Journal of Chemotherapy
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Role of Exogenous Interferons on Intrinsic Antiviral Activity of Macrophages from Patients Affected by Neoplasia R.A. Merendino, A. Arena, M.C. Liberto, M. Mesiti, S. Chillemi, D. Iannello & L. Bonina To cite this article: R.A. Merendino, A. Arena, M.C. Liberto, M. Mesiti, S. Chillemi, D. Iannello & L. Bonina (1990) Role of Exogenous Interferons on Intrinsic Antiviral Activity of Macrophages from Patients Affected by Neoplasia, Journal of Chemotherapy, 2:2, 116-122, DOI: 10.1080/1120009X.1990.11738994 To link to this article: https://doi.org/10.1080/1120009X.1990.11738994
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Journal of Chemotherapy
Role of Exogenous Interferons on Intrinsic Antiviral Activity of Macrophages from Patients Affected by Neoplasia R.A. MERENDINO - A. ARENA M.e. LIBERTO * M. MESITI ** - S. CHILLEMI ** D. IANNELLO - L. BONINA
Summary --------------------------------
Macrophages derived from in vitro cultured monocytes were infected with herpes simplex virus type 2. A marked impairment in the intrinsic antiviral activity was found in macrophages obtained from patients with breast cancer or melanoma. Moreover, the antiviral activity of macrophages from healthy donors, differentiated in serum from patients with neoplasia, was also impaired. The aim of this work was the evaluation of a, ~, y exogenous interferon in restoring the intrinsic antiviral activity of macrophages from patients affected by breast cancer or melanoma under different conditions. Pretreatment of macrophages with a, ~ interferons, but not y interferon, restored their impaired intrinsic antiviral activity. Key words: macrophage, neoplasia, HSV-2, interferons.
Institute of Medical Microbiology, University of Messina;
* Institute of Medical Microbiology, University of Reggio Calabria, Catanzaro; ** Institute of Oncology, University of Messina. Italy. Correspondence: Prof. R.A. Merendino, Institute of Medical Microbiology, Piazza XX Settembre 4, 98100 Messina. Italy.
Vol. 2 - n. 2 (116-122) - 1990
INTRODUCTION
It is now well established that interferons (IFNs) exhibit antiproliferative, antiviral and immunoregulatory activities 1; therefore they have been employed in a number of clinical trials 2,3. Clinical studies have shown that IFN s can be highly effective against certain types of cancer 4,5; moreover positive results have been reported for a number of herpes virus-associated diseases 6. Among the immunocompetent cells involved in host response to the development of both infectious diseases and neoplasia, macrophages are of crucial importance 7,8 due to their various functions and complex interactions with other components of the immune system 9. In previous studies 10,11 we detected a marked impairment of the intrinsic antiviral activity versus herpes simplex type 2 (HSV-2) of macrophages derived from monocytes isolated from the peripheral blood of patients with breast cancer or melanoma compared with those from normal subjects. The macrophages were differentiated under varying conditions; in particular, macrophages from tumor-bearing patients, differentiated in serum from healthy donors, showed more efficient antiviral activity than the same cells differentiated in autologous serum. These results are consistent with the hypothesis that the antiviral activity of mononuclear cells can be affected by the presence of factors in sera from patients with neoplasia. In view of the well-known role played by interferons on macrophage functions, we have focused our attention on the role of exogenous interferons (1, J3, y) in restoring the intrinsic antiviral activity of macrophages from patients affected by neoplasia under different conditions.
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ROLE OF EXOGENOUS INTERFERONS ON INTRINSIC ANTIVIRAL ACTIVITY ETC.
MATERIALS AND METHODS
Tissue culture media and cells Human aneuploid HEp-2 cells were obtained from Flow Laboratories Inc. (Asnieres). The cells were grown in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum and antibiotics. Human monocytes and macrophages were maintained in RPMI 1640 (Flow Laboratories Inc., Asnieres) containing 10% heat-inactivated human serum and antibiotics.
Virus A recent clinical isolate of herpes simplex virus type 2 was used to infect HEp-2 cells from which a stock suspension of virus was prepared and stored at - 80 0 C.
Interferons aFNs) Human IFNs type a (3.1 x 10 6 International Reference Units, IRU/mg) and 'Y (1 X 106 IRU/mg) purchased from Sigma, and type f3 (10 7 . 38 IRU/mg) purchased from Lee
Biomolecular (San Diego, CA) were used at a concentration of 30 and 75 IU/ml. The IFN preparations were titered in each experiment using a microassay based on the inhibition of cytopathic effect of vesicular stomatitis virus on HEp-2 cells 12.
Patients Five groups of donors were studied: a control group of five healthy adults of either sex; five patients with breast cancer; five patients with breast cancer which had metastasized; five patients with melanoma; five patients with melanoma which had metastasized. All the patients affected by breast cancer· were post-menopausal. The patients had not received any chemotherapy, hormonotherapy or radiotherapy. All the groups were tested for the presence of circulating anti-HSV-2 antibodies by means of a «micro-ELISA kit» for HSV (PBI International). A summary of the clinical information concerning the patients studied is reported in Table 1. The classifications for breast cancer and melanoma are based on the «Union International Contre Ie Cancer» 13 and Clark's reports 14.
TABLE 1 . Information about the patients studied. Patients
Sex
Age
Type of neoplasia *
A B C D E F G
F F F
62 56 58 61 55 55 60 57 60 61
LD.C. LD.C. LD.C. LD.C. LD.C. LD.C. LD.C. LD.C. LD.C. LD.C.
57 60 54 61 58 55 63 61
N.M. N.M. N.M. N.M. N.M. N.M. N.M. N.M. N.M. N.M.
Ii I L
M N
0 P
Q R S T U V
F F F F F F F F M M F M F M F F M
~~
* LD.C., infiltrating ductal carcinoma; N.M., nodular melanoma.
** Clark classification. *** UICC classification: «Union International Contre Ie Cancer». **** Antibody titers vs herpes simplex virus.
Stage (Clark) **
III III II II III III IV V IV III
Stage (TNM) ***
Ab ****
T2N-MO TlN-MO T2N-MO TlN-MO T3N-MO T2N-M1 T3N-Ml T3N-Ml T2N-M1 T2N-M1
32 32 16 64 4 4
T2N-MO T2N-MO TlN-MO TlN-MO T2N-MO T2N+M1 T3N+M1 T4N+M1 T3N+M1 T2N+M1
8
64 32 16 8
16 32 64 16 64 32 8 16 64
118
R.A. MERENDINO - A. ARENA - M.C. LIBERTO - M. MESITI - S. CHILLEMI - D. IANNELLO - L. BONINA
Isolation of monocytes and culture of macrophages
Intrinsic antiviral activity of macrophages
Human monocytes were collected from venous blood of the donors described above. The blood was collected, rapidly added to RPMI 1640 containing 10 IV/ml of heparin (Liquemin, Roche), and centrifuged on Ficoll Hypaque gradients 15. The mononuclear cells were washed twice in RPMI 1640 and placed in plastic multiwell tissue culture dishes (Nunc, Denmark) at a concentration of 5 X 10 5 per well. After incubating the cells for 4 h at 37° C in 5% CO 2 atmosphere, the non-adherent cells were removed by washing the cultures. The adherent cellular population consisted of monocytes as assessed by the phagocytosis of Candida albicans, by peroxidase activity in the cytoplasm and morphological criteria 16; the viability was 90% as assessed by the Trypan blue exclusion test. The in vitro differentiation of monocytes into macrophages was followed for 10 days by checking the improvement of phagocytic activity, the increase in cell size, as well as the level of peroxidase and esterase activity. Monocytes were differentiated in vitro under different conditions: a) monocytes from healthy donors differentiated in autologous serum; b) monocytes from healthy donors differentiated respectively in sera from patients with breast cancer or melanoma; c) monocytes from healthy donors differentiated in sera from patients with breast cancer which had metastasized or melanoma which had metastasized; d) monocytes from patients with breast cancer or melanoma differentiated in sera from healthy donors; e) monocytes from patients with breast cancer which had metastasized or melanoma which had metastasized differentiated in sera from healthy dortors; f) monocytes from patients with breast cancer or melanoma differentiated in autologous serum; g) monocytes from patients with breast cancer 'which had metastasized or melanoma which had metastasized differentiated in autologous serum. The adherent cells were counted with an ocular microscope equipped with a grid, and the number of cells per well was estimated. Between 25 and 30% of the total number of monocytes present in the original seeding susp~hsion were retained on the plastic surface after 10 days of incubation. This percentage did not depend on the group of donors studied. The media were changed every 3 days.
The intrinsic antiviral activity is considered the mechanism by which macrophages are able to destroy viruses intracellularly. This activity was expressed in terms of virus yield from macrophages 22 h post-infection. The macrophages from different donors were infected with HSV-2 at multiplicity of infection 0.1 and 2, calculated on the basis of the number of adherent cells after 10 days of culture. The multiplicity of infection expresses the ratio between the number of infectious viral particles and the number of viable target cells. HSV-2 was allowed to adsorb for 1 h at 37° C, then the cells were washed three times with RPMI 1640, covered with 1 ml of the same medium and incubated at 37° C in 5% CO 2 • Samples were collected 22 h postinfection, frozen and thawed three times in order to release intracellular virus and then clarified by centrifu- I gation at 1000 g for 10 min. The virus suspension obtained was titrated on HEp-2 cells according to the method of Dulbecco 17. Interferon treatment
To evaluate the effect of in vitro treatment with exogenous interferons on the antiviral activity, the macrophages were incubated for 18 hours with n, ~ or y interferon at two different concentrations (30 and 75 IV/ml). Before being infected with HSV-2, the cells were repeatedly washed in order to remove unadsorbed and extracellular IFN. Macrophages were then tested for intrinsic antiviral activity as described before. Calculations
Statistical evaluations were performed by means of analysis of variance.
RESULTS
Intrinsic antiviral activity of macrophages
Intrinsic antiviral activity was expressed in terms of virus yield from macrophages 22 hours after infection. Cells were infected with HSV-2 at multiplicity of infection 0.1 or 2. Results are shown in Tables 2-5.
119
ROLE OF EXOGENOUS INTERFERONS ON INTRINSIC ANTIVIRAL ACTIVITY ETC.
TABLE 2 - The growth of HSV-2 (multiplicity of infection: MOl 0.1) in macrophages from healthy donors (HD), patients affected by breast cancer (BCo) and breast cancer with metastasis (BCI), treated with interferons a, P and )I. log,. PFU/ml Interferon Treatment
Concentration IFN (IU/ml)
Macrophages* : Serum:
HD sHD
None
BCo sHD
BC1 sHD
HD sBCo
HD sBC1
BCo sBCo
BC1 sBC1
2.30
2.95
1.90
2.47
3.80
4.51
a
30 75
1.95 1.30
2.79 2.02
1.77 0.74
2.26 1.62
3.50 3.18
4.39 4.05
l3
30 75
1.23 0.69
2.17 1.45
0.93 0.39
1.53 0.47
3.46 2.55
4.05 3.19
Y
30 75
2.51 2.40
2.98 2.89
1.88 1.88
2.34 2.41
3.71 3.65
4.40 4.41
* Macrophages were obtained from HD, BCo and BC1, differentiated in presence of serum taken from healthy donors or patients affected by neoplasia. The results represent the mean of five experiments from five different donors, expressed in log,. PFU/ml. In all the experiments the S.D. was less than 20%.
TABLE 3 - The growth of HSV-2 (multiplicity of infection: MOl 2) in macrophages from healthy donors (HD) and patients affected by breast cancer (BCo) and breast cancer with metastasis (BCI), treated with interferons a, p and )I. log,. PFU/ml Interferon Treatment
Concentration IFN (IU/ml)
None
Macrophages *: Serum:
HD sHD
BCo sHD
BC1 sHD
HD sBCo
HD sBC1
BCo sBCo
BC1 sBC1
3.93
5.16
5.41
4.45
4.11
5.91
6.26
a
30 75
3.41 2.98
4.96 4.65
5.13 4.81
3.97 3.75
3.93 3.56
5.89 5.54
5.99 5.95
l3
30 75
2.81 1.92
4.72 4.21
4.99 4.51
3.72 2.79
3.48 2.61
5.72 5.21
5.86 5.62
Y
30 75
3.93 3.85
5.45 5.08
5.21 4.98
4.33 4.09
3.97 3.96
5.96 5.98
6.37 6.08
* Macrophages obtained from HD, BCo and BC1, differentiated in presence of serum taken from healthy donors or patients affected by neoplasia. The results represent the mean of five experiments from five different donors, expressed in log,. PFU/ml. In all the experiments the S.D. was less than 20%.
Table 2 shows that HSV-2 replication at multiciplity of infection 0.1 was completely inhibited by macrophages from healthy donors that were differentiated in the presence of autologous serum (p < 0.001). Conyersely, the same cells did not inhibit viral replieation when differentiated in vitro in the presence of serum from patients with breast cancer. On the other hand, the virus yield of macrophages from patients with breast cancer was significantly higher compared with controls differentiated in the presence of both autologous serum and normal serum (p 1( 0.005). In particular, the virus yields from cells differentiated
in the presence of autologous serum was significantly higher than the yield from the same cells differentiated in serum from healthy donors (p < 0.001). As shown in Table 4, the same results were obtained when the intrinsic antiviral activity of macrophages from patients with melanoma was studied under different conditions (see «Materials and Methods»). The results reported in Tables 3 and 5 show that the antiviral activity of macrophages was markedly influenced by the multiplicity of infection IS. In fact, higher virus yield was recovered when macrophages from controls and pa-
120
R.A. MERENDINO - A. ARENA - M.e. UBERTO - M. MEsm - s. CHILLEMI - D. IANNELLO - L. BONINA
TABLE 4 - The growth of HSV-2 (MOl 0.1) in macrophages from healthy donors and patients affected by melanoma (Mo) and melanoma with metastasis (Ml), treated with interferons a, p and y. log,. PFU/ml Interferon Treatment
Concentration IFN (IV/mll
Macrophages* : Serum:
HD sHD
None
Mo sHD
Ml sHD
HD sMo
HD sMI
Mo sMo
Ml sMI
2.87
3.19
2.91
3.18
3.85
4.62
a
30 75
2.63 1.93
2.92 2.51
2.71 2.09
2.91 2.41
3.64 3.45
4.39 4.21
~
30 75
1.94 1.37
2.26 1.69
2.01 0.81
2.26 1.02
3.48 2.65
4.22 3.62
Y
30 75
2.87 2.80
3.42 3.09
2.93 2.87
3.20 3.41
3.79 3.71
4.55 4.64
* Macrophages were obtained from HD, Mo and Ml, differentiated in presence of serum taken ftom healthy donors or patients affected by neoplasia. The results represent the mean of five experiments from five different donors, expressed in log,. PFU/ml. In all the experiments the S.D. was less than 20%.
TABLE 5 - The growth of HSV-2 (MOl 2) in macrophages from healthy donors (HD) and patients affected by melanoma (Mo) and melanoma with metastasis (Ml), treated with interferons a, p and y. log,. PFU/ml Interferon Treatment
Concentration IFN (IV/mll
None
Macrophages *: Serum:
HD sHD
Mo sHD
Ml sHD
HD sMo
HD sMI
Mo sMo
Ml sMI
3.93
5.41
5.71
4.62
4.55
6.16
6.33
a
30 75
3.41 2.98
5.19 4.87
5.51 4.96
4.33 3.98
4.33 3.93
5.98 5.81
6.09 5.91
~
30 75
2.81 1.92
5.09 4.51
5.39. 4.81
4.04 3.42
3.98 3.34
5.92 5.65
6.11 5.83
Y
30 75
3.93 3.85
5.33 5.37
5.75 5.61
4.51 4.62
4.33 4.38
6.09 6.08
6.39 6.37
* Macrophages obtained ftom HD, Mo and Ml, differentiated in presence of serum taken from healthy donors or patients affected by neoplasia. The results represent the mean of five experiments from five different donors, expressed in log,. PFU/ml. In all the experiments the S.D. was less than 20%.
tients with breast cancer and melanoma were infected at multiplicity of infection 2. In particular, the virus yield from macrophages of patients was significantly higher than virus yield from macrophages of healthy donors (p < c.: 0.002). . Effect of interferon on the antiviral activity of macrophages
A series of experiments was carried out to assess whethet IFN s could influence the intrinsic antiviral ~ctivity of human macrophages.
These cells, obtained from both healthy donors and patients, were preincubated with different concentrations of IFN and afterwards tested for intrinsic antiviral activity. As shown in Table 2, a preincubation with 75 IU/ml of a-IFN but not with 30 IV/ml, significantly improved the antiviral activity (p < 0.01) of macrophages obtained from patients with breast cancer with and without metastasis differentiated in the presence of serum from healthy donors. The same basic behavior was observed in macrophages from healthy donors differentiated in the sera of patients with breast cancer with and
121
ROLE OF EXOGENOUS INTERFERONS ON INTRINSIC ANTIVIRAL ACTIVITY ETC.
without metastasis (p < 0.005). The ~-IFN treatment appeared to be more effective than that with a-IFN; in fact, we observed an appreciable effect by adding 30 IU/ml of ~-IFN to macrophages from healthy donors differentiated in sera from patients with neoplasia and to macrophages from breast cancer patients with and without metastasis differentiated in the presence of sera from healthy donors (p < 0.001). Under all the experimental conditions a significant improvement of the antiviral activity is detectable, using a concentration of 75 IU/ml of ~-IFN (p < 0.001). The virus growth was not modified with respect to control cultures when the cells had been preincubated with y-IFN. As shown in Table 4, essentially the same results were obtained when the intrinsic antiviral activity of macrophages from patients with melanoma was studied under different conditions (see «Materials and Methods»). Tables 3 and 5 show the HSV-2 yield from the macrophages of the same groups of donors, infected at a multiplicity of infection of 2 and preincubated with a,~ or y-IFN. The viral titers were significantly higher with respect to the titers obtained from macrophages infected at a multiplicity of infection of 0.1. Nevertheless, it is possible to detect an appreciable effect of a-IFN (p < 0.001) and ~ IFN (p < 0.002) at the dose of 75 IU/ml on the antiviral activity of macrophages when compared to the untreated controls. These effects are evident in all the experimental conditions considered, except when macrophages from breast cancer patients with and without metastasis are differentiated with autologous serum and treated with 75 IU/ml of a-IFN. Moreover, it is possible to produce an improvement in antiviral activity by adding 30 IU/ml of a-IFN and ~-IFN to macrophages from healthy donors differentiated in autologous serum with respect to the untreated controls (p < 0.005 and p < 0.001 respectively). Conversely, the antiviral .. activity of macrophages from h~althy donors differentiated in sera from breast cancer patients with and without metastasis appears to be improved only when the treatment is performed with 30 IU/ml of ~-IFN (p < 0.01). Also in this case the treatment with y-IFN does not affect macrophage antiviral activity under any of the experimental conditions studied.
DISCUSSION
The experiments described in this paper were carried out to establish whether exogenous interferons affect the impaired intrinsic antiviral activity versus HSV -2 of macrophages from patients with neoplasia. In fact it has been widely reported that immunocompetent cells and, in particular, macrophages from patients affected by neoplasia or from tumor-bearing animals are functionally depressed 19. The impairment of the intrinsic antiviral activity of macrophages was demonstrated by our group using macrophages from patients affected by breast cancer, melanoma 10 and using tumor bearing rats as well 20. Moreover, we demonstrated that the multiplicity of infection greatly affects the virus yield of macrophages, obtained either from normal subjects or patients with neoplasia 10. We showed that factors present in the serum of patients with neoplasia inhibited the antiviral activity of macrophages from healthy donors 11. For this reason, in the present study, macrophages were cultured under different conditions (see «Materials and Methods») and infected at a multiplicity of infection of 0.1 and 2. This inhibitory activity of the serum on the antiviral activity is related to the clinical stage of the disease, namely to the presence of metastasis both in breast cancer and melanoma-bearing patients. The results were obtained by the study of homogeneous groups of patients selected on the basis of the clinical stage of the neoplasia and the absence of chemotherapy, hormonotherapy or radiotherapy. Moreover, no relationships between macrophage intrinsic antiviral activity and the humoral immune response versus the virus used were found in our study. In fact, as reported in Table 1, the behavior of macrophages seems to be independent of the presence of circulating antibody versus HSV-2 in healthy donors as well as in neoplastic patients. The inhibitory factors in sera have not been biochemically characterized. Several possibilities exist including prostaglandins, small molecular weight pep tides and other less defined substances that could account for the inhibitory activity of tumor extracts or sera from patients on the function of macrophages 11.
122
R.A. MERENDINO - A. ARENA - M.C. LIBERTO - M. MESITI - S.
The possibility of influencing viral replication, and, in particular, HSV replication in human macrophages by treatments with exogenous interferon has been described 12. In fact, treatment of human macrophage cultures with interferon resulted in the inhibition of viral replication_ Our experiments clearly demonstrate that the addition of a- and j3-interferon to cultures of macrophages from cancer-bearing patients leads to an enhancement of the antiviral activity. Moreover, the preincubation of macrophages from patients with exogenous interferon could partially restore their antiviral activity, depending on the kind of interferon, its concentration, the multiplicity of infection and the clinical stage of neoplasia. Moreover, the presence of metastasis appears to negatively influence the antiviral activity of macrophages in their sensitivity to interferons. The present results, related to a higher sensitivity of macrophages to a- and j3-interferon with respect to y-interferon are confirmed by other studies performed in a similar experimental model 12 • According to these authors, the difference in the activity of exogenous y-interferon in relation to a- and j3-interferon could probably be related to differences in the binding to receptors 21. The lack of effect of yinterferon on macrophages was comparable both in cells from normal subjects and from patients with neoplasia. It therefore appears that interferon is not the only factor involved in the regulation of macrophage antiviral activity and that probably other mechanisms are involved in the impairment of the antiviral activity of macrophages from patients with neoplasia. Moreover, the fact that the addition of aand.j3- interferon to cultures of macrophages from cancer-bearing patients leads to an enhancement of the antiviral activity, strongly·· implies that interferon is a key molecule. ACKNOWLEDGMENTS: This work was supported by a grant of the Italian National Research Council (CNR) n. 88.02031.04.
REFERENCES 1 Baron S, Dianzani F, Stonton GJ. General consideration of the interferon system. In: Baron S, Dianzani F, Ston-
CHILLEMI -
D. IANNELLO -
L.
BONINA
ton GJ, eds. The interferon system, University of Texas Medical Branch, Galveston, 1982: 1-12. 2 Cantell K. Clinical performance of natural human leukocyte interferon. Immunobiology 1986; 172: 231-242. 3 Tyrrel D. Interferons and their clinical value. Rev Infect Dis 1982; 9: 243-248. 4 Merigon TC. Pharmacokinetics and side effects of interferon in man. Tex Rep BiolMed 1977; 35: 441-447. 'Fleischmann WR Jr, Fleischmann CM, Gindhart TD. Effect of hyperthermia on the antiproliferative activities of murine a, ~ and y-interferon: Differential enhancement of murine y-interferon. Cancer Res 1986; 46: 8-13. • Jordan GW, Fried RP, Merigan TC. Administration of human leukocyte interferon in herpes zoster. 1. Safety, circulating antiviral activity and host responses to interferon. J Infect Dis 1974; 130: 56-62. 7 Mogensen SC. Role of macrophages in natural resistance to virus infections. Microbiol Rev 1979; 43: 1-26. • Morahan PS, Connor JR, Learly 1. Viruses and the versatile macrophage. Br Med Bull 1985; 41: 15-25. • Nelson DS, Nelson M, Farram E, Inoue Y. Cancer and subversion of host defenses. Aust J Exp BioI Med Sci 1981; 59: 229-262. 10 Merendino RA, Iannello D, Arena A et al. Evaluation of macrophage antiviral activity in patients affected by neoplasia. Med Oncol Tumor Pharmacother 1988; 5: 191-197. " Merendino RA, Arena A, Liberto MC, Iannello D, Bonina L, Mesiti M, Mastroeni P. Influence of sera from patients affected by neoplasia on some human macrophage functions. Cancer Detect Prevent 1988; 12: 73-80. 12 Domke-Optiz I, Straub P, Kirchner H. Effect of interferon on replication of herpes simplex virus types 1 and 2 in human macrophages. J Virol 1986; 60: 37-42. 13 Harmer A. TNM-Classification of Malignant Tumors. 2nd ed. Union International Contre Ie Cancer (VICC), Geneve, 1978. 14 Clark WH Jr. A classification of malignant melanoma in man correlated with histogenesis and biological behaviour. In: Montagna W, Hu F, eds. Advances in Biology of Skin and the Pigmentary System. London: Pergamon Press, 1967: 621647. " Boyum A. Separation of leukocytes from blood and bone marrow. Scand J Clin Lab Invest 1968: 94: 196-202. 16 Daems WTH, Roos D, Van Der Rhee HJ. The subcellular distribution and biochemical properties of peroxidase in monocytes and macrophages. In: Dingle JT, Shaw IH, Jacques PJ, eds. Lysosomes in Applied Biology and Therapeutics. Amsterdam: Elsevier, North Holland, 1979: 463-514. 17 Dulbecco R. Production of plaques in monolayer tissue cultures. Proc Nat! Acad Sci USA 1952; 38: 747-752. 18 Mastroeni P, Merendino RA, Arena A, Bonina L, Liberto MC, Gazzara D. In vitro study on human mononuclear phagocytic cells behaviour versus DNA and RNA viruses. Giorn Batt Virol Immunol 1985; 78: 262-273. "Evans R. Tumor macrophages in host immunity to malignancies. In: Fink ·MA, eds. The macrophage in neoplasia. New York: Academic Press Inc, 1976: 27-42. 20 Iannello D, Bonina L, Merendino RA, Arena A, Mastroeni P, Bizzini B. Evaluation of Corynebacterium granulosum derived P40 fraction on macrophage anti-herpes simplex virus type 1 functions. Antiviral Res 1985; 1: 167-172. 21 Orchansky P, Rubinstein M, Fischer DG. The interferon-y receptor in human monocytes is different from the one in non-hematopoietic cells. J Immunol 1986; 136: 169173.