Peroxidase Staining in Elicited and Nonelicited Mononuclear Peritoneal Cells from BCG-Sensitized and Nonsensitized Mice

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1 NFECTON AND MMUNTY, Aug. 1976, p Copyright X) 1976 American Society for Microbiology Vol. 14, No. 2 Printed in U.S.A. Peroxidase Staining in Elicited and Nonelicited Mononuclear Peritoneal Cells from BCG-Sensitized and Nonsensitized Mice A. W. D. LEPPERl* AND P. D'ARCY HART National nstitute for Medical Research, Mill Hill, London NW7 1AA, England Received for publication 7 April 1976 The peroxidase (PO) activity in nonelicited macrophages and in casein-elicited monocytes, obtained from peritoneal cavities of nonsensitized and BCG-sensitized mice and cultivated on glass for 1 or 2 h, was studied by light and electron microscopy, using the 3,3'-diaminobenzidine technique. These two types of glass-adherent peritoneal cells differed in PO activity. n macrophages, PO activity was predominantly in the nuclear envelope, rough endoplasmic reticulum, and occasionally in vesicles of the Golgi apparatus. n monocytes, PO activity was confined to cytoplasmic dense bodies resembling lysosomes, and was greater at 10 and 24 h after elicitation than at 96 h. The BCG sensitization did not significantly alter the proportion of cells with PO-positive granules in macrophages or monocytes from that observed in nonsensitized mice. From its lysosomal site, the PO in monocytes could come into contact with those microorganisms whose ingestion by these cells was followed by phagolysosome formation. The myeloperoxidase bactericidal system of polymorphonuclear (PMN) leukocytes (11) has been shown to be effective in the killing of some species of potentially pathogenic bacteria and fungi (12). An association of lysosomal peroxidase (PO) activity with ingested particles or microorganisms within the phagolysosomes of these leukocytes (2) and of guinea pig peritoneal mononuclear cells (4) has been demonstrated with enzyme cytochemical techniques by electron microscopy. t has also been shown (10) that the bactericidal properties both of the mouse polymorph and to a lesser extent of its monocyte-derived exu peritoneal macrophage ("elicited monocyte") are associated with a respiratory burst resulting in the production of H202 and the ability to fix iodide by an oxidative process mediated by endogenous PO. On the other hand, the nonelicited macrophage, presumed normally resident in the mouse peritoneum, has not been shown either to fix iodide or to possess significant levels of PO (15, 16). However, this cell does seem to be more competent in killing certain bacteria than the elicited monocyte (15). n guinea pigs (3, 4) and rats (14), endogenous PO activity of the "resident" peritoneal macrophages is localized to the nuclear envelope, endoplasmic reticulum, and occasional ' Present address: CSRO Division of Animal Health, Animal Health Research Laboratory, P.O. Parkville, Melbourne, Victoria 3052, Australia. Golgi vesicles; in the guinea pig monocyte-derived exu peritoneal macrophage, it is confined to cytoplasmic (lysosomal) granules. Both types of mononuclear cell of guinea pigs actively ingest small particles of latex, but PO is found only in the phagolysosomes of the monocyte-derived exu cell (6, 7). Although resident (nonelicited) peritoneal macrophages of mice have so far been found to be virtually devoid of PO activity, the promonocyte of mouse bone marrow, and the blood-derived macrophage of its peritoneum elicited in response to the injection of serum, both posse s PO activity in the form of lysosomal granules (16). n that study, a greater proportion of POcontaining monocytes was likely to be found in exus collected at 12 h than at 72 h after the injection of serum. Since PO systems might have a role in antibacterial defense by mononuclear as well as by PMN phagocytes, we examined the PO activity in nonelicited macrophages and in exu-derived (elicited) monocytes from the peritoneal cavities of BCG-sensitized and nonsensitized mice, using light and electron microscopy. MATERALS AND METHODS Mononuclear cells. Albino mice of the P strain that had received 4 x 105 to 9 x 105 bacteria of Mycobacterium bovis var. BCG (Glaxo Laboratories) in a volume of 0.1 ml, divided between hind footpad and neck, 6 months previously were further sensi- 522

2 VOL. 14, 1976 tized by intravenous inoculation of 2 x 10" to 3 x 108 bacteria. This schedule had been shown to give positive footpad tests to tuberculin. Seven days later, half of the group was injected intraperitoneally with 3 ml of 1.2% sodium caseinate in 0.9% NaCl (16). At 10, 24, and 96 h later, subgroups of five caseininjected mice were killed by cervical dislocation, and elicited peritoneal cells were harvested after injection into the peritoneum of 3 ml of medium NCTC 109 (containing penicillin [100 U/ml and heparin [5 U/ml]). Nonelicited cells were collected from nonstimulated peritoneal cavities of the other half of the group in a similar manner and at the same times. The elicited and nonelicited cells were washed with NCTC 109 (containing penicillin), resuspended in this medium plus 10% fetal calf serum, and allowed to settle in 5-ml portions on the bottoms of 50- mm-diameter glass petri dishes for 1 h at 37 C under an atmosphere of air containing 5% CO2. Nonadherent cells (mainly lymphocytes) were then removed from the glass by gentle agitation and aspiration of medium, and the adherent cells (granulocytes and mononuclear cells) were processed for light or electron microscopy. Monocytes and macrophages were collected from the peritoneal cavities of nonsensitized P strain mice in a similar manner and at similar times to those described above. Electron and light microscopy. Petri dishes containing adherent peritoneal cells after 1 h of incubation were chilled by standing on crushed ice. Medium was removed by aspiration and replaced by 5 ml of fixative. Cells were fixed for 5 min at 40C in 1. 5% purified charcoal-treated glutaraldehyde in 0.1 M cacodylate buffer, ph 7.4, after which they were mechanically dislodged and centrifuged at 200 x g for 5 min in conical centrifuge tubes. Fresh fixative was added, and cells were further fixed in suspension for 20 min at 4 C. They were then washed three times in cacodylate buffer and once in 0.05 M tris(hydroxymethyl)aminomethane buffer (ph 7.6). Cells were incubated for 30 min at room tempera- PEROXDASE STANNG N PERTONEAL CELLS 523 ture in 10 ml of the 0.05 M tris(hydroxymethyl)aminomethane buffer containing 10 mg of 3,3'-diaminobenzidine tetrahydrochloride (DAB; Polysciences nc., Warrington, Pa.) in the absence of H202. For enzyme detection, tubes containing half the quantity of cells were incubated for a further 30 min with the addition of 0.01% H202. For control purposes, the remaining half was incubated for 30 min in the absence of H202. Cells were pelleted in agar and postfixed for 90 min in 2% osmium tetroxide in distilled water. Preparations were then dehydrated and embedded in epoxy resin. For electron microscopy, thin sections were cut by using glass knives and examined with a Philips EM 300 microscope. For light microscopy, thick (2,um) sections were cut and stained with 1% alcoholic solution of toluidine blue, and the proportions of granulocytes and mononuclear cells were determined in a total of 1,000 adherent cells in representative sections from each preparation; counts of mononuclear cells containing PO-positive granules were also made. RESULTS Nonelicited peritoneal macrophages. Adherent cells from the nonstimulated peritoneal cavities of both nonsensitized and BCG-sensitized mice contained relatively few granulocytes (6.0 and 8.3%, respectively). Mononuclear cells (macrophages) predominated (94.0 and 91.7%), and only a small proportion of these (1.0 and 3.6%) contained PO-positive granules as seen by light microscopy (Table 1). The remaining cells were granulocytes, lymphocytes having been removed by the washing; PO staining was invariably observed in the azurophil granules of these. n the electron microscope, the most commonly observed type of peritoneal macrophage in the monolayers is illustrated in Fig. 1. t was relatively large and contained a reniform or TABLE 1. Light-microscopic examination of toluidine blue-stained epoxy sections of adherent peroxidasereacted peritoneal cells, from nonsensitized and BCG-sensitized mice. Mice Type of peritoneal exu Proportion of mono- Granulo- Mononu- nuclear cells concytes clear cells taining peroxidase- (%) (96) positive granules (%) Nonsensitized Nonelicited 6.0a h exu- 73.9b BCG-sensitized Nonelicited 8.3a h exu- 75.5b h exu- 44.0b h exu- 13.8b a Eosinophils and basophils only. b Mainly PMN leukocytes and eosinophils.

3 524 LEPPER AND HART NFECT. MMUN..1. ;;,41 t.. 0, "e,.i.".i-r '*.j, /-' e.~~~~~~~n 4 e "' 1. ".'i -i" t. ik 1. -t op....4.,..,. a 'k '' N FG. 1. Predominant type of nonelicited macrophage from the mouse peritoneal cavity. Peroxidase reaction product localized in the nuclear envelope and endoplasmic reticulum (arrows). DAB and lead citrate; x23,500. '. convoluted nucleus. PO reaction product was seen localized in the nuclear envelope, the rough endoplasmic reticulum, and very occasionally in small vacuoles of the Golgi region. Less common were smaller mononuclear cells with a more rounded nucleus. n these, a few PO-positive dense bodies with homogeneous contents were scattered in various parts of the cytoplasm, but DAB reaction product was not seen elsewhere in the cell. n control preparations in which H202 had been omitted, no reaction product was visible in either type of cell. Elicited monocytes. The adherent cell populations of peritoneal exus consisted of mononuclear cells and granulocytes (the presence of the latter being accounted for by the short period on glass and the mild washing of the monolayers formed). There was little difference, in 10-h exus, between the proportion of mononuclear cells from nonsensitized (26.1%) and BCG-sensitized (24.5%) mice (Table 1). The proportions of these mononuclear cells that contained PO-positive granules were also similar (18.5 and 23.4%). The greatest proportion (35.5%) of monocytes that contained PO-positive granules was seen in peritoneal exus collected 24 h after injection of caseinate. A much larger proportion of mononuclear cells was present in exus 4 days after casein injection, but very few ofthese (4.5%) contained PO-positive granules, this proportion being almost as low as in the nonelicited macrophages (3.6%). However, PMN leukocytes and eosinophils still contained abundant PO-positive granules, as in granulocytes of nonelicited cells. The main difference between monocytes and macrophages was the presence in the elicited cells of abundant membrane-bound dense bodies of varying size and having the general appearance and distribution of secondary lysosomes. ntense PO activity was associated with the medium- and larger-sized bodies (Fig. 2). The reaction product appeared as a dense floccular deposit, clearly contained within a limiting membrane in the smaller bodies; in the larger ones, diffusion of reaction product tended to obscure the limiting membrane. n preparations in which H202 had been omitted from the

4 VOL. 14, 1976 ~~~~k--~~~~~~~~~~~~~.~ S ~~~~:,;4 PEROXDASE STANNG N PERTONEAL CELLS 525 'A~~~~~~~~~~~~~~~i f -, - a:t. 9.;" f.;, 0 4 X. 14~ v '*'. FG. 2. Elicited monocyte from 24-h peritoneal exu after caseinate injection. Peroxidase reaction product contained in dense cytoplasmic bodies (secondary lysosomes) (L). DAB and lead citrate; x38,500. substrate, the product was absent from lysosomal areas. Little or no reaction product was found in monocyte preparations collected 4 days after the caseinate injection. DSCUSSON n the nonelicited macrophages and elicited monocytes of our study, which were both examined shortly after collection from the mouse peritoneal cavity, the light-microscopic and ultrastructural distributions of PO reaction product resembled those described for guinea pigs and rats (4, 7, 13, 14). Our observations also support the recent finding of Daems and his coworkers that at least two types of PO-positive mononuclear cell can be obtained from the mouse peritoneal cavity (5, 7). Although the exact cytochemical specificity of the DAB method has still to be fully valid in the cells of various animal species, the absence of DAB reaction product in our control preparations of macrophages or monocytes that lacked H202 strongly favors the interpretation that any reaction seen in the preparations with the complete reagent mixture was due to peroxidase. The injection of caseinate elicited a similar response to that of newborn calf serum administered intraperitoneally into mice (17); in both cases the proportion of monocytic cells with POpositive granules rose to a maximum 10 to 24 h after inoculation, falling to a low level in exus collected later. The previous studies were confined to cell preparations from nonsensitized animals. A notable observation in the present study is that, as judged from light microscopy, sensitization of the mice with BCG did not appreciably affect the proportions of granulocytes and mononuclear cells in the total adherent cell populations. Furthermore, this procedure did not affect the proportions of nonelicited POpositive cells, nor those in peritoneal exus examined 10 h after elicitation with caseinate. n particular, the level of PO-positive granules in nonelicited macrophages was very low in spite of prior BCG sensitization. t is difficult to conceive that this very low PO activity could signify any marked peroxidase-mediated antibacterial role by nonelicited mouse peritoneal macrophages. The greater activity apparent in

5 526 LEPPER AND HART freshly collected elicited mouse monocytes, and their lysosomal localization, could offer more scope for such a role, following possible fusion between lysosomes and bacterium-containing phagosomes. However, the failure of BCG sensitization to elevate appreciably the level of activity is not encouraging to this notion. An interesting facet of this problem is presented by Mycobacterium tuberculosis. There are indications that, in the complex situation obtaining in vivo, macrophages can kill tubercle bacilli after immunity has been naturally acquired or induced by BCG. We therefore made additional experiments, using peroxidase staining of cells infected in vitro. Monocytes from peritoneal exus collected 24 h after caseinate injection into BCG-sensitized mice were allowed to ingest M. microti (vole tubercle bacillus, pathogenic to mice) immediately after adherence to glass; the infected monolayers were then processed for electron microscopy. Dense bodies, resembling secondary lysosomes, in these freshly collected cells stained PO positive as usual; but full assessment of ultrastructural features was hampered by the difficulty, commonly experienced after glutaraldehyde fixation, of achieving good preservation of host membranes and mycobacterial morphology. Unfortunately, use of low concentrations of glutaraldehyde is a prerequisite for the preservation of enzyme activity. Despite this, an association was suggested between lysosomal PO reaction product and intraphagosomal bacilli when the latter were obviously damaged, but not when they appeared intact (presumed viable). This apparent nonexposure to lysosomal PO was inferred to be the consequence of inhibition of lysosome-phagosome fusion by viable tubercle bacilli, corresponding to the phenomenon demonstrated in well-established monolayers of nonelicited macrophages from normal mice by prelabeling their secondary lysosomes with ferritin before ingestion of M. tuberculosis or M. microti (1, 8, 9). Although these additional findings in vitro thus offer no support for the possibility that intracellular tubercle bacilli are exposed to a potentially bactericidal PO system, it does not follow that in a BCG-sensitized host the subcellular characteristics of mononuclear cells developing in and around a tuberculous granuloma are the same as those of an elicited peritoneal monocyte. Further clarification is required before dismissing a PO system as irrelevant to antituberculous cellular defense. ACKNOWLEDGMENT We thank J. A. Armstrong for helpful criticism. LTERATURE CTED NFECT. MMUN. 1. Armstrong, J. A., and P. D'Arcy Hart Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J. Exp. Med. 134: Bainton, D. F Origin, content, and fate of PMN granules, p n R. C. Williams and H. H. Fudenberg (ed.), Phagocytic mechanisms in health and disease. Georg Thieme, Stuttgart. 3. Cotran, R. S., and M. Litt Ultrastructural localization of horseradish peroxidase and endogenous peroxidase activity in guinea pig peritoneal macrophages. J. mmunol. 105: Daems, W. Th., and P. Brederoo Electron microscopical studies on the structure, phagocytic properties, and peroxidatic activity of resident and exu peritoneal macrophages in the guinea pig. Z. Zellforsch. Mikroskop. Anat. 144: Daems, W. Th., H. K. Koerten, and M. R. Soranzo On the differences between monocyte-derived and tissue macrophages. Seventh nt. Meet. RES Soc., Pamplona, Spain. 6. Daems, W. Th., R. E. Poelmann, and P. Brederoo Peroxidatic activity in resident peritoneal macrophages and exu monocytes of the guinea pig after ingestion of latex particles. J. Histochem. Cytochem. 21: Daems, W. Th., E. Wisse, P. Brederoo, and J. J. Emeis Peroxidatic activity in monocytes and macrophages, p n R. van Furth (ed.), Mononuclear phagocytes in immunity, infection and pathology. Blackwell, Oxford. 8. Draper, P., and P. D'Arcy Hart Phagosomes, lysosomes and mycobacteria: cellular and microbial aspects, p n R. van Furth (ed.), Mononuclear phagocytes in immunity, infection and pathology. Blackwell, Oxford. 9. Hart, P. D'Arcy, J. A. Armstrong, C. A. Brown, and P. Draper Ultrastructural study of the behavior of macrophages toward parasitic mycobacteria. nfect. mmun. 5: Karnovsky, M. L The biochemical basis of the functions of polymorphonuclear leukocytes and macrophages, p n L. Brent and J. Holborow (ed.), Progress in immunology, vol. 4. North-Holland, Amsterdam, Oxford; American Elsevier, New York. 11. Klebanoff, S. J odination of bacteria: a bactericidal mechanism. J. Exp. Med. 126: Lehrer, R Measurement of candidacidal activity of specific leukocyte types in mixed cell populations.. Normal, myeloperoxidase-deficient, and chronic granulomatous disease neutrophils. nfect. mmun. 2: Nichols, B. A., and D. F. Bainton Ultrastructure and cytochemistry of mononuclear phagocytes, p n R. van Furth (ed.), Mononuclear phagocytes in immunity, infection and pathology. Blackwell, Oxford. 14. Robbins, D., H. D. Fahimi, and F. S. Cotran Firne structural cytochemical localization of peroxidase activity in rat peritoneal cells: mononuclear cells, eosinophils and mast cells. J. Histochem. Cytochem. 19: Simmons, S. R., and M. L. Karnovsky lodinating ability of various leukocytes and their bactericidal activity. J. Exp. Med. 138: van Furth, R., J. G. Hirsch, and M. E. Fedorko Morphology and peroxidase cytochemistry of mouse promonocytes, monocytes and macrophages. J. Exp. Med. 132: