detectable TNF-a in the blood.29 In summary, an immunocytochemicalanalysis of TNF-a expression in monocytes has been developed and applied to healthy volunteers and a small group of weight-stable and weight-losing cancer patients. Whereas serum TNF-a levels were mildly elevated, monocyte immunostaining tended to be lower, particularly in relation to MNC supernatant levels, in some cancer patients with weight loss. These results suggest an altered state of TNF-a regulation in some cancer patients, possibly resulting in enhanced TNF-a production. A larger study, looking simultaneously at gene and protein expression, is required to evaluate these possibilities further.
Acknowledgements This work was supported by the Cancer Society of New Zealand Inc. JLM was recipient of the Royal Australasian College of Surgeons Foundation New Zealand Fellowship (1989). We thank Professor A. M. van Rij for critical review and advice.
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