Self-Renewal of Pulmonary Alveolar Macrophages - CiteSeerX

5 downloads 101 Views 817KB Size Report
by Strontium-89. Lab. Invest. 46, 165, 1982. 23. Shellito, i., Esparza,. C., and Armstrong,. C. Maintenance of the normal rat alveolar macrophage cell population.
Journal

of Leukocyte

Biology

42:443-446

(1987)

Self-Renewal of Pulmonary Alveolar Macrophages: Evidence From Radiation Chimera Studies John

D. Tarling,

Hsiu-san

Lin, and Shin

Radiation Oncology Center, Mallinckrodt institute of Radiology (J.D.T., partment of Microbiology and Immunology (H.L.), Washington University St. Louis, Missouri

Hsu H.L., S.H.) and DeSchool of Medicine,

Radiation-induced chimeric mice were used to study the origin of pulmonary alveolar macrophages. Unlike in other studies, these radiation chimeras were prepared by using a special fractionated irradiation regimen to minimize the killing of alveolar macrophage colony-forming cells, putative local stem cells. For this study CBA mice with or without T6 chromosome marker were used. Under this experimental condition, the majority of alveolar macrophages in mitosis are of host origin even after 45 weeks. These data suggest that alveolar macrophages are a self-renewing population under normal steadystate conditions. Key words:

alveolar

macrophages,

radiation

INTRODUCTION Pulmonary alveolar macrophages (PAM) serve as a first line of defense against airborne microorganisms and particles [12]. They are continuously lost, mainly through airways, and must be constantly replenished [121. It has been suggested that the majority if not all of PAM are derived from blood monocytes under normal steady-state conditions [2]. However, many recent studies using various techniques have failed to show any significant emigration of blood monocytes into the alveolar spaces unless the latter are stimulated [7,21,23,28]. Another approach that has often been used to demonstrate that PAM are derived from bone marrow stem cells is the use of radiation-induced chimera [4,8,13,15,251. In these studies, animals are given a single large dose of radiation (9 to 10 Gy) before bone marrow transplantation. Because the irradiation destroys not only the proliferative potential of the majority of bone marrow stem cells but also that of local stem cells that may be present in the alveolar spaces, the data can only be used to prove that PAM can come from bone marrow cells under the experimental condition used and should not be used to determine the origin of new PAM under the normal steady-state condition. We have recently discovered that about 20% of murine PAM are capable of extensive replication and colony forming in vitro (alveolar macrophage colony-forming cells, AL-CFC) [16-18, 201. The dose-response curve of AL-CFC to ionizing irradiation has an initial shoulder of about 1 Gy [17]. Because the dose-response curve of bone marrow pluripotent hemopoietic stem cells has no © 1987 Alan

R. Liss,

Inc.

chimera,

bone

marrow

transplant

or very small initial shoulder [26], one can take advantage of this difference to devise a fractionated irradiation schedule that selectively damages the proliferative potential of all hemopoietic stem cells without causing a significant killing of AL-CFC. In this study, we have used a fractionated irradiation regimen to prepare chimeric mice and investigate the source of PAM under the condition that host AL-CFC are minimally damaged by the irradiation. Our studies have shown that the majority of PAM in chimeras are of host origin even after 11 months.

MATERIALS

AND METHODS

Animals Reciprocal radiation chimeras were produced using 8to 12-week-old CBA/J and CBA/T6 male mice. The CBA/T6 mice carry two easily recognizable minute chromosomes that can be observed in mitotic cells [19]. All mice were obtained from The Jackson Laboratory, Bar Harbor, Maine. Irradiation A fractionated bone Received

marrow March

regimen transplant. 13, 1987;

was This

accepted

used

to prepare

consisted May

mice

of three

for daily

14, 1987.

Reprint requests: Hsiu-san Lin, Mallinckrodt Institute of Radiology, Washington University, 510 South Kingshighway, St. Louis, MO 63110.

444

Tarling,

Lin, and Hsu

exposures of 1 Gy on 4 consecutive days (12 dose); the duration between exposures was at For control and comparison, we also irradiated mice with a single dose of 9 Gy. A 137Cs employed at a dose rate of 1 Gy/min (17).

Assays

for Hemopoietic

Stem

Cells

Gy total least 4 h. groups of unit was

and AL-CFC

The number of pluripotent hemopoietic stem cells (CFU-S) and committed stem cells for both granulocytes and monocytes (GM-CFC) in femoral marrow and ALCFC in bronchial washings was assayed by the methods described previously [3,16,26]. Bone

Marrow

Transplantation

Fourteen to sixteen h after the final irradiation, 5 x 106 donor bone marrow (BM) cells, suspended in 0.5 ml alpha medium (Difco, Detroit, MI), were injected into the tail vein of recipient mice. Karyotype At times up to 45 weeks after bone marrow transplant, the karyotypes of PAM and BM cells were determined. Because of the low mitotic index of PAM [5,6], all mice were injected with 1 g/g body weight vincristine sulphate 2.5 h before killing to increase in the number of PAM in mitosis [291. BM cells were obtained from femurs and PAM by lung lavage [16,18]. Chromosome spreads of these populations were prepared as described previously [19,29]. Chromosome spreads were viewed, under oil, at x 1,000 magnification and cells of CBA/T6 and CBA/J karyotype determined by the presence or absence, respectively, of the two minute chromosomes. Cells of host or donor origin could consequently be distinguished. Karyotype

of AL-CFC

Alveolar macrophages were grown on microscopic glass slides placed inside 100 mm-petri dishes. The growth medium contained 10% fetal calf serum and 10% L-cell conditioned medium (source of colony-stimulating factor) in alpha medium [16-18]. Seven days after plating, colonies were prepared for chromosome spreading in situ. Colonies were incubated in I /2g/nll of vincristine sulfate for 2.5 h before they were processed for karyotyping.

RESULTS Effects of Fractionated Whole Body Irradiation on Bone Marrow Hemopoietic Stem Cells and AL-CFC To ensure that the fractionation irradiation regimen, 12 Gy in 12 fractions, is as effective as 9 Gy given in single dose in killing hemopoietic stem cells, we determined the number of CFU-S and GM-CFC in femoral marrow 1 day after the last dose of irradiation. We did not see any

TABLE 1. Survivals of Various Colony-Forming Different Whole Body Irradiation Regimensa Surviving Single Cell

large

Cells

fraction

dose

Fractionated

(9 Gy)

(12

irradiation

Gy/12

fractions/4

CFU-S

0.0015

± 0.OOI1C

0.0011

± 0.0009

GM-CFC

0.0011

± 0.0007

0.0008

±

0.0003

AL-CFC

0.023

± 0.0075

±

0.22

aMice

were

bCFU..S

killed

(pluripotent

or AL-CFC cMn ±

1 day

after

0.86 the

hemopoietic

last stem

After

dose

days)

of radiation.

cells)

or

GM-CFC

per

femur

per mouse. SD (4 experiments).

statistically significant difference between these two groups (Table 1). In fact, the fractionated irradiation regimen was more effective in reducing the number of CFU-S per femur. The survival of AL-CFC was reduced to 0.02 after 9 Gy, whereas only a small reduction in the survival of AL-CFC to 0.86 was noted in the mice given fractionated irradiation (Table 1). Posttransplant Populations

Karyotype

of PAM

and BM

Chimeras were killed at various times after irradiation and the karyotypes of their BM cells and PAM were determined. We examined between 25 and 100 mitoses for each mouse. Data from three to five separate experiments using two to nine mice for each experimental point were combined and are shown in Figure 1. No appreciable difference was noted with BM cells whether the mice received either a single large dose or fractionated irradiation. The proportion of BM cells of host origin decreased rapidly and reached less than 10% within 2 weeks (Fig. 1). In sharp contrast, a dramatic difference was noted with PAM cells when different whole body irradiation regimens were used. For the mice that received a single large dose of irradiation, the proportion of cells of host origin was about 50% by the seventh week, and by the tenth week, it was less than 10%. After the fractionated irradiation, the proportion of host cells remained high throughout the experimental period, and the value was 60% after 45 weeks. Between the thirteenth and twentieth weeks after the transplantation, the drop in the proportion of mitotic cells of host origin was partly due to the small fraction of mice that gave low values. For example, in one of six mice at the thirteenth week and in three of nine mice at the sixteenth week, the proportion of mitotic cells of host origin was less than 20%. If the median value rather than the mean was used, the proportion of mitotic cells of host origin was more than 90% at these two time points. Karyotype

of AL-CFC

in Radiation

Because only a small fraction are in mitosis at any given time,

Chimeras

of alveolar macrophages the information acquired

Alveolar

Macrophages

Self-Renewal

445

100

z 0

IU) 0

I 0

401 0. >-

I0 >-

0.

20 I

20

JO TIME

Fig. types

1.

AFTER

30 TRANSPLANTATION

50 (weeks)

The proportion of cells in mitosis that carry host karyoPAM; 0, BM cells) or one large in chimeras prepared by the fractionated irradiation (#{149}, BM cells). Mean ± SD.

by examining the freshly obtained alveolar cells in mitosis may not accurately reflect the real composition of alveolar cells. Moreover, the newly recruited blood monocytes may not undergo mitosis in vivo, but monocytes could be induced to proliferate in vitro under appropriate culture conditions [14]. For these reasons, we cultured alveolar cells to allow colony-forming cells to replicate and form colonies in vitro and then studied the karyotype of these colony-forming cells to determine their origin. Between 1 and 4 mitoses were usually present in each colony. We examined the karyotype of ALCFC obtained 4 and 16 weeks after the transplantation. The proportion of AL-CFC of host origin was very close to the values obtained by examining the karyotype of freshly obtained alveolar cells. For chimeras prepared by the fractionated irradiation, 99% of 109 evaluable colonies derived from PAM cells obtained 4 weeks after the transplantation carried host karyotype. After 16 weeks, 79% of 326 colonies examined had karyotype of host origin. The proportion of AL-CFC of host origin in chimeras prepared by a single large dose of irradiation was 98% and 6% after 4 and 16 weeks, respectively.

DISCUSSION Our studies have shown that when the putative local macrophage stem cells, AL-CFC, are protected from the irradiation used to prepare animals for radiation chimeras, the majority of PAM in chimeras remain host origin even after 11 months. The turnover time of PAM

dose

of irradiation

(A,

PAM;

,

has been estimated to be about 10 to 40 days [2,5,8]. If BM-derived circulating blood monocytes are the only antecedent of PAM, cells of donor origm would be expected to move in and repopulate the lungs of recipient mice within a short time after bone marrow transplantation. The failure of donor BM-derived cells to replace all host cells in the lungs, together with the presence of host PAM in mitosis, indicates that PAM population is capable of self-renewal. The result of our experiments using a single large dose of radiation to prepare recipient mice for chimeras is in good agreement with previous studies using similar experimental design [4,8,19]. When the majority of local stem cells are killed, there is no choice but to recruit blood monocytes to the lung. Blood monocytes may mature into alveolar macrophages when they arrive at alveolar spaces or become new local stem cells before they replicate to produce new alveolar macrophages in the lung. Mouse blood monocytes have recently been found to possess an extensive proliferative capacity [14]. In addition to mice, the presence of alveolar macrophages that are capable of replication has been well documented in humans, rats, and hamsters under both normal steady state and pathological conditions [1,9,10,181. It should be noted that the time required for the disappearance of mitotic cells of host origin is about 8 weeks even with this radiation regimen. Moreover, the appearance of donor cells coincides with the repopulation of colony forming cells in alveolar spaces [15]. By following the disappearance of macrophages with a Y body, the life span of alveolar macrophages is estimated to be 81 days

446

Tarling,

Lin, and Hsu

after allogeneic marrow transplantation in man [25]. Even with this fractionated irradiation regimen to minimize the killing of AL-CFC, the proportion of host cells in the alveolar spaces was slowly reduced to about 60% after 11 months. One possible explanation is that all local stem cells have a limited life span as described for other normal mammalian cells [11] and they have to be replaced gradually by new cells, most likely blood monocytes. Another explanation is that the lungs of chimeric mice may be infected time to time and monocytes are recruited into the alveolar spaces. As a result, the proportion of macrophages of host origin is reduced. The recruitment of blood monocytes into the alveolar spaces can occur when inflammatory stimuli are given [27]. The whole body irradiation used to prepare chimeras may also induce inflammation in the lung. Our data presented here, supports the conclusion made by other investigators using a variety of experimental approaches that PAM are a self-renewing population under normal steady state conditions [6,7,21-24,28]. However, bone marrow-derived cells (monocytes) can be recruited into the alveolar spaces when a significant number of local stem cells such as AL-CFC are damaged or inflammation is induced.

ACKNOWLEDGMENTS This work was supported and Al 15542. We thank Connie Povilat for editing

by U.S.PHS grants HL 19746 Connie Huycke for typing and the manuscript.

REFERENCES 1. Bitterman, PB., Saltzman, L.E., Adelberg, S., Ferrans, Vi., and Crystal, R.G. J. Clin. Invest. 74, 460, 1984. 2. Blusse van Oud Alblas, A., and van Furth, R. Origin, kinetics and characteristics of pulmonary macrophages in the normal steady state. J. Exp. Med. 149, 1504, 1979. 3. Bradley, T.R., and Metcalf, D. The growth of mouse bone marrow cells in vitro. Aust. i. Exp. Biol. Med. Sci. 44, 287, 1966. 4. Brunstetter, M., Hardie, J.A., Schiff, R., Lewis, J.P., and Cross, CE. The origin of macrophages: Studies of stem cells using the ES-2 marker of mice. Arch. Intern. Med. 127, 1064, 1971. 5. Coggle, i.E., and Tarling, J.D. Cell kinetics of pulmonary alveolar macrophages in the mouse. Cell Tissue Kinet. 15, 139, 1982. 6. Coggle, i.E., and Tarling, i.D. The population kinetics of pulmonary alveolar macrophages. J. Leuk. Biol. 35, 317, 1984. 7. Collins, F.M., and Auclair, L.K. Mononuclear phagocytes within the lungs of unstimulated parabiotic rats. J. Reticuloendoth. Soc. 27,429, 1980.

8. Godleski, i.J., and Brain, J.D. The origin of alveolar macrophage in mouse radiation chimeras. J. Exp. Med. 136, 630, 1972. 9. Golde, D.W., Byers, L.A., and Finley, T.N. Proliferative capacity of human alveolar macrophages. Nature 247, 373, 1974. 10. Golde, D.W., Finley, T.N., and Cline, M.J. The pulmonary macrophage in acute leukemia. N. Engl. J. Med. 290, 875, 1974. II. Hayflick, L. The cell biology of human aging. N. EngI. J. Med. 295, 1302, 1976. 12. Hocking, W.G., and Golde, D.W. The pulmonary-alveolar macrophage. N. Engl. J. Med. 301, 580, 1979. 13. Johnson, K.i., Ward, PA., Striker, G., and Kunkel, R. A study of the origin of pulmonary macrophages using the ChediakHigashi marker. Am. i. Pathol. 101, 365, 1980. l4. Lin, H-S. Colony formation in vitro by mouse blood monocytes. Blood 49, 593, 1977. 15. Lin, H-S. Repopulation of murine alveolar macrophage colonyforming cells after whole body irradiation. Br. i. Cancer 53 (Suppl. 7), 363, 1986. 16. Lin, H-S., Hsu, S. Effects of dose rate and dose fractionation of irradiation on pulmonary alveolar macrophage colony-forming cells. Radiat. Res. 103, 260, 1985. 17. Lin, H.S., Kuhn, C., and Chen, D.M. Radiosensitivity of pulmonary alveolar macrophage colony-forming cells. Radiat. Res. 89, 290, 1982. 18. Lin, H-S., Kuhn, C., and Kuo, T.T. Clonal growth of hamster free alveolar cells in soft agar. J. Exp. Med. 142, 877, 1975. l9. Pinkett, MO., Cowdrey, C.R., and Nowell, P.C. Mixed hematopoietic and pulmonary origin of alveolar macrophages as demonstrated by chromosome markers. Am. i. Pathol. 48, 859, 1966. 20. Reppun, T.S., Lin, H-S., and Kuhn, C. III. Isokinetic separation and characterization of mouse pulmonary alveolar colony-forming cells. J. Reticuloendoth. Soc. 25, 379, 1979. 21. Sawyer, R.T. The ontogeny of pulmonary alveolar macrophages in parabiotic mice. J. Leuk. Biol. 40, 347, 1986. 22. Sawyer, R.T., Strausbauch, PH., and Volkman, A. Resident macrophage proliferation in mice depleted of blood monocytes by Strontium-89. Lab. Invest. 46, 165, 1982. 23. Shellito, i., Esparza, C., and Armstrong, C. Maintenance of the normal rat alveolar macrophage cell population. Am. Rev. Respir. Dis. 135, 78, 1987. 24. Tarling, i.D., and Coggle, i.E. The absence of effect on pulmonary alveolar macrophage numbers during prolonged periods of monocytopenia. J. Reticuloendoth. Soc. 31, 22l, 1982. 25. Thomas, E.D., Ramberg, R.E., Sale, G.E., Sparkes, R.S., and Golde, D.W. Direct evidence for a bone marrow origin of the alveolar macrophages in man. Science 192, 10l6, 1976. 26. Till, J.E., and McCulloch, E.A. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14, 213, 1961. 27. Velo, G.P., and Spector, W.G. The origin and turnover of alveolar macrophage in experimental pneumonia. J. Pathol. 109, 7, 1973. 28. Volkman, A. Disparity in origin of mononuclear phagocyte populations. J. Reticuloendothel. Soc. 19, 249, 1976. 29. Wright, N., and Appleton, D.R. The metaphase arrest technique-a critical review. Cell Tissue Kinet. 13, 643, l980.