Using the Ch6diak-Higashi Marker

Transcription

1 A Study of the Origin of Pulmonary Macrophages Using the Ch6diak-Higashi Marker Kent J. Johnson, MD, Peter A. Ward, MD, Gary Striker, MD, and Robin Kunkel, MS Using bone marrow reconstitution techniques with cells bearing the Chediak-Higashi marker, the authors have been able to demonstrate in mice that both interstitial and intraalveolar macrophages of the lung are derived from bone marrow precursor cells. The morphologic approach (transmission electron microscopy) employed in this study provides direct evidence and confirmation of earlier reports, in which entirely different techniques were used to study cell traffic in the lung. The use of the Chediak-Higashi marker has great advantages over other more cumbersome and difficult techniques. (Am J Pathol 1980, 101: ) IN RECENT YEARS there has been much experimental work performed to ascertain the origin and kinetics of alveolar macrophages. There is good evidence from animal studies that progenitors of the alveolar macrophage derive from the bone marrow.' These studies have involved whole-body radiation in mice to suppress their bone marrow, followed by marrow transplantation of cells with distinctive markers, in order to permit specific identification of donor cells in the lungs of recipient mice. The markers that have been used to identify the donor macrophages have included enzymatic,2 antigenic,3 or, most commonly, karyotypic markers.4 These approaches are difficult to perform and interpret and may, in the case of enzyme markers, interfere with the morphologic character of the lung tissue. With these data in mind we choose to study the Chediak-Higashi (beige) mouse, using the giant lysosomes as markers of marrow-derived cells. These lysosomes are easily identified by conventional light and transmission electron microscopy. Using beige mouse marrow transplants into syngeneic mice lacking the lysosomal defect, we were able to identify that pulmonary monocytes are marrow-derived cells that populate both the interstitial and intraalveolar compartments of the lung. From the Departments of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, and University of Washington School of Medicine, Seattle, Washington. Supported in part by NIH Grants AI and HL and a grant from the Connecticut Lung Association. Accepted for publication June 7, Address reprint requests to Kent J. Johnson, MD, Department of Pathology, University of Michigan Medical School, Ann Arbor, MI /80/ $ American Association of Pathologists

2 366 JOHNSON ET AL American Journal of Pathology Materials and Methods Marrow Transplantation Normal C57BL/6J mice and the syngeneic beige (C57BL/6[bg/bg]) mice were obtained from a breeding colony maintained at the University of Washington. The age of the mice at the time of the study was 2 months; both males and females were used. Details of the marrow transplantation are provided elsewhere.5 The marrow cells were aspirated from donor femurs. C57BL/6J mice received marrow from either isologous donors or from beige mice, and beige mice received marrow from C57BL/6J mice. At least three mice were used in each group. Graft survival was assessed by the findings of adequate numbers (< 2,000/cu mm) of neutrophils on peripheral blood smears. The animals were killed 14 days after bone marrow transplantation. Electron Microscopy Studies on Lungs After exsanguination, the lungs were removed from anesthetized mice. The lungs were gently perfused with a 6.25% glutaraldehyde solution in 0.1 M sodium cacodylate buffer (ph 7.0). After 24 hours, the lungs were then placed in a solution of 7.5% sucrose in 0.1 M sodium cacodylate buffer. For electron-microscopic studies the lungs were then postfixed for 2 hours in 2.0% osmium tetroxide buffered in 0.2 M sodium collidine (ph 7.35) and then embedded in Epon epoxy resin. Ultrathin sections were then obtained and examined with a Philips EM 300 transmission electron microscope. Results In control C57BL/6J mice irradiated and given syngeneic bone marrow, the appearance of the lungs was essentially normal, with intact vascular (capillary), interstitial, and intraalveolar components (Figure 1). Fine villous projections of alveolar epithelial cells were recognizable. No giant lysosomal granules were recognized in any of the compartments of the lung. These findings were in striking contrast to those of C57BL/6J mice that were reconstituted with bone marrow of C57BL/6(bg/bg) mice. In these animals, the presence of macrophages bearing the giant lysosomes was evident both in an intraalveolar and interstitial location (Figures 2-5). The presence of macrophages bearing the ultrastructural markers on the 14th day after marrow infusion indicates that the traffic from the blood to the pulmonary interstitial and intraalveolar compartments occurs in a relatively brief period of time. As was anticipated, none of the alveolar epithelial cells revealed the presence of the lysosomal marker, indicating that, at least under the conditions employed, these cells do not derive from precursors present in the bone marrow. Radiation of the C57BL/6(bg/bg) mice did not result in the disappearance of the preexisting macrophages in the lung 14 days after the radiation and infusion of C57BL/6J marrow. Macrophages with the distinctive lysosomal marker could still be observed both in the interstitial and in the intraalveolar compartments 2 weeks after the marrow infusion (Figure 6).

3 Vol. 101, No. 2 ORIGIN OF PULMONARY MACROPHAGES 367 November 1980 Discussion The use of the beige mouse as a donor of bone marrow cells provides a direct morphologic approach to a study of cell traffic in the lung. This experimental system does not require the use of indirect approaches such as enzyme histochemistry, the technical features of which may be associated with distortion of tissues and difficulty in a precise morphologic classification of cells bearing the histochemical stain. Posing additional difficulties is the reliance on antigenic markers, the demonstration of which usually requires the use of frozen sections with all their attendant artifacts and difficult use in fine morphologic discrimination. Finally, the use of karyotype or Barr body markers is a definitive approach but is extremely limited by the small number of samples that can be analyzed and the requirement in the former that cells be actively proliferating. Although studies in beige mice are limited by the numbers of animals available, the other drawbacks of the techniques described above are avoided and excellent morphologic resolution is possible. The data described in this paper provide direct evidence that under the experimental conditions employed bone marrow cells differentiate into both interstitial and intraalveolar macrophages within the lung and that this process occurs within a 2-week period and over, perhaps, a much shorter duration of time. Under the conditions employed, the data also indicate that lung cells other than the macrophages are neither reconstituted nor replaced by circulating cells derived from the bone marrow. Thus, in inflammatory diseases of the lung seen in man, it seems likely that the accumulation of interstitial and intraalveolar macrophages (especially in cases of usual interstitial pneumonia with a desquamative component) is perhaps largely via recruitment of circulating monocytes with local differentiation rather than proliferation of macrophages perexisting within the lung. The variety of chemotactic factors (such as C5 fragmentation products, bacterial chemotactic factors, and lymphokines,6) demonstates that chemotactic activity for macrophages may represent the signals that govern the traffic of monocytes from the blood into the lung. In addition, there may also be a natural "homing" mechanism for monocytes into these interstitial and intraalveolar areas, analogous to the homing of lymphocytes to lymphoid organs. References 1. Hocking WG, Golde DW: The pulmonary-alveolar macrophage. N Engl J Med 1979, 301: Weiden PL, Storb R, Tsai M: Marrow origin of canine alveolar macrophages. J Reticuloendothel Soc 1975, 17:

4 368 JOHNSON ET AL American Journal of Pathology 3. Godleski JJ, Brain JD: The origin of alveolar macrophages in mouse radiation chimeras. J Exp Med 1972, 136: Pinkett MO, Cowdrey CR, Nowell PC: Mixed hematopoietic and pulmonary origin of (alveolar macrophages) as demonstrated by chromosome markers. Am J Pathol 1966, 48: Striker GE, Mannik M, Tung MT: Role of marrow-derived monocytes and mesangial cells in removal of immune complexes from renal glomeruli. J Exp Med 1979, 149: Ward PA, Becker EL: Biology of leukotaxis. Rev Physiol, Biochem Pharmacol 1977, 77:

5 v /1<j I a). I-i Am 0 FI Figure 1-A representative section of lung taken from a control C57BL/6J mouse given isologous bone marow showing normal lung architecture. (x2630)

6 -M WMM,.~ i*is. 1;.. *V 2,..-N 6.,1, c4. Figures 2 and 3-Sections of lungs from C57BL/6J mice given C57BL/6(bg/bg) marrow showing macrophages with the characteristic giant lysosomes (arrows) in interstitial and intraalveolar locations (x2630)

7 10. i,.'if % ill,.",: 3 0 A.

8 i9 4.I '' A >. jr :., -:'._ ;. "o 4...,.. :T *, ' rlf-,a 1. -;...C. -l-% ;.a Figure 4-Higher power view of a macrophage in an interstitial location. Basement membrane separates the interstitial macrophage from a Type 11 alveolar epithelial cell. Section from a C57BL/6J mouse given C57BL/6(bg/bg) marrow. (x7020)

9 9N "-.: At it Si._ '' 'N-.&. "RM'-.. Figure 5-An intraalveolar macrophage containing giant lysosomes. Section from a C57BL/6J mouse given C57BL/6(bg/bg) marrow. (xl 1,200)

10 M-')i ': AL '*.% p..e:a ^,, Se ]^.. ft'..5s..2.4 s '* e.. B. < ws " > ':^.*..hk >t..,,g,g... W T' ::{;.'.i i. F.Bp I, S, t'lf :.i. ffi, "!4S"'4t *AIt7w.. Figure 6-Lung section from a C57BL/6(bg/bg) mouse given C57BL/6J marrow showing persistence of an intraalveolar macrophage with giant lysosomes. (x 1 1,200)