neoplasms were tubular and papillary in histologic type ..... Figure 7-Papillary adenocarcinoma induced transplacentally by ENU showing Clara cell antigen in ...
Immunocytochemical Localization of the Surfactant Apoprotein and Clara Cell Antigen in Chemically Induced and Naturally Occurring Pulmonary Neoplasms of Mice From the Tumor Pathology and Pathogenesis and Perinatal Carcinogenesis Sections, Laboratory of Comparative Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Frederick, Maryland; Veterans Administration Medical Center and University of Pittsburgh, Pittsburgh, Pennsylvania; and the Pathology Histotechnology Laboratory, Program Resources, Inc., NCI-Frederick Cancer Research Facility, Frederick, Maryland
JERROLD M. WARD, DVM, PhD,
GURMUKH SINGH, MBBS, PhD, SIKANDAR L. KATYAL, PhD, LUCY M. ANDERSON, PhD., and ROBERT M. KOVATCH, DVM
The localization of surfactant apoprotein (SAP) and the Clara cell antigen(s) (CCA) was studied in naturally occurring and experimentally induced pulmonary hyperplasias and neoplasms by avidin-biotin peroxidase complex (ABC) immunocytochemistry. Lungs of B6C3F1 and A strain mice with naturally occurring lesions, B6C3F1 mice given injections of N-nitrosodiethylamine (DEN), BALB/c nu/nu or nu/ + mice exposed transplacentally on Day 16 of gestation to ethylnitrosourea (ENU), or BALB/c nu/ + mice exposed to ENU at 8-12 weeks of age were preserved in formalin or Bouin's fixative. After ABC immunocytochemistry, SAP was found in the cytoplasm of normal alveolar Type II cells; in the majority of cells in focal alveolar and solid hyperplasias originating in peribronchiolar or peripheral locations; and in
solid, tubular, papillary, and mixed adenomas and carcinomas. The larger mixed-pattern neoplasms and small or large tubular neoplasms usually had the least namber of cells with SAP. The majority of large papillary adenomas and carcinomas in BALB/c mice exposed to ENU and in untreated A strain mice contained SAP in the nuclei of many neoplastic cells but only in the cytoplasm of a few neoplastic cells. CCA was found in normal Clara cells of bronchi and bronchioles but not in any hyperplastic or neoplastic lesion of any mouse studied. This study provided immunocytochemical evidence that the vast majority of naturally occurring and experimentally induced pulmonary neoplasms of mice are alveolar Type II cell adenomas and carcinomas. (Am J Pathol 1985, 118:493-499)
PULMONARY TUMORS OF MICE were first described in 1940 as originating from alveolar lining cells. I Subsequently, many investigators provided additional morphologic and ultrastructural evidence that naturally occurring and experimentally induced alveolar-bronchiolar neoplasms of mice were of alveolar Type II cell origin.2'-6 Several studies, however, noted that some neoplasms arose in or adjacent to bronchioles, suggesting a bronchiolar epithelial origin of at least some neoplasms. ,6,8,14,17-20 Kauffman et all8 first described significant morphologic evidence for the development of Clara cell neoplasms in Swiss mice transplacentally exposed to ethylnitrosourea (ENU). These Clara cell neoplasms were tubular and papillary in histologic type and had ultrastructural evidence of Clara cell differentiation.7'18'19 Katyal et al described an antibody which immunocytochemically demonstrated surfactant apoprotein (SAP) localized in the cytoplasm of normal rat pulmo-
nary alveolar Type II cells21 and human pulmonary disorders and neoplasms.2224 Others have also developed a similar antibody.25 More recently, Singh et al26 characterized an antibody to the secretory products of rat Clara cells (CCA) with which the antigen(s) were shown to be localized to rat Clara cells. In this report, we demonstrated the localization of SAP and CCA in normal lung and naturally occurring
Supported in part by federal funds from the Department of Health and Human Services, under Contract NOI-CO23910 with Program Resources, Inc., by PHS Grant CA33717 awarded to Dr. Singh by the National Cancer Institute, a grant from the Veterans Administration to Dr. Singh, and NIH Grant HL289193 to Dr. Katyal. Accepted for publication October 23, 1984. Address reprint requests to Dr. J. M. Ward, NCI-Frederick Cancer Research Facility, Building 538, Frederick, MD 21701.
493
494
WARD ET AL
AJP * March 1985
and chemically induced pulmonary hyperplasias and neoplasms of mice.
(Fl mice derived from parents from the Jackson Laboratory, Bar Harbor, Maine) were sacrificed at 22 weeks and 16 months of age.
Materials and Methods Pathology Mice were sacrificed, and complete necropsies were done. Generally, lungs were fixed in formalin and embedded in paraffin, and several representative sections were prepared of each lung. For one large group given DEN and sacrificed 6 months later, whole lung sections were prepared from lungs perfused with formalin. Lungs from some of the ENU-exposed mice were preserved in Bouin's fixative, and representative sections were prepared.
Animals and Chemicals B6C3F1 male mice were obtained from the Animal Genetics and Production Branch of the Division of Cancer Treatment, National Cancer Institute, Frederick, Maryland. At 28 days of age, they received a single intraperitoneal injection of N-nitrosodiethylamine (DEN) in tricaprylin at 80 mg/kg. The mice were part of a large liver tumor promotion study." At 6, 9, and 18 months after injection, groups of mice were sacrificed. They were free of serum antibodies to all known murine viruses, including Sendai virus and pneumonia virus of mice and to Mycoplasma sp. BALB/c nu/+ females and nu/nu males were from a colony derived from mice obtained from GIBCO Animal Resources Laboratory (Madison, Wisconsin).28 ENU was synthesized according to the method of Arnt as described previously.28 Female nu/+ mice impregnated by nu/nu males were given intraperitoneal injections on the 16th day of gestation of 10 or 50 mg/kg of ENU in trioctanoin. The nu/nu and nu/+ offspring were sacrificed at 8-24 months of age. The treated dams were sacrificed 12 months after treatment. Other results obtained with these mice have been previously de-
ABC Immunocytochemistry Selected lungs fixed in formalin or Bouin's fixatives from the mice noted above were sectioned and the avidin-biotin peroxidase complex (ABC) immunocytochemical procedure was followed as previously described.29'30 Rabbit antibodies to SAP2" and control normal rabbit sera were diluted 1:100, 200, and 400 with phosphate-buffered saline (PBS) and used for all cases. Rabbit antibodies to CCA26 and control normal rabbit sera were diluted at 1:400, 800, and 1600 with PBS and used for all cases. The CCA antibody was prepared from immunization of rabbits from serum-free pulmonary lavage in Freund's adjuvant. The antibody was characterized by immunodiffusion, immunoelectrophoresis, and immunocytochemistry.26 The Vectastain ABC Kit (Vector Laboratories, [tic., Burlingame, Calif) was used for immunocytochemical localization of SAP and CCA in tissue sections.
scribed.28
Eighteen male or female B6C3F1 mice 1-2 years of with naturally occurring pulmonary proliferative lesions were originally obtained from the NCI Animal Genetics and Production Branch, Frederick, Maryland, and maintained to 2 years of age as controls as part of carcinogenesis experiments. Untreated strain A mice
age
Table 1-Morphologic Characteristics of Proliferative Pulmonary Lesions in Mice
Treatment
Age at Strain treatment or stock
None None
-
B6C3F1 A
DEN
4 weeks
B6C3F1
ENU
Day 16 of gestation
ENU
-
8-12 weeks
BALB/ct
Age at necropsy 2 years 22 weeks 16 months 6 months 9 months 18 months 8-24 months
nu/nu and nu/+ BALB/c 12 months
Number and location of lesions Morphologic type Number of Mice Peribronchiolar Peripheral* Alveolar Solid Papillary Tubular Mixedt 18 5 6 21 9 9 6
6 0 3 13 8 3 0
12 6 6 25 19 28 17
9 0 2 18 17 0 0
3 5 5 9 2 11 5
0 0 0 1 0 3 12
0 1 0 10 8 14 0
6 0 2 0 0 3 0
6
5
11
0
9
2
2
3
Peripheral, not obviously connected to bronchioles, or too large to determine original location.
t Mixed epithelial patterns.
t Since there were no clear differences related to age or genotype, results from these mice are grouped together.
VOl. 118 * No. 3
ALVEOLAR TYPE II CELL NEOPLASMS OF MICE
495
w
-
*4o,,,
,4
W %sb . %
*
t
%0
.^. I
as
A
a
'.:
d 49f
..e,
0
Figure 1-Small papillary lesion extending into bronchiole from alveoli (or vice versa) of B6C3F1 mouse injected with DEN. Note immunoreactive Clara cell antigen in normal bronchiolar Clara cells but not in cells of papillary lesion. (Immunoperoxidase-hematoxylin, x 220) Figure 2-Solid adenoma with papillary focus involving a bronchiole. Tumor cells contain immunoreactive surfactant apoprotein. Antigen is also seen on surface of bronchiolar cells. (Immunoperoxidase-hematoxylin, x 220) Figure 3-Solid adenoma adjacent to and growing within a bronchiole. (H&E, x 330) Figure 4-A tubular adenoma without immunoreactive surfactant apoprotein. Note normal alveolar Type II cells with antigen. (Immunoperoxidase-hematoxylin, x 220)
496
WARD ET AL
AJP * March 1985
Table 2-Immunoperoxidase Localization of Surfactant Apoprotein (SAP) in Selected Proliferative Pulmonary Lesions of Mice
Treatment None None DEN
ENU-Tt ENU-Pt *
Strain or stock B6C3F1 A
B6C3F1 BALB/c BALB/c
Alveolar 6/9 2/2 10/10
Morphologic type (number positive for SAP/number tested) Tubular Solid Papillary 3/3 6/6 9/11 5/5 7/10
Mixed* 5/6
2/6 10/12 2/2
1/1
1/1
3/9
5/8
2/2
2/2
Mixed epithelial patterns.
t Exposed to ENU transplacentally on the 16th day of gestation. t Exposed to ENU at 8-12 weeks of age.
Results Localization of Clara Cell Antigen and Surfactant Apoprotein in Normal Mouse Lung The use of rabbit anti-rat SAP and anti-rat CCA with ABC immunocytochemistry on normal mouse lung and normal areas of lungs with tumors revealed antigen localization patterns similar to those in rats.2"'26 SAP was localized in alveolar Type II cells of normal lung and could be readily detected at antibody dilutions up to 1:200. Weak and inconsistent staining was seen at 1:400. The Type II cells appeared vacuolated after immunocytochemical staining, and the immunoreactive SAP was found throughout the cytoplasm. Some SAP was found on the surface of alveolar macrophages and bronchiolar epithelial cells. CCA was localized in bronchial and bronchiolar Clara cells. The immunoreactive CCA was seen in a granular, focal and/or diffuse distribution in the cytoplasm of Clara cells at dilutions up to 1:1600. A weak background staining of alveolar Type II cells was usually seen at a dilution of 1:400, but not at 1:800. Sections treated with normal rabbit serum did not have any specific staining even at dilutions of 1:100. Morphologic Classification of Proliferative Pulmonary Lesions Lesions of the mouse lung were classified according to the authors' experience and findings in other
studies.4''7831 Some authors described all focal proliferative alveolar lesions as neoplastic,'0"'1 while others describe the small focal lesions which do not compress adjacent normal parenchyma as focal hyperplasia and small nodular lesions, which compress adjacent normal tissues as adenomas.41' For the purposes of this study we classified the lesions as to morphologic type: alveolar, solid, tubular, papillary, or of a mixed epithelial pattern. Small lesions were either alveolar, solid, or tubular. Alveolar hyperplastic lesions maintained the normal alveolar wall structure but contained
enlarged lining cells. Small nodular lesions were solid, tubular, or papillary. Some small papillary foci were seen within tubular lesions. The largest tumor masses were most often mixed, containing tubular, papillary, and solid areas in varying proportions. Details of the histogenesis and morphology for each experimental group are given below.
Focal Hyperplasias and Lung TImors Induced by DEN in B6C3F1 Mice At 6 and 9 months after DEN injection, pulmonary tumors in B6C3F1 mice were very small (microscopic
only or less than 1 mm in diameter) and originated in alveolar walls in peripheral locations or in association with bronchioles and adjacent to alveoli (Table 1). Twenty-one of 65 lesions at 6 or 9 months were found in close association with bronchioles. None of these lesions were similar to those caused by murine respiratory viruses. Early bronchiolar hyperplastic lesions were frequently papillary and connected to normalappearing bronchiolar cells and hyperplastic or neoplastic alveolar cells (Figure 1). The cells in these lesions were cuboidal and covered a thin connective-tissue core. Immunocytochemical staining revealed that cells in these small papillary lesions contained cytoplasmic SAP antigen distributed in a granular pattern (Figure 2), but no CCA was detectable (Figure 1). Larger lesions adjacent to bronchioles were usually solid (Figure 3) or had an alveolar pattern, and the majority of tumor cells contained SAP in a foamy or vacuolated distribution but lacked CCA. Hyperplastic alveolar lesions in peripheral locations were composed of rounded or cuboidal cells that usually contained SAP but not CCA. Larger alveolar, tubular, or solid lesions (several millimeters in diameter), especially seen in mice at 18 months, were more often mixed. A few tumors had solid foci in which tumor cells contained lipid droplets of varying sizes. The majority of adenomas contained varying amounts of SAP and no CCA (Table 2). A few
Vol. 118 * No. 3
adenomas similar in morphology to those described above contained neither antigen; these included especially the larger papillary, tubular, or mixed tumors. Lesions classified as papillary or tubular (Figure 4) alveolar hyperplasias and tumors and large mixed pattern tumors (usually papillary-solid or tubular-papillary) contained areas with SAP and other areas without the antigen (Figure 5). Areas of mixed tumors lacking in SAP usually appeared cytologically malignant and in some sections invaded adjacent parenchyma and bronchioles. Often, focal areas of these lesions contained some tumor cells with SAP in a cytoplasmic granular or vacuolar distribution but always lacked CCA.
Lung Tumors Induced by ENU Three morphologic types of lung tumors (adenomas and carcinomas) were seen in mice transplacentally exposed to ENU. Solid, tubular, and papillary patterns, as described previously, were found in most mice.8"7'18 The cytoplasm of variable numbers of tumor cells in areas of solid and tubular tumors were immunoreactive in a granular pattern with SAP but not CCA, when fixed in Bouin's fixative. Tumor cells in papillary tumors contained many intranuclear inclusions immunoreactive with SAP (Figure 6) but not CCA (Figure 7). Only a rare papillary tumor cell contained SAP in a cytoplasmic granular pattern. Tumors induced postnatally by ENU and fixed in Bouin's were more solid and less papillary than transplacentally induced tumors but 13/16 tumors contained SAP. Some intranuclear inclusions contained SAP. Several formalin-fixed lungs contained alveolar Type II cells and tumors that stained poorly for SAP. These lungs had been stored in fixative for over 1 year.
Naturally Occurring Tumors of B6C3F1 and Strain A Mice
Naturally occurring tumors of B6C3F1 mice were in peribronchiolar (6 tumors), peripheral, or undetermined (due to large tumor size) locations (Table 1). Tumors were of the alveolar, solid, or of mixed patterns. Small lesions were of the alveolar or solid types. Almost all tumors contained SAP but not CCA (Table 2). One solid area of a mixed tumor had SAP immunoreactive tumor cells with lipid droplets and large vacuoles. Strain A mice had alveolar, solid and mixed lesions similar to those of other strains. All tumors contained variable numbers of tumor cells with intracytoplasmic SAP in a granular distribution, and three tumors also contained intranuclear inclusions immunoreactive for SAP (Figure 8).
ALVEOLAR TYPE II CELL NEOPLASMS OF MICE
497
Discussion The present study of naturally occurring and experimentally induced hyperplasias and neoplasms of mice clearly demonstrated that most lesions contained SAP and not CCA, even when they arose in closed association with bronchioles. It is not surprising that proliferative cells in close association with bronchiolar epithelium contained SAP because alveoli are immediately adjacent to bronchioles. The small lesions in peribronchiolar or peripheral locations almost always contained SAP, and the largest mixed tumors usually had large areas without SAP. Thus, SAP, an antigen found in normal alveolar Type II cells, was often absent from areas of mixed pattern tumors, which must have been less differentiated. Because almost all small lesions contained SAP, we would suggest that these hyperplasias and tumors arise from alveolar Type II cells, and that diagnoses of alveolar Type II cell hyperplasia, adenoma, and carcinoma are warranted. Although the less common and small tubular hyperplasias and adenomas were most often lacking in SAP, some clearly contained SAP but always lacked CCA. These lesions were never seen originating within bronchioles. They may represent a less differentiated lesion of Type II cells or could arise from alveolar Type I cells, alveolar brush cells,32 lipid-containing cells,33 or other pulmonary cells, including Clara cells. The lipid found in some focal areas of tumors may have resulted from cellular degeneration or differentiation of pulmonary stem cells into cells resembling the alveolar fatcontaining cell.33 Our histologic findings appear to agree with those of others."'-0"'1 Small (early) lesions arose from alveolar Type II cells, either peribronchiolar or peripheral, most often with an alveolar or solid pattern. As the lesions enlarged they often appeared mixed, with new morphologic types appearing,6 some of which appeared cytologically malignant and which were deficient in SAP. Naturally occurring and experimentally induced lesions were in general strikingly similar with regard to cellular localization of SAP. An exception to this similarity was the appearance of SAP in the nuclei of ENU-induced papillary tumors. Nuclear SAP is not seen in normal Type II alveolar cells but does occur in adenocarcinomas of human lung and appears to represent antigen associated with tubular nuclear inclu-
sions.22,25 Kauffman examined ENU-induced papillary lung tumors in Swiss (Bagg-Webster) mice and concluded, on the basis of ultrastructural characteristics, that some of these were of Clara cell origin.'8 Because we rarely found CCA and always detected SAP within similar tumors, we conclude that these probably arose from alveolar Type II cells. The findings of CCA in normal
498
WARD ET AL
AJP * March 1985
Figure 5-Portion of a large mixed carcinoma showing immunoreactive surfactant apoprotein in areas of the tumor. (Immunoperoxidase-hematoxylin, x 330) Figure 6-Surfactant apoprotein in nuclei of a papillary adenocarcinoma induced transplacentally by ENU. (Immunoperoxidase-hematoxylin, x 240) Figure 7-Papillary adenocarcinoma induced transplacentally by ENU showing Clara cell antigen in normal bronchiolar Clara cells but not in tumor cells. (Immunoperoxidase-hematoxylin, x 220) Figure 8-Surfactant apoprotein in nuclei and cytoplasm (arrows) of tumor cells in spontaneous solid carcinoma of an A strain mouse. (Immunoperoxidase-hematoxylin, x 540)
ALVEOLAR TYPE II CELL NEOPLASMS OF MICE
Vol. 118 * No. 3
Clara cells in the same lung sections confirmed our assumption that CCA was lacking in the tumors. Possible reasons for these discrepant conclusions include special propensity for Bagg-Webster mice to develop Clara cell tumors, acquisition of Clara cell ultrastructural features during progression of tumors of Tyrpe II cell origin, or loss of CCA and acquisition of nuclear SAP during progression and dedifferentiation of Clara cell tumors. The finding of different numbers and sizes of secretory granules in normal mouse Clara cells and in papillary tumors of mice"8 suggests antigenic and chemical differences may exist as well. It would, however, be extremely unusual for a characteristic cell-specific antigen (CCA) to be lost from all tumors because SAP was frequently found in small and large alveolar Type II cell tumors. Experiments are planned to distinguish between these possibilities.
15. 16.
17. 18. 19. 20. 21. 22.
References 1. Grady HG, Stewart HL: Histogenesis of induced pulmonary tumors in strain A mice. Am J Pathol 1940, 16:417-432 2. Biancifiori C, Giornelli Santilli FE, Milia U, Bucciarelli E: Histogenesis of pulmonary tumors by hydrazine sulphate in Balb/C Cb/Se substrain mice, Lung Tumors in Animals. Edited by L Severi. Division of Cancer Research, University of Perugia, Italy, 1966, pp 881-894 3. Brooks RE: Pulmonary adenoma of strain A mice: An electron microscopic study. J Natl Cancer Inst 1968, 41:719-742 4. Goodman DG, Bates RR, Ward JM, Frith CH, Sauer RM, Jones SR, Strandberg JD, Squire RA, Montali RJ, Parker GA: Common lesions in aged B6C3F1 (C57BL/6N x C3H/HeN)Fl and Balb/CStCrlfC3H/NcTr mice. Washington, DC, Armed Forces Institute of Pathology, 1981, pp 3-7 5. Mori K: Further studies on the histogenesis of pulmonary tumors in mice induced by 4-nitroquinoline 1-oxide. Gann 1964, 55:315-323 6. Mori K, Hirafuku I: Histogenesis of lung carcinoma in mice induced by 4-nitroquinoline 1-oxide: carcinoma arising from areas of adenoma. Gann 1964, 55:205-209 7. Rijhsinghani KS, Krakower C, Abrahams C, Swerdlow M, Rao KVN, Ghose T: A light and electron microscopic study of liver and lung tumors induced by a single low dose of diethylnitrosamine (DEN) in C57BL x C3HF1 mice. Cancer Detection and Prevention 1981, 4:85-97 8. Sato T, Kauffman SL: A scanning electron microscopic study of the type II and Clara cell adenoma of the mouse lung. Lab Invest 1980, 43:28-36 9. Shimkin MB, Stoner GD: Lung tumors in mice: Application to carcinogenesis bioassay. Adv Cancer Res 1975, 21:1-58 10. Stewart HL: Comparative aspects of certain cancers, Cancer, Vol 4. Edited by FF Becker. New York, Plenum Press, 1975, pp 303-374 11. Stewart HL, Dunn TB, Snell KC, Deringer MK: Tumours of the respiratory tract, Pathology of Tumours in Laboratory Animals. Vol II, TUmours of the Mouse. Edited by VS Turusov. Lyon, IARC, 1979, pp 251-287 12. Suzuki Y: Neoplastic effect of vinyl chloride in mouse lung: Lower doses and short-term exposure. Environ Res 1983, 32:91-103 13. Svoboda DL: Ultrastructure of pulmonary adenomas in mice. Cancer Res 1962, 22:1197-1201 14. Takayama S, Oota K: Induction of malignant lung tumors
23.
24.
25. 26.
27.
28. 29.
30. 31.
32. 33.
499
in various strains of mice by oral administration of Nnitrosodimethylamine and N-nitrosodiethylamine,2 pp 677-694 Ward JM, Goodman DG, Squire RA, Chu KC, Linhart MS: Neoplastic and nonneoplastic lesions in aging B6C3F1 mice. J Natl Cancer Inst 1979, 63:849-854 Witschi H, Haschek WM: Cells of origin of lung tumors in mice (corres). J Natl Cancer Inst 1983, 70:991 Kauffman SL: Histogenesis of the papillary Clara cell adenoma. Am J Pathol 1981, 103:174-180 Kauffman SL, Alexander L, Sass L: Histologic and ultrastructural features of the Clara cell adenoma of the mouse lung. Lab Invest 1979, 40:708-716 Parsa I, Kauffman SL: Malignant Clara cell line derived from ethylnitrosourea-induced murine lung adenoma. Cancer Lett 1983, 18:311-316 Choudhury C, Kauffman SL, Serravalli E, Durkin H: Lymphocytic infiltration of bronchoalveolar adenomas in mice. Cancer Lett 1983, 20:299-304 Katyal SL, Singh G: An immunologic study of the apoproteins of rat lung surfactant. Lab Invest 1979, 40:562-567 Singh G, Katyal SL, Torikata C: Carcinoma of type II pneumocytes. Immunodiagnosis of a subtype of "bronchio-alveolar carcinomas." Am J Pathol 1981, 102:195-208 Singh G, Katyal SL, Ordonez NG, Dail DH, Negishi Y, Weedn VW, Marcus PB, Weldon-Linne CM, Axiotis CA, Alvarez-Fernandez E, Smith WI: Type II pneumocytes in pulmonary tumors: Implications for histogenesis. Arch Pathol Lab Med 1984, 108:44-48 Singh G, Katyal SL: Surfactant apoprotein in nonmalignant pulmonary disorders. Am J Pathol 1980, 101:51-62 Dairaku M, Sueishi K, Tanaka K, Horie A: Immunohistological analysis of surfactant-apoprotein in the bronchiolo-alveolar carcinoma. Virchows Arch [Pathol Anat] 1983, 400:223-234 Singh G, Katyal SL: An immunological study of the secretory products of rat Clara cells. J Histochem Cytochem 1984, 32:49-54 Ward JM, Rice JM, Creasia D, Lynch P, Riggs C: Dissimilar patterns of promotion by di(2-ethylhexyl)phthalate and phenobarbital of hepatocellular neoplasia initiated by diethylnitrosamine in B6C3F1 mice. Carcinogenesis 1983, 4:1021-1029 Anderson LM, Last-Barney K, Budinger JM: Sensitivity to carcinogenesis in nude mice: skin tumors caused by transplacental exposure to ethylnitrosourea. Science 1982, 218:682-684 Hsu SM, Raine L, Fanger H: Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody PAP procedures. J Histochem Cytochem 1981, 29:577-580 Ward JM, Reynolds CW, Argilan F: Immunoperoxidase localization of large granular lymphocytes in normal tissues and lesions of athymic nude rats. J Immunol 1983, 131:132-139 Ward JM: Background data and variations in tumor rates of control rats and mice. Prog Exp Tumor Res 1983, 26:241-258 Meyrick B, Reid L: The alveolar brush cell in rat lung: A third pneumocyte. J Ultrastruct Res 1968, 23:71-80 Maksvytis HJ, Vaccaro C, Brody JS: Isolation and characterization of the lipid-containing interstitial cell from the developing rat lung. Lab Invest 1981, 45:248-259
Acknowledgments We are grateful for the aid of Fred Argilan, Kim Forsman, and Cindy Harris in immunohistotechnique, Joyce Vincent and Kathy Breeze in typing, and Larry Ostby in photomicrography.
