Journal of Chemotherapy
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Modulation of the Intrinsic Antiviral Activity by Escherichia coli Endotoxin in Macrophages from Patients with Neoplasia R.A. Merendino, A. Arena, G. Mancuso, S. Zummo, S. Chillemi, M. Mesiti & L. Bonina To cite this article: R.A. Merendino, A. Arena, G. Mancuso, S. Zummo, S. Chillemi, M. Mesiti & L. Bonina (1991) Modulation of the Intrinsic Antiviral Activity by Escherichia coli Endotoxin in Macrophages from Patients with Neoplasia, Journal of Chemotherapy, 3:1, 16-22, DOI: 10.1080/1120009X.1991.11739057 To link to this article: http://dx.doi.org/10.1080/1120009X.1991.11739057
Published online: 15 Jul 2016.
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Citing articles: 2 View citing articles
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Date: 20 August 2017, At: 15:30
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Journal of Chemotherapy
Vol. 3 - n. 1 (16-22) - 199 1
Modulation of the Intrinsic Antiviral Activity by Escherichia coli Endotoxin in Macrophages from Patients with Neoplasia R.A. MERENDINO - A . ARENA G . MANCUSO - S. ZUMMO - S. M . MESITI ,., - L. BONINA
CHILLEMI ~,
Summary - - - Macrophages from patients with breast cancer showed an impairment of their antiviral activity. The capability to hinder herpes simplex virus type 2 replication of macrophages from healthy donors and from patients with breast cancer was compared to the in-vitro treatment with Escherichia coli lipopolysaccharide (LPS). The LPS showed a dose-dependent effect on the different macrophage populations studied. Nevertheless, macrophages from healthy donors appeared to be more sensitive to LPS in comparison with macrophages from the patients under our observation. On these cells LPS treatment was not able to modify the antiviral property, when these macrophages were differentiated in autologous serum. Key words: LPS, macrophage, antiviral activity, neoplasia.
Instit ute of Microbiology and '' Institute o f Onco logy , Medical School, Uni versit y o f Messina , P.zza XX Se ttembre, 98 100 Messina, Italy. Correspondence: Prof. Rosaria Alba Mere ndino, Institut e of Med ical M icrobiology, Piazza XX Settembre 4, 98 100 Messina, It aly.
© Edizioni Ri visre Scientifiche - Firenze
INTRODUCTION
It is well known that endotoxins modify a number of immune functions in humans and have an effect on many different cell types including fibroblasts 1 , lymphocytes 2 and macrophages 3 • They serve as mitogens for B cell proliferation 4 and as adjuvants for antibody production 5 • They can stimulate properdin levels 6 , enhance resistance to infections\ and contribute to the regression of malignant disease in patients treated with bacterial extracts 8 . The endotoxin effect on macrophages is extremely varied since it induces the secretion of biologically active products such as IL-l 9 and increases glucosamine incorporation 10 and prostaglandin production 11 • Therefore the main effect of lipopolysaccharide (LPS) on macrophages is the activation of several biochemical pathways and functions, including the ability to restrict the replication of infectious agents. Among the immunocompetent cells involved in limiting the spreading of several viruses, the . cells belonging to the mononuclear-phagocytic system are of crucial importance 12 • 13 • In particular, the role of macro phages in the natural resistance of mice to herpes simplex virus infection has been well studied 14 • 1 5 • In an earlier study 16 we were able to demonstrate that macrophages derived from monocytes cultured in vitro, could restrict the growth of adenovirus, measles virus and HSV-2. However, macrophages from patients with breast cancer or melanoma had impaired antiviral activity_ Moreover we have shown that the intrinsic antiviral activity of macrophages appeared to be strongly affected by the multiplicity of infection 1 7 used and that the serum from patients ISSN 11 20-099X
17
MODULATION OF THE INTRIN SIC ANTIVIRAL ACTIVITY BY ESCI-IERICHTA COLI, ETC.
with neoplasia determined a marked impairment of the antiviral activity of macrophages obtained from healthy donors 18 . The aim of this work was to study the possibility of influencing in vitro the antiviral activity of macrophages from healthy adults and patients with neoplasia by the LPS obtained from Escherichia coli, strain 0128:B12.
MATERIALS AND METHODS
Downloaded by [Australian Catholic University] at 15:30 20 August 2017
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 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 an tibia tics. · All the sera and media were tested for endotoxin content using a colorimetric Limulus lysate gelation assay (PBI, Milan, Italy); samples that contained detectable levels of endotoxin were either absorbed overnight with polymixin B or discarded.
TABLE 1 -
Patient
A B
c
D
E F G H I L
Healthy donors M N 0 p
Q R
s T
u v
Information concerning the patients studied.
Sex
Age
Type of neoplasia ''
Stage (TNM) §
Ab
F F F F F F F F F F
62 56 58 61 55 55 60 57 60 61
I. D .C. I. D.C. I. D.C. !.D.C . !.D .C. !.D.C . !.D.C. !.D.C . !.D.C. !.D.C.
T2N-MO TlN-MO T2N -MO TlN-MO T3N-MO T2N-M1 T3N-M1 T3N-M1 T2N -M1 T2N-M1
32 32 16 64 4 4 8 64 32 16
F F F F F F F F F F
65 58 56 60 56 55 61 57 60 59
64 32 4 16 8 32 32 4 8 16
* !.D .C., infiltrating ductal carcinoma. § UICC classification: «Union Intern ati onal Centre le
Cancer».
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°C.
Patients Three groups of donors were studied: a control group of 10 healthy female adults; 5 patients with breast cancer, and 5 patients with breast cancer which had metastasized. All the patients studied were in post-menopause age. The patients had not yet undergone any kind of chemotherapy, hormonotherapy or radiotherapy. All groups were tested for the presence of circulating anti-HSV-2 antibodies by means of a «micro-ELISA kit» for HSV (PBI International, Milan, Italy). A summary of the clinical information concerning the patients studied is reported in Table 1. The classification for breast cancer is based on the «Union International Contre le Cancer» 19 •
Isolation of monocytes and culture of macrophages. Blood was drawn from volunteer donors with informed consent. Human monocytes were collected from venous blood of the donors described above. The blood was collected rapidly; added to RPMI 1640 (Gibco SpA, Milan, Italy) containing 10 IU/ml of preservative-free heparin (Sigma Chemical Company, Milan, Italy) and centrifuged on Ficoll Hypaque (Pharmacia SpA, Milan, Italy) gradients 20 • The mononuclear cells were washed twice in RPMI 1640 and resuspended in RPMI 1640 containing 10% heat inactivated fetal calf serum_ The cells were placed in plastic multi-well plates (Nunc, Milan, Italy) at a concentration of 5x1 0 5 per well. After 4h of incubation at 37°C in a 5% C02 atmosphere, non-adherent cells were removed by washing. The adherent cell population consisted of monocytes as judged by phagocytosis
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18
R. A. MERENDINO - A. ARENA - G. MA NCU SO - S. ZUMMO - S. CHILLEMI - M . MESITI - L. BON!N A
of Candida albicans, the presence of peroxidase activity in the cytoplasm 21 and by morphological criteria; the viability was 90% as assessed by trypan blue exclusion. The differentiation of monocytes into macrophages was followed for 10 days in culture 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 in serum from patients with breast cancer; c) monocytes from healthy donors differentiated in serum from patients with breast cancer which had metastasized; d) monocytes from patients with breast cancer differentiated in serum from healthy donors; e) monocytes from patients with breast cancer which had metastasized differentiated in serum from healthy donors; f) monocytes from patients with breast cancer differentiated in autologous serum; g) monocytes from patients with breast cancer 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 suspension, was retained on the plastic surface after 10 days of incubation. This percentage was independent of group donors studied. The media were changed every 3 days.
Intrinsic antiviral activity of macrophages The intrinsic antiviral activity is considered the mechanism by which macrophages are able to destroy viruses intracellularly 18 . On the basis of a previous study on the influence of serum factors on intrinsic antiviral activity of macrophages 17 , monocytes were differentiated in vitro in the presence of sera from patients with neoplasia and of sera from healthy donors. Then macrophages from different donors were infected with HSV-2 at a multiplicity of infection of 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 1h at 37°C; then the cells were washed three times with RPMI 1640, covered with 1ml of the same medium and incubated at 37°C in 5% C02. Samples were collected at different times post infection (5, 10, 20 hours), frozen and thawed three times to release intracellular virus and clarified by centrifugation at 1000 X g for 10 min. The virus suspension obtained was titrated on HEp-2 cells according to the method of Dulbecco 22 .
LPS preparations LPS, from Escherichia coli strain 0128:B12 extracted with phenol by the Westphal method, was purchased from Difco laboratoires (Detroit, Michigan USA).
LPS treatment Before treatment with LPS, macrophages were washed twice in RPMI 1640 medium. Then the mononuclear cells were incubated for 3h at 37°C in RPMI 1640 medium with LPS (0.1-1-10 Jlg/ml) . The medium was then removed and the cultures of macrophages were infected with HSV-2 at a multiplicity of infection (MOI) 0.1 and 2. HSV-2 was allowed to adsorb for 1h at 37°C, then the cells were washed three times with RPMI 1640, covered with 1 ml of the same medium with the different concentrations of LPS and incubated at 37°C in 5% C02. Samples were collected at different times (5, 10, 20 hours) post infection and frozen at - 80°C until virus titration was performed on permissive HEp-2.
Statistics Statistical evaluation was performed by analysis of variance.
RESULTS
Effects of neoplasia on macrophage behavior versus HSV-2. The capability of hindering HSV-2 replication of macrophages from healthy donors and from patients with breast cancer, was checked after the in vitro challenge with two different MOis 0.1 and 2.
19
MODULATION OF Til E I NTR INS IC ANTI VIR AL ACTIV ITY BY ESCJJ ER ICIIIA COLI, ETC.
A
5
c
4
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3
Figure 1. HSV-2 yield at different hours post-infection at a MOl 0.1 in macrophages under different conditions. Section A represe nts the untrea ted controls: M 0 from HD differenti ated in sHD (- - - •) ; M 0 fr om HD different iated in sBCo (- -- .A.) ; M 0 from HD differentiated in sBC 1 (-- - • ); M 0 from BCo differenti ated in sH D ( - - • ); M 0 from BC1 differe nti ated in sHD ( - - .A.); M 0 from BC 0 differe nti ated in sBCo (- - •) ; M 0 from BCl differenti ated in sBC l (- - X). The sec ti ons B, C, D represent the sa me conditions as in section A but in vitro trea ted respectively with 0.1, 1 and 10 J.L g/ml of LPS.
-E
2
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;-
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:::>
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5
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In Figure 1 A and 2 A the behaviors of different macrophage populations versus HSV-2 are illustrated. In particular, macrophages from healthy donors differentiated in autologous serum and infected with MOl 0 .1 completely res trict HSV-2; whereas macrophages from healthy donors and patients with breast cancer, differentiated under various conditions, replicate the virus. Among these populations (see materials and methods), differe nces in the degree of permissiveness versus HSV-2 were noted. In particular, the virus yield was significantly higher (P < 0.005) in condition BC 1 differentiated in sBC 1 compared to the control condition (HD differentiated in sHD). It is important to point out that when the macrophages were infected at MOl 2, no virus restriction was observed in all the conditions studied. Also in this case the degree of permissiveness varies according to the in vitro circumstances of differentiation.
EHect of LPS treatment on macrophage intrinsic antiviral activity. In Figures 1 B,C,D and 2 B,C,D, are reported the results of a series of experiments carried out
in order to assess whether in vitro treatmen t with LPS could influence the intrinsic antiviral activity of human macrophages. Figures Band C illustrate the effects of 0.1 and 1 1-lg/ml of LPS respectively on the antiviral function of different macrophage populations. It is possible to evidence an improvement in an tiviral ac tivity of macrophages from patients with breas t cancer and patients who had metas tasized , differentiated under different conditions. This improvement is particularly evident on macrophages from healthy donors differentiated in sera from patients affected by breast cancer (P < 0.005) . The effect of LPS treatment is dose-dependent , in fact , as reported in section D of the Figu res, the highes t ac tivation of viral restriction being noted at 10 1-lg/ml of LPS. In particular in Figure 1D the macrophages from healthy donors under different conditions were able to completely hinder the virus replication . Macrophages from healthy donors appeared to be more sensitive to LPS in comparison with macrophages from patients (P < 0 .002). Nevertheless, LPS treatment of macrophages from patients, differentiated in autologous serum, did not influence the antiviral ac tivity of these cells. In Figures 1 and 2 it is possible to illustrate
20
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the overall feature of the importance of the MOI on the ability of macrophages to restrict HSV-2, as well as on the modulatory effect of LPS on this function . DISCUSSIO N
The experiments reported in this paper were carried out in order to study the effect of in vitro treatment with LPS on the intrinsic antiviral activity of macrophages from healthy donors or patients with breast cancer. It is well known that macrophages play a crucial role in the defence of the host against many infectious agents, and in particular against several viruses 12 • On the other hand, it has been repeatedly reported that several functions of immunocompetent cells such as macrophages are compromised in animals bearing experimental tumors and in patients with neoplasia 23 • The depression of functions of macrophages in the host affected by neoplastic diseases can be a factor that facilitates the development of infections by opportunistic microorganisms. Previous studies, carried out using the experimental model reported in this paper, had confirmed that several funct.ions of macro-
20 h
Figure 2. HSV-2 yield at different hours post-infection at a MOl 2 in macrophages under different conditions. Section A represe nts the un treated controls: M 0 from HD differentiated in sHD (- - - •); M 0 from HD differentiated sBCo(- - - .A.; M0 from HD differentiated in sBC1 (- -- •); M 0 from BCo differenti ated in sHD (- - •); M 0 from BC 1 differentiated in sHD (- - .A.) ; M 0 from BCo differentiated in sBC 0 (- - •); M0 from BC1 differenti ated in sBC 1 (- - X). The sections B, C, D represent the sa me conditions as in section A but in vitro treated res pectively with 0.1, 1 and 10 ~ g/ml of LPS.
phages were severely impaired in patients with breast cancer or melanoma. In particular, phagocytic and microbicidal activities as well as antiviral activity were significantly lower with respect to healthy donors 1 6 • 18 • Moreover, the results obtained in our previous studies supported the repeated demonstration of circulating inhibitory factors in the host affected by neoplasia 18 • Tumor extracts and sera from patients with neoplasia inhibit numerous functions of mononuclear-phagocytic cells. Such inhibitory activity has been alternatively attributed to small molecular weight peptides , prostaglandins and other less defined substances 23 • 24 • On this basis, and in consideration of the modulatory role of LPS on immunocompetent cell function 2 5 , the intrinsic antiviral activity of macrophages from patients affected by breast cancer, differentiated in the presence of autologous serum or in serum from healthy donors, was studied in comparison with macrophages from healthy donors. The experiments described in this paper demonstrate that the presence of LPS can restore the intrinsic antiviral activity of macrophages obtained from patients with neoplasia. In fact, the incubation with 10
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MODULATION OP Til E INTRINSIC ANTIVIRAL ACTIVITY BY ESCJ/ERICJ/IA COLI, ETC.
and 1 ~g/ml of LPS, during viral infection at MOl 0.1, significan tly improved the antiviral activity of macrophages obtained from healthy donors and patients with breast cancer and which had metastasized, differentiated in vitro under different conditions. In contrast, at MOl 2 this improvement was evident only on macrophages from healthy donors, differentiated under different conditions, and from patients with neoplasia, differentiated in the presence of serum of healthy donors, but not in autologous serum. Therefore, the antiviral activity of cells belonging to the mononuclear phagocytic system seems to be markedly influenced by factors such as the multiplicity of infection used 17 and the presence of circulating inhibitory factors 18 • The inhibitory factors present in serum from patients with neoplasia have not been characterized in our study. Even though it has not yet been completely elucidated, the interaction of LPS with mononuclear phagocytes can probably be attributed to the presence of specific receptors on the membrane of these cells 26 • The consequence of this interaction is the triggering of mechanisms ultimately leading to the activation of macrophages. It is possible, therefore, to hypothesize, at this stage of our studies, that the interaction of macrophages from patients differentiated in the presence of autologous serum with a well known activating stimulus such as LPS, is different with respect to normal macrophages and macrophages from patients with neoplasia, differen tiated in the presence of sera from healthy donors. This difference was particularly evident when macrophages were infected at MOl 2. This study was undertaken on a homogeneous population 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, the behavior of macrophages seems to be independent with respect to the presence of circulating antibody vs HSV-2, in controls as well as in neoplastic patients, as reported in Table 1. The multifarious effects of LPS represent an intriguing problem, particularly on the phagocytic cells endowed with the capacity to resist and control neoplasia and pathogens.
21
ACKNOW LEDGMENTS - This work was supported by grant 87 .01349.44 and 88. 0074 8.44 fr om th e Italian Co nsigli o Nazionale delle Ri ce rche.
REFEREN CES ' Vaheri A, Ruoslahti E, Sarvas M, Nurminen M . Mitogenic effect by lipopolysaccharide and pokeweed lec tin on density- inhibited chick embryo fibrobla sts. J E xp Med 19 73; 138: 1356-1364. 2 Andersso n J , Melch ers F, G alanos C, Luderit z 0 . The mitoge ni c affec t o f lipopolysacch aride o n bone marrow-d erived mou se lymph ocy tes. ] E xp Med 197 3; 137 : 943 -953. 1 Wahl LM , W ahl SM , Merge nh age n SE, M artin GR . Co ll agenase production by endo tox in -ac ti va ted mac roph ages. Proc Nat! Acad Sci 197 4 ; 7 1: 3598-3606. • G ery I , Kruger K , Spiese l SZ. Stimulati on of B-lymphocytes by endotoxin: reactions of thymu s-d eprived mi ce and karyot ype analy sis of dividing cells in mi ce bearing T6 T6 th ymu s graft s. ] Immunol 19 72; 108: 1088- 109 1. ' Neter E. Endoroxins and the immune res ponse. Curr Top Microbia l Immunol 1969; 47 : 82-90 . ' Pillemer L , Schoe nberg MD , Blum L, Wurz L. Properdin sys tem and immunity. II . In te rac tion o f the properdin system with polysaccharides . Science 1955 ; 122: 54 5-5 56 . 7 Landy M , Pillemer L. Increased resistance to infectio n and accompanying alteratio n in properdin levels follow ing admini str ation of bacteri al lipopolysaccharides. J Exp M ed 195 6; 104 : 383-4 0 9. ' Pelner L. H os t-tumor ant ago ni sm. XV. Th e apparent benefici al effects of acute bacteri al infec tions or of toxin th erapy on th e course of malignant melanoma. ] Am G eri atr Soc 1960; 8: 378-382. ' Cavaillon JM , Fitting C, C aro ff M , Ca vaill o n NH . Dissoc iati on o f cell -associ ated Interleukin-l (IL-l) and IL-l release induced by lipopolysaccharide and Lipid A . Infect Immun 1989; 57 : 79 1-797. 0 ' WilronJM , Rosenstreich DL, Oppe nheim]. Ac ti va tio n of guinea pig macrophages by bacterial lipopoly saccharide requires bone marrow d erived lymphocy tes. ] Immunol 1975; 114: 388-393. " Rosenstreich DL, Gla de LM, W ahl LM, Sandberg AL, Mergc nh agen SE. Anal ysis of the cellular defects ef end oto xin unres ponsive C3 H/HeJ mice . In: D . Schless inger eds. Microbi ology. Am erican Soc iety for Microbiology, W ashingto n. D C ; 1977 ; 314-320 . 12 Mogense n SC. Role o f macrophages in natural res istance to virus infections. Microbia l Rev 19 79: 43 : 1-26. 13 Morahan PS , Morse SS , McGeorge MB . M acroph age ex trin sic anti viral acti vity during herpes simplex virus infection. ] Gen Vira l 1980; 46 : 29 1-300 . 14 Kirchner H . Immunobiology of infec ti o n with herpes simplex virus. Monogr Vira l 1982; 13: 1-28. " Pederse n EB , H aahr S, Mogense n SC. X -Linked res istance o f mice to high doses o f herpes simplex virus type 2 correlates with ea rl y interfero n producti on . Infec t Immun 1983 ; 42 : 740-74 6. •• M as troeni P , Merendino RA , Are na A, Bonina L, Liberto MC , G azz ara D. «In vitro>> study on hum an mononu clear ph agocy ti c cells behavi our versus D N A and RNA viruses. Gi or Batt Vira l Immun ol 1985; LXX VIII : 262-273. 17 Merendino RA , I ann ello D , Arena A, et a!. Eva lu ati on of macrophage antiviral ac tivity in patie nts affec ted by neoplasia . Med Oneal Tumor Ph armaco ther 1988; 5: 19 1-19 7. " Merendino RA , Arena A , Liberto MC, Iannello D ,
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R.A. MERENDINO • A. ARENA
G. MANCUSO · S. ZUMMO · S. CHILLEMI - M. MESITI - L. BONINA
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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 . 19 Harmer ed. TNM-Classification of malignant tumors, 2nd edn. Geneva, Union International Contre le Cancer (UICC) 1978. 20 Boyum A. Separation of leucocytes from blood and bone marrow . Scand J Clin Lab Invest 1968; 94: 196-202. 21 Daems WTH, Roos D, Vander Rhee HJ. The subcellular distribution and biochemical properties of peroxidase in monocytes and macrophages. In: Dingle JT, Shaw IH, J acques PJ eds. Lysosomes in Applied Biology and Therapeutics. Amsterdam: Elsevier North Holland; 1979: p. 463-514. 22 Dulbecco R. Production of plaques in monolayer tissue
cultures by single particles of an anim al virus. Proc Nat! Acad Sci USA 1952; 38: 747-752. "Nelson DS , Nelson M, Farram E, Inoe Y. Cancer and subversion of host defences. Aust J Exp Bioi Med Sci 198 1; 59: 229-262 . " Nelson DS , Nelson M, Inoue Y. Some aspec ts of macrophage-tumor relationships. In Reichard S, Kojima M, eds. Macrophage biology. New York: Alan R Liss, Inc 1985; 833844. " Morrison DC , Ryan JL. Bacterial endotoxins and hos t immune responses. Adv Immunol 1979; 28: 293-450. "Wright SD, Jong MTC. Adhesion-promoting receptors on human macrophages recogni ze Escherichia coli by binding to lipopolysaccharide. J Exp Med 1986; 164: 1876-1888.
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Modulation of the Intrinsic Antiviral Activity by Escherichia coli Endotoxin in Macrophages from Patients with Neoplasia R.A. Merendino, A. Arena, G. Mancuso, S. Zummo, S. Chillemi, M. Mesiti & L. Bonina To cite this article: R.A. Merendino, A. Arena, G. Mancuso, S. Zummo, S. Chillemi, M. Mesiti & L. Bonina (1991) Modulation of the Intrinsic Antiviral Activity by Escherichia coli Endotoxin in Macrophages from Patients with Neoplasia, Journal of Chemotherapy, 3:1, 16-22, DOI: 10.1080/1120009X.1991.11739057 To link to this article: http://dx.doi.org/10.1080/1120009X.1991.11739057
Published online: 15 Jul 2016.
Submit your article to this journal
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yjoc20 Download by: [Australian Catholic University]
Date: 20 August 2017, At: 15:30
Downloaded by [Australian Catholic University] at 15:30 20 August 2017
Journal of Chemotherapy
Vol. 3 - n. 1 (16-22) - 199 1
Modulation of the Intrinsic Antiviral Activity by Escherichia coli Endotoxin in Macrophages from Patients with Neoplasia R.A. MERENDINO - A . ARENA G . MANCUSO - S. ZUMMO - S. M . MESITI ,., - L. BONINA
CHILLEMI ~,
Summary - - - Macrophages from patients with breast cancer showed an impairment of their antiviral activity. The capability to hinder herpes simplex virus type 2 replication of macrophages from healthy donors and from patients with breast cancer was compared to the in-vitro treatment with Escherichia coli lipopolysaccharide (LPS). The LPS showed a dose-dependent effect on the different macrophage populations studied. Nevertheless, macrophages from healthy donors appeared to be more sensitive to LPS in comparison with macrophages from the patients under our observation. On these cells LPS treatment was not able to modify the antiviral property, when these macrophages were differentiated in autologous serum. Key words: LPS, macrophage, antiviral activity, neoplasia.
Instit ute of Microbiology and '' Institute o f Onco logy , Medical School, Uni versit y o f Messina , P.zza XX Se ttembre, 98 100 Messina, Italy. Correspondence: Prof. Rosaria Alba Mere ndino, Institut e of Med ical M icrobiology, Piazza XX Settembre 4, 98 100 Messina, It aly.
© Edizioni Ri visre Scientifiche - Firenze
INTRODUCTION
It is well known that endotoxins modify a number of immune functions in humans and have an effect on many different cell types including fibroblasts 1 , lymphocytes 2 and macrophages 3 • They serve as mitogens for B cell proliferation 4 and as adjuvants for antibody production 5 • They can stimulate properdin levels 6 , enhance resistance to infections\ and contribute to the regression of malignant disease in patients treated with bacterial extracts 8 . The endotoxin effect on macrophages is extremely varied since it induces the secretion of biologically active products such as IL-l 9 and increases glucosamine incorporation 10 and prostaglandin production 11 • Therefore the main effect of lipopolysaccharide (LPS) on macrophages is the activation of several biochemical pathways and functions, including the ability to restrict the replication of infectious agents. Among the immunocompetent cells involved in limiting the spreading of several viruses, the . cells belonging to the mononuclear-phagocytic system are of crucial importance 12 • 13 • In particular, the role of macro phages in the natural resistance of mice to herpes simplex virus infection has been well studied 14 • 1 5 • In an earlier study 16 we were able to demonstrate that macrophages derived from monocytes cultured in vitro, could restrict the growth of adenovirus, measles virus and HSV-2. However, macrophages from patients with breast cancer or melanoma had impaired antiviral activity_ Moreover we have shown that the intrinsic antiviral activity of macrophages appeared to be strongly affected by the multiplicity of infection 1 7 used and that the serum from patients ISSN 11 20-099X
17
MODULATION OF THE INTRIN SIC ANTIVIRAL ACTIVITY BY ESCI-IERICHTA COLI, ETC.
with neoplasia determined a marked impairment of the antiviral activity of macrophages obtained from healthy donors 18 . The aim of this work was to study the possibility of influencing in vitro the antiviral activity of macrophages from healthy adults and patients with neoplasia by the LPS obtained from Escherichia coli, strain 0128:B12.
MATERIALS AND METHODS
Downloaded by [Australian Catholic University] at 15:30 20 August 2017
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 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 an tibia tics. · All the sera and media were tested for endotoxin content using a colorimetric Limulus lysate gelation assay (PBI, Milan, Italy); samples that contained detectable levels of endotoxin were either absorbed overnight with polymixin B or discarded.
TABLE 1 -
Patient
A B
c
D
E F G H I L
Healthy donors M N 0 p
Q R
s T
u v
Information concerning the patients studied.
Sex
Age
Type of neoplasia ''
Stage (TNM) §
Ab
F F F F F F F F F F
62 56 58 61 55 55 60 57 60 61
I. D .C. I. D.C. I. D.C. !.D.C . !.D .C. !.D.C . !.D.C. !.D.C . !.D.C. !.D.C.
T2N-MO TlN-MO T2N -MO TlN-MO T3N-MO T2N-M1 T3N-M1 T3N-M1 T2N -M1 T2N-M1
32 32 16 64 4 4 8 64 32 16
F F F F F F F F F F
65 58 56 60 56 55 61 57 60 59
64 32 4 16 8 32 32 4 8 16
* !.D .C., infiltrating ductal carcinoma. § UICC classification: «Union Intern ati onal Centre le
Cancer».
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°C.
Patients Three groups of donors were studied: a control group of 10 healthy female adults; 5 patients with breast cancer, and 5 patients with breast cancer which had metastasized. All the patients studied were in post-menopause age. The patients had not yet undergone any kind of chemotherapy, hormonotherapy or radiotherapy. All groups were tested for the presence of circulating anti-HSV-2 antibodies by means of a «micro-ELISA kit» for HSV (PBI International, Milan, Italy). A summary of the clinical information concerning the patients studied is reported in Table 1. The classification for breast cancer is based on the «Union International Contre le Cancer» 19 •
Isolation of monocytes and culture of macrophages. Blood was drawn from volunteer donors with informed consent. Human monocytes were collected from venous blood of the donors described above. The blood was collected rapidly; added to RPMI 1640 (Gibco SpA, Milan, Italy) containing 10 IU/ml of preservative-free heparin (Sigma Chemical Company, Milan, Italy) and centrifuged on Ficoll Hypaque (Pharmacia SpA, Milan, Italy) gradients 20 • The mononuclear cells were washed twice in RPMI 1640 and resuspended in RPMI 1640 containing 10% heat inactivated fetal calf serum_ The cells were placed in plastic multi-well plates (Nunc, Milan, Italy) at a concentration of 5x1 0 5 per well. After 4h of incubation at 37°C in a 5% C02 atmosphere, non-adherent cells were removed by washing. The adherent cell population consisted of monocytes as judged by phagocytosis
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of Candida albicans, the presence of peroxidase activity in the cytoplasm 21 and by morphological criteria; the viability was 90% as assessed by trypan blue exclusion. The differentiation of monocytes into macrophages was followed for 10 days in culture 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 in serum from patients with breast cancer; c) monocytes from healthy donors differentiated in serum from patients with breast cancer which had metastasized; d) monocytes from patients with breast cancer differentiated in serum from healthy donors; e) monocytes from patients with breast cancer which had metastasized differentiated in serum from healthy donors; f) monocytes from patients with breast cancer differentiated in autologous serum; g) monocytes from patients with breast cancer 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 suspension, was retained on the plastic surface after 10 days of incubation. This percentage was independent of group donors studied. The media were changed every 3 days.
Intrinsic antiviral activity of macrophages The intrinsic antiviral activity is considered the mechanism by which macrophages are able to destroy viruses intracellularly 18 . On the basis of a previous study on the influence of serum factors on intrinsic antiviral activity of macrophages 17 , monocytes were differentiated in vitro in the presence of sera from patients with neoplasia and of sera from healthy donors. Then macrophages from different donors were infected with HSV-2 at a multiplicity of infection of 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 1h at 37°C; then the cells were washed three times with RPMI 1640, covered with 1ml of the same medium and incubated at 37°C in 5% C02. Samples were collected at different times post infection (5, 10, 20 hours), frozen and thawed three times to release intracellular virus and clarified by centrifugation at 1000 X g for 10 min. The virus suspension obtained was titrated on HEp-2 cells according to the method of Dulbecco 22 .
LPS preparations LPS, from Escherichia coli strain 0128:B12 extracted with phenol by the Westphal method, was purchased from Difco laboratoires (Detroit, Michigan USA).
LPS treatment Before treatment with LPS, macrophages were washed twice in RPMI 1640 medium. Then the mononuclear cells were incubated for 3h at 37°C in RPMI 1640 medium with LPS (0.1-1-10 Jlg/ml) . The medium was then removed and the cultures of macrophages were infected with HSV-2 at a multiplicity of infection (MOI) 0.1 and 2. HSV-2 was allowed to adsorb for 1h at 37°C, then the cells were washed three times with RPMI 1640, covered with 1 ml of the same medium with the different concentrations of LPS and incubated at 37°C in 5% C02. Samples were collected at different times (5, 10, 20 hours) post infection and frozen at - 80°C until virus titration was performed on permissive HEp-2.
Statistics Statistical evaluation was performed by analysis of variance.
RESULTS
Effects of neoplasia on macrophage behavior versus HSV-2. The capability of hindering HSV-2 replication of macrophages from healthy donors and from patients with breast cancer, was checked after the in vitro challenge with two different MOis 0.1 and 2.
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MODULATION OF Til E I NTR INS IC ANTI VIR AL ACTIV ITY BY ESCJJ ER ICIIIA COLI, ETC.
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Figure 1. HSV-2 yield at different hours post-infection at a MOl 0.1 in macrophages under different conditions. Section A represe nts the untrea ted controls: M 0 from HD differenti ated in sHD (- - - •) ; M 0 fr om HD different iated in sBCo (- -- .A.) ; M 0 from HD differentiated in sBC 1 (-- - • ); M 0 from BCo differenti ated in sH D ( - - • ); M 0 from BC1 differe nti ated in sHD ( - - .A.); M 0 from BC 0 differe nti ated in sBCo (- - •) ; M 0 from BCl differenti ated in sBC l (- - X). The sec ti ons B, C, D represent the sa me conditions as in section A but in vitro trea ted respectively with 0.1, 1 and 10 J.L g/ml of LPS.
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In Figure 1 A and 2 A the behaviors of different macrophage populations versus HSV-2 are illustrated. In particular, macrophages from healthy donors differentiated in autologous serum and infected with MOl 0 .1 completely res trict HSV-2; whereas macrophages from healthy donors and patients with breast cancer, differentiated under various conditions, replicate the virus. Among these populations (see materials and methods), differe nces in the degree of permissiveness versus HSV-2 were noted. In particular, the virus yield was significantly higher (P < 0.005) in condition BC 1 differentiated in sBC 1 compared to the control condition (HD differentiated in sHD). It is important to point out that when the macrophages were infected at MOl 2, no virus restriction was observed in all the conditions studied. Also in this case the degree of permissiveness varies according to the in vitro circumstances of differentiation.
EHect of LPS treatment on macrophage intrinsic antiviral activity. In Figures 1 B,C,D and 2 B,C,D, are reported the results of a series of experiments carried out
in order to assess whether in vitro treatmen t with LPS could influence the intrinsic antiviral activity of human macrophages. Figures Band C illustrate the effects of 0.1 and 1 1-lg/ml of LPS respectively on the antiviral function of different macrophage populations. It is possible to evidence an improvement in an tiviral ac tivity of macrophages from patients with breas t cancer and patients who had metas tasized , differentiated under different conditions. This improvement is particularly evident on macrophages from healthy donors differentiated in sera from patients affected by breast cancer (P < 0.005) . The effect of LPS treatment is dose-dependent , in fact , as reported in section D of the Figu res, the highes t ac tivation of viral restriction being noted at 10 1-lg/ml of LPS. In particular in Figure 1D the macrophages from healthy donors under different conditions were able to completely hinder the virus replication . Macrophages from healthy donors appeared to be more sensitive to LPS in comparison with macrophages from patients (P < 0 .002). Nevertheless, LPS treatment of macrophages from patients, differentiated in autologous serum, did not influence the antiviral ac tivity of these cells. In Figures 1 and 2 it is possible to illustrate
20
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the overall feature of the importance of the MOI on the ability of macrophages to restrict HSV-2, as well as on the modulatory effect of LPS on this function . DISCUSSIO N
The experiments reported in this paper were carried out in order to study the effect of in vitro treatment with LPS on the intrinsic antiviral activity of macrophages from healthy donors or patients with breast cancer. It is well known that macrophages play a crucial role in the defence of the host against many infectious agents, and in particular against several viruses 12 • On the other hand, it has been repeatedly reported that several functions of immunocompetent cells such as macrophages are compromised in animals bearing experimental tumors and in patients with neoplasia 23 • The depression of functions of macrophages in the host affected by neoplastic diseases can be a factor that facilitates the development of infections by opportunistic microorganisms. Previous studies, carried out using the experimental model reported in this paper, had confirmed that several funct.ions of macro-
20 h
Figure 2. HSV-2 yield at different hours post-infection at a MOl 2 in macrophages under different conditions. Section A represe nts the un treated controls: M 0 from HD differentiated in sHD (- - - •); M 0 from HD differentiated sBCo(- - - .A.; M0 from HD differentiated in sBC1 (- -- •); M 0 from BCo differenti ated in sHD (- - •); M 0 from BC 1 differentiated in sHD (- - .A.) ; M 0 from BCo differentiated in sBC 0 (- - •); M0 from BC1 differenti ated in sBC 1 (- - X). The sections B, C, D represent the sa me conditions as in section A but in vitro treated res pectively with 0.1, 1 and 10 ~ g/ml of LPS.
phages were severely impaired in patients with breast cancer or melanoma. In particular, phagocytic and microbicidal activities as well as antiviral activity were significantly lower with respect to healthy donors 1 6 • 18 • Moreover, the results obtained in our previous studies supported the repeated demonstration of circulating inhibitory factors in the host affected by neoplasia 18 • Tumor extracts and sera from patients with neoplasia inhibit numerous functions of mononuclear-phagocytic cells. Such inhibitory activity has been alternatively attributed to small molecular weight peptides , prostaglandins and other less defined substances 23 • 24 • On this basis, and in consideration of the modulatory role of LPS on immunocompetent cell function 2 5 , the intrinsic antiviral activity of macrophages from patients affected by breast cancer, differentiated in the presence of autologous serum or in serum from healthy donors, was studied in comparison with macrophages from healthy donors. The experiments described in this paper demonstrate that the presence of LPS can restore the intrinsic antiviral activity of macrophages obtained from patients with neoplasia. In fact, the incubation with 10
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MODULATION OP Til E INTRINSIC ANTIVIRAL ACTIVITY BY ESCJ/ERICJ/IA COLI, ETC.
and 1 ~g/ml of LPS, during viral infection at MOl 0.1, significan tly improved the antiviral activity of macrophages obtained from healthy donors and patients with breast cancer and which had metastasized, differentiated in vitro under different conditions. In contrast, at MOl 2 this improvement was evident only on macrophages from healthy donors, differentiated under different conditions, and from patients with neoplasia, differentiated in the presence of serum of healthy donors, but not in autologous serum. Therefore, the antiviral activity of cells belonging to the mononuclear phagocytic system seems to be markedly influenced by factors such as the multiplicity of infection used 17 and the presence of circulating inhibitory factors 18 • The inhibitory factors present in serum from patients with neoplasia have not been characterized in our study. Even though it has not yet been completely elucidated, the interaction of LPS with mononuclear phagocytes can probably be attributed to the presence of specific receptors on the membrane of these cells 26 • The consequence of this interaction is the triggering of mechanisms ultimately leading to the activation of macrophages. It is possible, therefore, to hypothesize, at this stage of our studies, that the interaction of macrophages from patients differentiated in the presence of autologous serum with a well known activating stimulus such as LPS, is different with respect to normal macrophages and macrophages from patients with neoplasia, differen tiated in the presence of sera from healthy donors. This difference was particularly evident when macrophages were infected at MOl 2. This study was undertaken on a homogeneous population 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, the behavior of macrophages seems to be independent with respect to the presence of circulating antibody vs HSV-2, in controls as well as in neoplastic patients, as reported in Table 1. The multifarious effects of LPS represent an intriguing problem, particularly on the phagocytic cells endowed with the capacity to resist and control neoplasia and pathogens.
21
ACKNOW LEDGMENTS - This work was supported by grant 87 .01349.44 and 88. 0074 8.44 fr om th e Italian Co nsigli o Nazionale delle Ri ce rche.
REFEREN CES ' Vaheri A, Ruoslahti E, Sarvas M, Nurminen M . Mitogenic effect by lipopolysaccharide and pokeweed lec tin on density- inhibited chick embryo fibrobla sts. J E xp Med 19 73; 138: 1356-1364. 2 Andersso n J , Melch ers F, G alanos C, Luderit z 0 . The mitoge ni c affec t o f lipopolysacch aride o n bone marrow-d erived mou se lymph ocy tes. ] E xp Med 197 3; 137 : 943 -953. 1 Wahl LM , W ahl SM , Merge nh age n SE, M artin GR . Co ll agenase production by endo tox in -ac ti va ted mac roph ages. Proc Nat! Acad Sci 197 4 ; 7 1: 3598-3606. • G ery I , Kruger K , Spiese l SZ. Stimulati on of B-lymphocytes by endotoxin: reactions of thymu s-d eprived mi ce and karyot ype analy sis of dividing cells in mi ce bearing T6 T6 th ymu s graft s. ] Immunol 19 72; 108: 1088- 109 1. ' Neter E. Endoroxins and the immune res ponse. Curr Top Microbia l Immunol 1969; 47 : 82-90 . ' Pillemer L , Schoe nberg MD , Blum L, Wurz L. Properdin sys tem and immunity. II . In te rac tion o f the properdin system with polysaccharides . Science 1955 ; 122: 54 5-5 56 . 7 Landy M , Pillemer L. Increased resistance to infectio n and accompanying alteratio n in properdin levels follow ing admini str ation of bacteri al lipopolysaccharides. J Exp M ed 195 6; 104 : 383-4 0 9. ' Pelner L. H os t-tumor ant ago ni sm. XV. Th e apparent benefici al effects of acute bacteri al infec tions or of toxin th erapy on th e course of malignant melanoma. ] Am G eri atr Soc 1960; 8: 378-382. ' Cavaillon JM , Fitting C, C aro ff M , Ca vaill o n NH . Dissoc iati on o f cell -associ ated Interleukin-l (IL-l) and IL-l release induced by lipopolysaccharide and Lipid A . Infect Immun 1989; 57 : 79 1-797. 0 ' WilronJM , Rosenstreich DL, Oppe nheim]. Ac ti va tio n of guinea pig macrophages by bacterial lipopoly saccharide requires bone marrow d erived lymphocy tes. ] Immunol 1975; 114: 388-393. " Rosenstreich DL, Gla de LM, W ahl LM, Sandberg AL, Mergc nh agen SE. Anal ysis of the cellular defects ef end oto xin unres ponsive C3 H/HeJ mice . In: D . Schless inger eds. Microbi ology. Am erican Soc iety for Microbiology, W ashingto n. D C ; 1977 ; 314-320 . 12 Mogense n SC. Role o f macrophages in natural res istance to virus infections. Microbia l Rev 19 79: 43 : 1-26. 13 Morahan PS , Morse SS , McGeorge MB . M acroph age ex trin sic anti viral acti vity during herpes simplex virus infection. ] Gen Vira l 1980; 46 : 29 1-300 . 14 Kirchner H . Immunobiology of infec ti o n with herpes simplex virus. Monogr Vira l 1982; 13: 1-28. " Pederse n EB , H aahr S, Mogense n SC. X -Linked res istance o f mice to high doses o f herpes simplex virus type 2 correlates with ea rl y interfero n producti on . Infec t Immun 1983 ; 42 : 740-74 6. •• M as troeni P , Merendino RA , Are na A, Bonina L, Liberto MC , G azz ara D. «In vitro>> study on hum an mononu clear ph agocy ti c cells behavi our versus D N A and RNA viruses. Gi or Batt Vira l Immun ol 1985; LXX VIII : 262-273. 17 Merendino RA , I ann ello D , Arena A, et a!. Eva lu ati on of macrophage antiviral ac tivity in patie nts affec ted by neoplasia . Med Oneal Tumor Ph armaco ther 1988; 5: 19 1-19 7. " Merendino RA , Arena A , Liberto MC, Iannello D ,
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G. MANCUSO · S. ZUMMO · S. CHILLEMI - M. MESITI - L. BONINA
Downloaded by [Australian Catholic University] at 15:30 20 August 2017
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 . 19 Harmer ed. TNM-Classification of malignant tumors, 2nd edn. Geneva, Union International Contre le Cancer (UICC) 1978. 20 Boyum A. Separation of leucocytes from blood and bone marrow . Scand J Clin Lab Invest 1968; 94: 196-202. 21 Daems WTH, Roos D, Vander Rhee HJ. The subcellular distribution and biochemical properties of peroxidase in monocytes and macrophages. In: Dingle JT, Shaw IH, J acques PJ eds. Lysosomes in Applied Biology and Therapeutics. Amsterdam: Elsevier North Holland; 1979: p. 463-514. 22 Dulbecco R. Production of plaques in monolayer tissue
cultures by single particles of an anim al virus. Proc Nat! Acad Sci USA 1952; 38: 747-752. "Nelson DS , Nelson M, Farram E, Inoe Y. Cancer and subversion of host defences. Aust J Exp Bioi Med Sci 198 1; 59: 229-262 . " Nelson DS , Nelson M, Inoue Y. Some aspec ts of macrophage-tumor relationships. In Reichard S, Kojima M, eds. Macrophage biology. New York: Alan R Liss, Inc 1985; 833844. " Morrison DC , Ryan JL. Bacterial endotoxins and hos t immune responses. Adv Immunol 1979; 28: 293-450. "Wright SD, Jong MTC. Adhesion-promoting receptors on human macrophages recogni ze Escherichia coli by binding to lipopolysaccharide. J Exp Med 1986; 164: 1876-1888.
