Immunology 1990 69 402-408

Differential effects of LPS, IFN-y and TNFa on the secretion of lysozyme by individual human mononuclear phagocytes: relationship to cell maturity C. E. LEWIS, S. P. McCARTHY, J. LORENZEN & J. O'D. McGEE Nuffield Department of Pathology and Bacteriology, University of Oxford, John Radeliffe Hospital, Headington, Oxford Acceptedfor publication 29 November 1989

SUMMARY Human mononuclear phagocytes can be activated to perform a variety of complex functions by exposure to the immunomodulators, lipopolysaccharide (LPS), interferon-gamma (IFN-y) and tumour necrosis factor alpha (TNFa). Although such activation often involves the release of various cytokines by monocytes and macrophages, little is known of the effects of such signals on their secretion of lysozyme (LZM). In this study, a reverse haemolytic plaque assay for LZM secretion is coupled with immunocytochemistry for the pan macrophage (CD68) marker, EBM/1 1. This enabled the direct effects of LPS, IFN-y and TNFa on the secretion of LZM by individual, immunoidentified human mononuclear phagocytes to be investigated. The overall secretion of this peptide by populations of freshly isolated or 3-day cultured monocytes was augmented by exposure for 6 hr to bacterial LPS, recombinant human IFN-y or recombinant human TNFaC. Extension of the culture period for monocytes from 3 to 7 days prior to use in the assay resulted in higher levels of LZM secretion, which could be further increased by TNFa but not by LPS or IFN-y. Individual peritoneal macrophages activated by inflammation in vivo were uniform in their augmented LZM responses to TNFa, but a small subpopulation of human peritoneal macrophages, which may represent younger 'inflammatory' exudate macrophages, was seen to be preferentially responsive to the LZMstimulating effects of LPS and IFN-y. These studies suggest that (i) secretion of LZM by human mononuclear phagocytes can be regulated by LPS and IFN-y, although the effects of these agents may be dependent upon the state of maturation and/or differentiation of the cells, and (ii) TNFac is a potent stimulant of LZM secretion by monocytes and macrophages irrespective of cell maturity.

feron-gamma (IFN-y) and tumour necrosis factor a (TNFa), which act either independently, sequentially or simultaneously to elicit various states of activation (Hamblin, 1988; Hamilton, 1988). Monocytes and macrophages have a remarkable secretory repertoire, with most of these products being released only in response to specific extracellular signals. By contrast, fibronectin (Fn) and the anti-bacterial enzyme, lysozyme (LZM), are secreted continuously by macrophages in the absence of exogenous stimulation (Gordon, Todd & Cohn, 1974; Johansson et al., 1979). However, the finding that both LPS and IFN-y modulate the secretion of Fn by rodent peritoneal macrophages (Cofano et al., 1984; Trotter, Beezhold & Lause, 1989) has fuelled speculation that these stimulants may also influence the secretion of other so-called constitutively secreted products by mononuclear phagocytes, such as LZM. Indeed, lengthy exposure to LPS and IFN (a and ,B) has been shown to modulate the secretion of this product by murine peritoneal macrophages (Warfel & Zucker-Franklin, 1986). To date, most studies have measured LZM using a bio-assay involving lysis of the bacterium Micrococcus lysodeikticus

INTRODUCTION Mononuclear phagocytes are multifunctional cells that can be activated to enhance their microbicidal and tumouricidal activity, chemotaxis, antigen presentation, expression of various receptors and/or surface antigens, and secretion of a range of products such as lysosomal hydrolases, eicosanoids and lymphokines (Adams & Hamilton, 1984; Gordon, 1986). The intracellular events involved in such functional changes by macrophages are thought to be regulated, in part, by signals encountered in the tissue microenvironment during their development from monocyte precursors and immature macrophages. These local signals include the bacterial cell-wall component, lipopolysaccharide (LPS), and the cytokines interAbbreviations: CDM, culture-derived macrophages; IFN-y, interferon-gamma; LPS, lipopolysaccharide; LZM, lysozyme; RHPA, reverse haemolytic plaque assay; TNFa, tumour necrosis factor alpha. Correspondence: Dr C. E. Lewis, Nuffield Dept. of Pathology and Bacteriology, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K.


Stimulated LZM secretion and cell maturity (Gordon et al., 1974). This assay simply measures the total LZM secreted by entire cell cultures, and gives no indication of the secretory activity of individual cells within such populations. Recent reports have used a combination of techniques to demonstrate heterogeneity amongst individual mononuclear phagocytes in their ability to secrete interleukin-1 (IL-1) (Elias et al., 1985), TNFa and macrophage colony-stimulating factor (Becker, Devlin & Haskill, 1989). With this in mind, the reverse haemolytic plaque assay (RHPA) has been adapted to permit the measurement of LZM secretion by single human monocytes and macrophages (Lewis et al., 1990a). In this immunoassay, the area of haemolysis (plaque) which forms around a secretory cell is directly proportional to the amount ofproduct secreted (Neill et al., 1987). Use of the RHPA has revealed that individual human mononuclear phagocytes are, in fact, markedly heterogenous in their ability to secrete LZM (Lewis et al., 1990a). The use of this powerful new experimental tool in the measurement of such cytokines as interleukins 1, 2 and 6, IFN-y and granulocyte-macrophage colony-stimulating factor has been demonstrated recently (Lewis et al., 1990b). The present report investigates the effects of cell maturation on the secretion of LZM by both human monocytes and peritoneal macrophages stimulated by LPS and IFN-y, as well as another macrophage-activating factor, TNFa. It is demonstrated that all three stimulants are able to augment LZM release by these cells, but that as monocytes mature into macrophages they lose their ability to elaborate increased levels of this peptide after exposure to LPS and IFN-y. MATERIALS AND METHODS Isolation of human peripheral blood monocytes and peritoneal

macrophages Monocytes were isolated from blood of normal human donors by density sedimentation on a Nycodenz gradient, as described previously by Boyum (1983). Briefly, this involved the removal of erythrocytes with Dextran 500 in 0 09% (w/v) NaCl solution, and centrifugation of the remaining leucocyte-enriched plasma -on a gradient of Nycodenz solution (Nyegaard U.K. Ltd, Birmingham, Warks) at 600g for 15 min. After several washes in serum-free RPMI-1640 medium (Gibco Ltd, Uxbridge, Middlesex), monocytes were resuspended to a final concentration of 1 x 106/ml and either used in the RHPA immediately or incubated in 16-mm plastic culture wells for 2 hr at 37°. In the latter case, non-adherent cells were removed by vigorous pipetting with warm medium, and the adherent cells harvested using a rubber policeman. Macrophages, activated in vivo by peritoneal inflammation (peritonitis), were obtained from the dialysate of patients undergoing continuous ambulatory peritoneal dialysis (CAPD) for renal failure. Peritoneal fluid was spun at 1000 g for 12 min and the cell pellet resuspended in 20 ml of serum-free RPMI. This procedure was repeated three times and the macrophages were separated from attendant neutrophils, fibroblasts and lymphocytes by adherence to plastic culture wells for 2 hr. After removal of non-adherent cells by washing, adherent cells were mechanically harvested as described above. Preparation of culture-derived macrophages (CDM) Peripheral blood monocytes were isolated on a Ficoll-Hypaque gradient according to the method-of Boyum (1968). Cells were


washed three times in serum-free RPMI-1640 medium and diluted to a final concentration of 5 x 106 cells/ml in RPMI-1640 supplemented with 10% fetal calf serum, 1% glutamine, penicillin (100 U/nil) and streptomycin (100 pg/ml) (Gibco Ltd). The cell suspension was aliquoted into plastic culture wells and washed after 2 hr to remove non-adherent cells. Adherent cells were cultured for 3 ('3-day CDM') or 7 ('7-day CDM') days under 5% C02/95% air at 370, after which time they displayed the characteristic morphology and phenotype of macrophages (Kaplan & Gaudernack, 1982). Reverse haemolytic plaque assay The reverse haemolytic plaque assay (RHPA) was performed as previously described for the detection of LZM secretion by human monocytes and macrophages (Lewis et al., 1989a). Freshly isolated monocytes. CDM or peritoneal macrophages were mixed thoroughly with an equal volume of sheep RBC (SBRC) previously coupled to protein A. The majority of any ovine white blood cells present had been removed prior to this conjugation procedure using the Ficoll-Hypaque method outlined above. The cell mixture was aliquoted into Cunningham chambers and allowed to settle on the glass slides for 30-45 min in an atmosphere of 5% C02/95% air at 370. Excess unattached cells were removed by rinsing each chamber with warm medium (Dulbecco's modified essential medium, DMEM; Gibco Ltd) containing 0-1 mg/ml bovine serum albumin and penicillin (100 U/ml) and streptomycin (100 pg/ml), to leave a confluent monolayer of cells attached to the glass floor of the chamber. Chambers were then filled with a test solution of either a 1:60 dilution of rabbit anti-human LZM (Dakopatts Ltd, High Wycombe, Bucks) in either the absence or presence of such stimulants as LPS (Salmonella minnesota; Sigma Ltd, Poole, Dorset), or human recombinant IFN-y or TNFa (Genzyme, Boston, MA). When present, sodium azide was removed from antisera before use on the RHPA using an Amicon Concentrator (Amicon Ltd, Stonehouse, Glos). The slides were then incubated at 370 for 6 hr, after which the contents of chambers were washed with medium to remove any unbound antibody or secretagogues, and chambers filled with 1/50 guinea-pig complement (Gibco Ltd) to initiate plaque formation. After 15-30 min, cells were exposed to either 0-2% (v/v) trypan blue solution (Sigma Ltd) and incubated for 5 min at 370 (to test the viability of the mononuclear phagocytes), or 4% (v/v) glutaraldehyde (Taab Ltd, Reading, Berks) in phosphate buffer (pH 7.4). Chambers were left intact and the slides stored at 40 for later analysis. Each test condition was duplicated on separate slides and repeated in at least two different assays.

Quantitative analysis ofplaque size The area of haemolytic plaques formed under each test condition was measured using a Wild M20 (Microinstruments Ltd, Oxford, Oxon) to which a forward-projecting drawing attachment had been attached. This permitted the image of a computer (Apple Macintosh) screen to be superimposed on the image of each plaque in the Cunningham chamber. Morphometry of plaque sizes was then achieved by tracing the diameter of plaques with the mouse linked to the computer. A program written in [email protected] by Dr J. Lorenzen, Nuffield Department of Pathology and Bacteriology, University of Oxford, measured the area of each plaque image and stored the data in a file which could be accessed by the [email protected] package. The standard

C. E. Lewis et al.


50 40 30 20 0 0 0.










0 0,

20U 10




Plaque size Figure 1. Photomicrograph of an immunoidentified monocyte (arrowhead) at the centre of a LZM plaque formed after a 6 hr exposure to control medium in the RHPA (erythrocyte ghosts can be seen in the plaque). Magnification bar= 20 pm.

deviation of measurements was++19 pm. The first 100-200 plaques on duplicate slides were systematically traced and measured by this computer-assisted morphometry. Plaque sizes (in pm2) were then allocated to size classes in 250 or 500 pm2 increments and arranged into frequency distributions for each test condition. Statistical analysis of the data was performed by the Mann-Whitney U-test.

Immunolabelling of human monocytes and macrophages At the end of the RHPA, cells were identified as mononuclear phagocytes by immunolabelling with EBM/ 1 (Dakopatts Ltd), a pan macrophage monoclonal antibody that has been assigned to the CD68 group. This monoclonal marker does not label cells other than those of the human monocyte/macrophage series (Kelly et al., 1988). Monolayers of sheep red blood cells (SRBC; Serotec, Kidlington, Oxon) and mononuclear phagocytes were exposed to 1% (v/v) H202 in methanol for 30 min, 1: 5 normal sheep serum in Tris-buffered saline (TBS; pH 7-3) for 30 min, 1: 50 culture supernatant containing the monoclonal antibody, EBM/l 1, for 2 hr, peroxidase-conjugated rabbit anti-mouse IgG (Dakopatts Ltd) at a dilution of 1:50 in TBS for 30 min and, lastly, the reaction product revealed in 0 5 mg/ml diaminobenzidine/H202 (Sigma Ltd) for 1-2 min. Immunocytochemical controls consisted of the substitution of primary or secondary antibodies by either control culture supernatant, irrelevant monoclonal antibodies or normal mouse/rabbit serum. RESULTS The trypan blue exclusion test for cell viability indicated that > 80% of monocytes or macrophages were viable at the end of 6 hr in the RHPA. Formation of haemolytic plaques (Fig. 1) was dependent on the secretion of LZM by the human cells present, since haemolytic plaques were not evident if: (i) human mononuclear phagocytes were omitted and the assay performed using SRBC alone; (ii) LZM antibody was omitted and replaced



(/m 2x 103)

Figure 2. Percentage frequency distributions ofthe sizes of LZM plaques formed by freshly isolated monocytes exposed to either control medium alone (-), 30 ug/ml LPS (0), 10 U/ml IFN-y (0) or 100 ng/ml TNFa (-) for 6 hr.

with 1: 50 normal (non-immune) rabbit serum; (iii) complement was omitted during the second incubation period; (iv) SRBC not coated with protein A were used; or (v) the LZM antibody was used in the assay after preabsorption overnight at 40 with excess human LZM (prepared from human lacrimal fluid). Although data from single experiments have been presented in this report, essentially similar results were obtained in replicate assays. The total number of LZM plaques formed by monocytes or macrophages did not vary between groups incubated in control medium and those exposed to LPS, IFN-y and TNFa (data not shown). In general, there was considerable variation amongst individual mononuclear phagocytes in the amount of LZM secreted per cell after exposure to control medium alone. This is seen in the wide range of LZM plaque sizes formed by single cells in each group for this treatment (from 250 to over 10,000 Mm2 in some cases), illustrated in Figs 2, 3, 4 and 6. Effects of LPS, IFN-y and TNFa on the release of LZM Monocytes and 3-day CDM. The shift to the right in the frequency distributions of plaque sizes formed by freshly isolated monocytes (Fig. 2a) and 3-day CDM (Fig. 3a, b) exposed to LPS and IFN-y for 6 hr indicates a significant (P

Differential effects of LPS, IFN-gamma and TNF alpha on the secretion of lysozyme by individual human mononuclear phagocytes: relationship to cell maturity.

Human mononuclear phagocytes can be activated to perform a variety of complex functions by exposure to the immunomodulators, lipopolysaccharide (LPS),...
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