THE ANATOMICAL RECORD 233:415-428 (1992)
Macrophage Development: IV. Effects of Blood Factors on Macrophages From Prenatal Rat Lung Cultures SERGE1 P. SOROKIN, NANCY A. McNELLY, AND RICHARD F. HOYT, JR. Laboratory of Pulmonary Cell Biology, Department of Anatomy and Neurobiology, Boston University School of Medicine, Boston, Massachusetts
ABSTRACT Effects of colony-stimulating factors M-CSF, GM-CSF, G-CSF, and IL-3 were assessed on cells of macrophage lineage present in organ cultured 14-day prenatal r a t lungs. Treatment groups were compared between one another and against control lungs grown on standard medium containing 40% fetal bovine serum without added factors, where a monoculture of macrophages rapidly develops from precursors present a t explantation, leading to appearance of a large mature population on the pleural surface outside the lungs. Studies were carried out in living cultures and by light and electron microscopy using peroxidase-coupled isolectin B, of Griffonia simplicifolia to identify macrophages and their precursors. In the first experiment, 14-day prenatal lung explants (14 + 0 days) containing macrophage precursors but not matured cells were exposed to individual CSFs for 7 days in an attempt to determine whether precursors are committed irrevocably to the macrophage line or can be altered by exposure to factors promoting significant granulocyte development. In succeeding experiments, 4- and 7-day-old cultures (14 + 4, 14 + 7 days) containing matured macrophages were targeted to see whether macrophage survival can be extended beyond expectations in controls and whether mitotic activity is stimulated. Recombinant CSFs were used at dosage levels known to promote colony formation in vitro (200-1,000 CFUlml). Cultures exposed fromprenatal day 14 to M-, GM-, G-CSF, or IL-3 yielded a monoculture of macrophages without exception. Populations developed in the presence of M- or GM-CSF were much larger than in controls or cultures grown with the other blood factors. GM-CSF-exposed cultures produced by far the largest macrophages, among them many multinucleate giant cells. Macrophages developed in the presence of G-CSF were also significantly larger than controls. Growth of the mature macrophage population was greatly stimulated by exposure to M-CSF or GM-CSF but not by IL-3 or G-CSF. Mitotic figures were noted in the coronas of emerged cells surrounding stimulated cultures, compared to none in the controls. Ultrastructurally, macrophages stimulated by M-CSF retained a mature appearance like macrophages in control, IL-3, and G-CSF treatment groups, whereas many in the GM-CSF group became less differentiated. As to long-term survival, a single 14day explant was grown for 8 days on standard medium (the equivalent date for birth), then placed in a soft agar medium containing M-CSF. Supplemented irregularly by M-CSF and GM-CSF, the culture remained viable until fixed on the 137th “postnatal” day and retained a small population of macrophages. Conclusions: (1)the macrophage lineage from embryonic rat lungs can be manipulated in culture; (2) macrophage precursors in these lungs seem committed to the macrophage line; (3) replication of both immature and mature macrophages is stimulated by M-CSF and GM-CSF; (4) with M-CSF, however, retention of mature characteristics and longevity are favored, whereas with GM-CSF maturity is partly lost and formation of giant cells emphasized. o 1992 Wiley-Liss, Inc. The experiments reported here were carried out on prenatal lung explants grown in organ culture, gur model system for studying developmental dynamics of pulmonary macrophages. At 14 days of gestation, prenatal rat lungs are being colonized b y macrophage precursors but as yet contain no mature celis*whereas the situation is reversed after the lungs are cultured for 4 or more days (Sorokin et al., 1992a,b). The investiga0 1992 WILEY-LISS, INC
tion was undertaken for three purposes: (1)to extend our studies on the functional properties of early-resident pulmonary macrophages to include responses to Received August 19, 1991; accepted December 19, 1991. Address reprint requests to Sergei P. Sorokin, Pulmonary Cell Biology, Department of Anatomy and Neurobiology, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118.
416
S.P. SOROKIN ET AL.
Fig. 1. Large phagocytic macrophages (dark rims) in the connective tissue separating epithelium (below) from the pleura (above) of 14 + 2-day lung organ cultures. Whether exposed to a: G-CSF or b GM-CSF, only macrophages and no granulocytes are produced. GSA I-B,-peroxidase and toluidine blue. x 400.
TABLE 1. CSFs from (a) MacroDhaee size measured at
CSF None M-CSF IL-3 G-CSF GM-CSF
CSF None IL-3 G-CSF M-CSF GM-CSF
+ 3 davs
N 100 50 50 50
Mean cell diameter (pm) 16.5 18.4 18.8 20.8
50
23.6
(b) Coronal areas measured at
N 13 2
2 5 8
+ 0 to + 7 days in culture
SEM 0.4 0.5 0.6 0.8 0.7
Comparison with controls T DF 1.92 2.18 3.77
6.41
148 148 148 148
P NS P
Macrophage Development: IV. Effects of Blood Factors on Macrophages From Prenatal Rat Lung Cultures SERGE1 P. SOROKIN, NANCY A. McNELLY, AND RICHARD F. HOYT, JR. Laboratory of Pulmonary Cell Biology, Department of Anatomy and Neurobiology, Boston University School of Medicine, Boston, Massachusetts
ABSTRACT Effects of colony-stimulating factors M-CSF, GM-CSF, G-CSF, and IL-3 were assessed on cells of macrophage lineage present in organ cultured 14-day prenatal r a t lungs. Treatment groups were compared between one another and against control lungs grown on standard medium containing 40% fetal bovine serum without added factors, where a monoculture of macrophages rapidly develops from precursors present a t explantation, leading to appearance of a large mature population on the pleural surface outside the lungs. Studies were carried out in living cultures and by light and electron microscopy using peroxidase-coupled isolectin B, of Griffonia simplicifolia to identify macrophages and their precursors. In the first experiment, 14-day prenatal lung explants (14 + 0 days) containing macrophage precursors but not matured cells were exposed to individual CSFs for 7 days in an attempt to determine whether precursors are committed irrevocably to the macrophage line or can be altered by exposure to factors promoting significant granulocyte development. In succeeding experiments, 4- and 7-day-old cultures (14 + 4, 14 + 7 days) containing matured macrophages were targeted to see whether macrophage survival can be extended beyond expectations in controls and whether mitotic activity is stimulated. Recombinant CSFs were used at dosage levels known to promote colony formation in vitro (200-1,000 CFUlml). Cultures exposed fromprenatal day 14 to M-, GM-, G-CSF, or IL-3 yielded a monoculture of macrophages without exception. Populations developed in the presence of M- or GM-CSF were much larger than in controls or cultures grown with the other blood factors. GM-CSF-exposed cultures produced by far the largest macrophages, among them many multinucleate giant cells. Macrophages developed in the presence of G-CSF were also significantly larger than controls. Growth of the mature macrophage population was greatly stimulated by exposure to M-CSF or GM-CSF but not by IL-3 or G-CSF. Mitotic figures were noted in the coronas of emerged cells surrounding stimulated cultures, compared to none in the controls. Ultrastructurally, macrophages stimulated by M-CSF retained a mature appearance like macrophages in control, IL-3, and G-CSF treatment groups, whereas many in the GM-CSF group became less differentiated. As to long-term survival, a single 14day explant was grown for 8 days on standard medium (the equivalent date for birth), then placed in a soft agar medium containing M-CSF. Supplemented irregularly by M-CSF and GM-CSF, the culture remained viable until fixed on the 137th “postnatal” day and retained a small population of macrophages. Conclusions: (1)the macrophage lineage from embryonic rat lungs can be manipulated in culture; (2) macrophage precursors in these lungs seem committed to the macrophage line; (3) replication of both immature and mature macrophages is stimulated by M-CSF and GM-CSF; (4) with M-CSF, however, retention of mature characteristics and longevity are favored, whereas with GM-CSF maturity is partly lost and formation of giant cells emphasized. o 1992 Wiley-Liss, Inc. The experiments reported here were carried out on prenatal lung explants grown in organ culture, gur model system for studying developmental dynamics of pulmonary macrophages. At 14 days of gestation, prenatal rat lungs are being colonized b y macrophage precursors but as yet contain no mature celis*whereas the situation is reversed after the lungs are cultured for 4 or more days (Sorokin et al., 1992a,b). The investiga0 1992 WILEY-LISS, INC
tion was undertaken for three purposes: (1)to extend our studies on the functional properties of early-resident pulmonary macrophages to include responses to Received August 19, 1991; accepted December 19, 1991. Address reprint requests to Sergei P. Sorokin, Pulmonary Cell Biology, Department of Anatomy and Neurobiology, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118.
416
S.P. SOROKIN ET AL.
Fig. 1. Large phagocytic macrophages (dark rims) in the connective tissue separating epithelium (below) from the pleura (above) of 14 + 2-day lung organ cultures. Whether exposed to a: G-CSF or b GM-CSF, only macrophages and no granulocytes are produced. GSA I-B,-peroxidase and toluidine blue. x 400.
TABLE 1. CSFs from (a) MacroDhaee size measured at
CSF None M-CSF IL-3 G-CSF GM-CSF
CSF None IL-3 G-CSF M-CSF GM-CSF
+ 3 davs
N 100 50 50 50
Mean cell diameter (pm) 16.5 18.4 18.8 20.8
50
23.6
(b) Coronal areas measured at
N 13 2
2 5 8
+ 0 to + 7 days in culture
SEM 0.4 0.5 0.6 0.8 0.7
Comparison with controls T DF 1.92 2.18 3.77
6.41
148 148 148 148
P NS P
