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Morphological identification of alpha-1-antitrypsin in pulmonary macrophages
Medical Intelligence MORPHOLOGICAL IDENTIFICATION OF ALPHA-1-ANTITRYPSlN IN PULMONARY MACROPHAGES* PRABODH K. GUPTA, M.D.,t JOHN K. FROST, M.D.,:I: SUSAN GEDDES, C.T.(A.S.C.P.),w BONNm ARACIL, C.T.(A.S.C.P.),[[ AgO FLORENCEDAVlnOVSKI, B.A.82
Abstract
Pulmonary macrophages, as seen in the routine sputum samples obtained from smokers, reveal bipolar orangophilic spurs. By use of histochemical and immunofluorescence techniques, these spurs have been found to contain alpha-l-antitrypsin. Morphological localization of this substance w#hin the pulrnonary macrophages may be significant. Alpha-l-antitrypsin, a glycoprotein, is an alpha-l-globulin synthesized in the human liver. ~ Present in serum, along alveolar walls, and within pulmonary macrophages, it inhibits proteolytic enzymes, including trypsin, chymotrypsin, elastase, and collagenase. Abnormally elevated serum levels of alpha1-antitrypsin have been noted in lung cancer patients with concomitant pulmonary infections, z Significantly lowered serum levels indicate a genetic deficiency, in which the body is incapable of protecting itself against
*This work was supported in part b}' National Cancer Institute contract no. N01-CB-92172 and a grant from the William Penn Foundation, Phi|adelphia. -~Assistant Professor, Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland. SProfessor, Department of l'athology, Johns ttopkins University School of Medicine, Baltimore, Maryland. w Johns Hopkins University School of Medicine, Bahimore, Maryland. IlStaff, St. Agnes Hospital, Baltimore, Mat3'land. 82 Johns Hopkins University School of Cytotechnology, Baltimore, Maryland.
unchecked proteases. Persons with alpha-lantitrypsin deficiency, both homozygous and heterozygous, develop emphysema prematurely, and smokers from either group have an alarmingly increased incidence of chronic obstructive pulmonary disease, s By identifying sites of location of alpha-l-antitrypsin within the lung, one may begin to speculate about the mechanism of its action and its relationship to chronic obstructive pulmonary disease, smoking, and lung cancer. 4 Alpha-l-antitrypsin can be stained with periodic acid-Schiff and Masson's trichrome stains, and is specifically detectable by fluorescein isothiocyanate labeled antisera. ~'6 It appears as orangophilic droplets in hepatocytes when stained by the modified Papanicolaou methodY Tictorially similar polar orangophilic structures occnr in pulmonary macrophages in Papanicotaou stained sputum specimens obtained from smokers, s'9 T h e present study was designed to morphologically identifyand localize alpha-l-antitrypsin within alveolar macrophages. MATERIALS AND METHODS Saccomanno fixed sputum specimens obtained from 50 smokers, !0 nonsmokers, and 20 patients with chronic obstructive pulmonary disease, as evidenced by chest roentgenograms, were stained by the modified Papanicolaou procedure, periodic acid-Schiff stain with diastase treatment, Masson's trichrome stain, and fluorescein isothiocyanate labeled antisera against alpha-l-antitrypsin by the direct immunofluorescence technique. Z~ Fluorescein labeled material was examined by using a Leitz Orthoplan microscope equipped with 9 Ploem epi-illumination; KP490 rim., BG38 exciter, and 530 nm. barrier filters; and an HBO 100 light source. RESULTS AND DISCUSSION Alveolar macrophages from smokers and patients with chronic obstructive pulmonary disease revealed a golden yellow fluorescence related to the tobacco pigment. The polar orangophilic structures of pulmonary macrophages from smokers, seen in Papanicolaou stained specimens, gave specific fluorescence staining reactions with flttorescein isothiocyanate labeled antiserum against alpha-Iantitrypsin (Fig. 1). Immunofluorescence also 3 : 4 5
HUMAN PATHOLOGY--VOLUME 10, NUMBER 3 May 1979
.jtO., "-'t .-~
Figure 1. Pulnaonary macrophage in spunnn. Fluorescence of FITC labeled antiserum against at-AT localizes its bipolar distribution in tire macrophage. revealed the presence of alpha-l-antitrypsin on the periphery of and scattered irregularly over the pulmonary macrophages (Figs9 2, 3). In a few sputum specimens containing antorpltous orangophilic material in the background, alpha-l-antitrypsin was detectable at these sites b)' immunofluorescence. Tiffs orangophilic material stained pink witlt periodic acid-Schiff stain after diastase treatment, and bright red with Masson's trichrome stain. No alplm-l-antitrypsin was identified in sputum specimens from nonsmokers. Qualitative differences among tlte various other groups were insignificant.
Figure 3. Same group of cells as in Figure 2, shown after restaining with Papanicolaou stain. The granules previously labeled b)*F1TC as arAT appear brigltt orange (dark in figure).
Our investigations establish the morphological identification of alpha-l-antitrypsin in pulmonary macrophages. Alpha-l-antitrypsin can be recognized in pulmonary macropbages of Saccomanno fixed, Papanicolaou stained sputum specimens.~,It stains orangophilic or eosinopbilic materi,q] o ~ u r r i n g at the poles, the periphery, or randomly scattered over the cell. Identification and morpttological localization of alpha-l-antitrypsin within alveolar macrophages in Saccomanno fixed sputnm may have significant implications not only in relation to its role in cttronic obstructive pulmonary disease and puhnonary emphysema, but also in ptdmonary neoplasm. References
,ip
346
Figure 2. Ptdmonary macropltages in spt, tunl. FITC labeled antiseruna identifies my-ATwitltin the cells.
1. Laurell, C. B., and Ericksson, S.: The electrophoretic alpha- l-globulin pattern of serum alpha- l-antltrypsin deficiency. Scand. J. Clin. Lab. Invest., 15:132-140, 1963. 2. ttarris, C. C., Primack, A., and Cohen, M.: Elevated alpha-l-antitr)'psin serum levels in lung cancer patients. Cancer, 34:280-281, 1974. 9 3. Lieberman, J.: t]eterozygous and homozygous alphar antitrypsin deficiency in patients with pulmonary emphysema. New Eng. J. Med., 281:279-284, 1969. 4. Caplin, M., and Festenstein, F.: Relation between lung cancer, chronic bronchitis and airways obstruction. Brit. Med.J.,3:678-680, 1975. 5. Cohen, A. B.: huerrelationships between tim human alveolar macrophage and alpha-l-antitrypsin. J. Clin. Invest., 52:2793-2799, 1973. 6. Tuttle, S. C., and Jones, R. K.: Fluorescent antibody studies of alpha-l-antitrypsin in adult human lung. Amer. J. Clin. Path., 64:477--482, 1975. 7. Cooper, H. C., and Gupta, P. K.: Hepatic t3topathoIogy in alpha-l-antitrypsin deficiency. Amer. J. Clin. Path., 62 : 118-122, 1974. 8. Frost, J. K., Gupta, P. K., Erozau, Y. S., C~rter, D.,
MEDICAL INTELLIGENCE Hollander, D. It., Levin, M. L., and Ball, W. C., Jr.: Pulmonary c)tologlc alterations in toxic environmental inhalation, ttuman Path., 4:521-536, 1973. 9. Walker, K. R., and Fulhner, C. D.: Observations of eosinophilic extracytoplasmic processes in pulmonarT macrophages. Acta Cytol.,15:363-364, 1971. 10. Saccomanno, G., Saundcrs, R. P., Ellis, tt., Archer, V. E., Wood, B. G., and Beckler, P. A.: Concentration of carcinoma or atypical ceils in sputum. Acta Cytol., 7:305-310, 1963.
JUXTAPOSITION OF THE ATRIAL APPENDAGES
PAULJ. BOOR,M.D.,* AND PATRICK R. McALLISTER, P n . D . t
Abstract
Juxtaposition of the atrial appendages is a rare congenital cardiac anomaly ahnost universally associated with severe conotruncal abnormalities, especially transposition of the aorta. This presentation describes a case of right sided juxtaposition of the atrial appendages without concomitant cdnotruncal malformations. This finding contradicts previous hypotheses concerning the genesis of jlLxtaposition. Juxtaposition o f the atrial a p p e n d a g e s is a rare cardiac anomaly in which both atrial appendages lie to one side o f the great arteries. This malformation may be diagnosed clinically b)' cardiac catbeterization and in tile majority o f cases is associated with other severe conotruncal cardiac anomalies, especially transposition o f the aorta. 15 Because o f this association, most authors consider juxtaposition to be a consequence o f the m o r e severe associated cardiac abnormalities. As an exception to this concept, this communication describes a case o f right sided juxtaposition of the atrial a p p e n d a g e s without severe conotruncal malformation. CASE H I S T O R Y A 930 gin. infant boy was delivered by cesarean section to a l~rimagravida I.atin American female in severe eclampsia. T h e patient was i m m a t u r e (estimated gestational age: 28 weeks), fed poorly, and was noted d u r i n g the second day o f life to have a g r a d e 3/6 systolic m u r m u r consistent with a patefit ductns *Assistant Professor of Pathology, University of Texas Medical Branch. Staff Pathologist, University of Texas Medical Branch Hospitals. Galveston, Texas. "LMedical Student, University of Texas Medical Branch, Galveston, Texas.
arteriosus. He was transferred to tile John Seal)" Hospital where surgical r e p a i r o f the patent ductus arteriosus was p e r f o r m e d at 15 days of age. Following surgery, the patient did well. However, a persistent softer bigh pitched systolic m u r m u r raised the diagnostic possibility o f ventricular septal defect, At 19 days o f age the patient suffered an episode p r e s u m e d to be sepsis and developed Enterobacter cloacae meningitis, which r e s p o n d e d to therapy. Subsequentl)' cardiac catheterization confirmed the diagnosis o f ventricular septal defect. T h e patient became severely hypotensive immediately following the procedure, develo p e d necrotizing enterocolitis and sepsis, and died at the age o f two months.
PATHOLOGY At autopsy the patient weighed 1000 gill. and showed no external congenital abnormalities. T h e normal orientation of the abdominal organs, atria, and inferior and s u p e r i o r venae cavae was indicative o f a solitus type o f visceroatrial situs. Tile surgical ligation o f the ductus arteriosus was intact. During the in situ examination o f tile thorax, both atrial a p p e n d a g e s were f o u n d to tile right o f the great vessels. ~ a c h a p p e n d a g e retained its characteristic m0r,'Ohology; i.e., the right atrial a p p e n d a g e was ~0rgad based, flat, and cuboidal, whereas the left atrial a p p e n dage was thin, finger-like, and curved. T h e left a p p e n d a g e bent toward the left a n d thus was overlying tile aorta (Fig. I). Dissection o f the h e a r t confirmed tile connection o f each atrial a p p e n d a g e with its respective atrium. T i l e foramen ovale was minimally patent (0.2 cm. in diameter). Tile right ventricle was moderately h)'pertrophic and a p p r o x i m a t e d the left ventricle in size. A small ventricular septal defect (1.5 ram. in diameter) was located just b e l o w the tricuspid valve in the muscular tissue o f the septal band (Fig. 2). T h e p u l m 0 n a r y artery and aorta were o f normal size and orientatiou. Tile left atrinm and ventricle showed no abnormalities o t h e r than the ventrictflar septal defect beneath the aortic vah'e. All cardiac valves were norlnal. Other significant pathologic findings that presumably led to the patient's demise included riglit middle and lower Iobc h e m o r r h a g i c p n e u m o n i a and severe necrotizing ileocolitis. Postmortem cuhures o f lung and meninges grew Enlerobacter cloacae. Neuropathologic changes included unusually severe nutritional d a m a g e in the hrain, presumably related to the patient's long p e r i o d o f intravenous nourishment.
347
Abstract
Pulmonary macrophages, as seen in the routine sputum samples obtained from smokers, reveal bipolar orangophilic spurs. By use of histochemical and immunofluorescence techniques, these spurs have been found to contain alpha-l-antitrypsin. Morphological localization of this substance w#hin the pulrnonary macrophages may be significant. Alpha-l-antitrypsin, a glycoprotein, is an alpha-l-globulin synthesized in the human liver. ~ Present in serum, along alveolar walls, and within pulmonary macrophages, it inhibits proteolytic enzymes, including trypsin, chymotrypsin, elastase, and collagenase. Abnormally elevated serum levels of alpha1-antitrypsin have been noted in lung cancer patients with concomitant pulmonary infections, z Significantly lowered serum levels indicate a genetic deficiency, in which the body is incapable of protecting itself against
*This work was supported in part b}' National Cancer Institute contract no. N01-CB-92172 and a grant from the William Penn Foundation, Phi|adelphia. -~Assistant Professor, Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland. SProfessor, Department of l'athology, Johns ttopkins University School of Medicine, Baltimore, Maryland. w Johns Hopkins University School of Medicine, Bahimore, Maryland. IlStaff, St. Agnes Hospital, Baltimore, Mat3'land. 82 Johns Hopkins University School of Cytotechnology, Baltimore, Maryland.
unchecked proteases. Persons with alpha-lantitrypsin deficiency, both homozygous and heterozygous, develop emphysema prematurely, and smokers from either group have an alarmingly increased incidence of chronic obstructive pulmonary disease, s By identifying sites of location of alpha-l-antitrypsin within the lung, one may begin to speculate about the mechanism of its action and its relationship to chronic obstructive pulmonary disease, smoking, and lung cancer. 4 Alpha-l-antitrypsin can be stained with periodic acid-Schiff and Masson's trichrome stains, and is specifically detectable by fluorescein isothiocyanate labeled antisera. ~'6 It appears as orangophilic droplets in hepatocytes when stained by the modified Papanicolaou methodY Tictorially similar polar orangophilic structures occnr in pulmonary macrophages in Papanicotaou stained sputum specimens obtained from smokers, s'9 T h e present study was designed to morphologically identifyand localize alpha-l-antitrypsin within alveolar macrophages. MATERIALS AND METHODS Saccomanno fixed sputum specimens obtained from 50 smokers, !0 nonsmokers, and 20 patients with chronic obstructive pulmonary disease, as evidenced by chest roentgenograms, were stained by the modified Papanicolaou procedure, periodic acid-Schiff stain with diastase treatment, Masson's trichrome stain, and fluorescein isothiocyanate labeled antisera against alpha-l-antitrypsin by the direct immunofluorescence technique. Z~ Fluorescein labeled material was examined by using a Leitz Orthoplan microscope equipped with 9 Ploem epi-illumination; KP490 rim., BG38 exciter, and 530 nm. barrier filters; and an HBO 100 light source. RESULTS AND DISCUSSION Alveolar macrophages from smokers and patients with chronic obstructive pulmonary disease revealed a golden yellow fluorescence related to the tobacco pigment. The polar orangophilic structures of pulmonary macrophages from smokers, seen in Papanicolaou stained specimens, gave specific fluorescence staining reactions with flttorescein isothiocyanate labeled antiserum against alpha-Iantitrypsin (Fig. 1). Immunofluorescence also 3 : 4 5
HUMAN PATHOLOGY--VOLUME 10, NUMBER 3 May 1979
.jtO., "-'t .-~
Figure 1. Pulnaonary macrophage in spunnn. Fluorescence of FITC labeled antiserum against at-AT localizes its bipolar distribution in tire macrophage. revealed the presence of alpha-l-antitrypsin on the periphery of and scattered irregularly over the pulmonary macrophages (Figs9 2, 3). In a few sputum specimens containing antorpltous orangophilic material in the background, alpha-l-antitrypsin was detectable at these sites b)' immunofluorescence. Tiffs orangophilic material stained pink witlt periodic acid-Schiff stain after diastase treatment, and bright red with Masson's trichrome stain. No alplm-l-antitrypsin was identified in sputum specimens from nonsmokers. Qualitative differences among tlte various other groups were insignificant.
Figure 3. Same group of cells as in Figure 2, shown after restaining with Papanicolaou stain. The granules previously labeled b)*F1TC as arAT appear brigltt orange (dark in figure).
Our investigations establish the morphological identification of alpha-l-antitrypsin in pulmonary macrophages. Alpha-l-antitrypsin can be recognized in pulmonary macropbages of Saccomanno fixed, Papanicolaou stained sputum specimens.~,It stains orangophilic or eosinopbilic materi,q] o ~ u r r i n g at the poles, the periphery, or randomly scattered over the cell. Identification and morpttological localization of alpha-l-antitrypsin within alveolar macrophages in Saccomanno fixed sputnm may have significant implications not only in relation to its role in cttronic obstructive pulmonary disease and puhnonary emphysema, but also in ptdmonary neoplasm. References
,ip
346
Figure 2. Ptdmonary macropltages in spt, tunl. FITC labeled antiseruna identifies my-ATwitltin the cells.
1. Laurell, C. B., and Ericksson, S.: The electrophoretic alpha- l-globulin pattern of serum alpha- l-antltrypsin deficiency. Scand. J. Clin. Lab. Invest., 15:132-140, 1963. 2. ttarris, C. C., Primack, A., and Cohen, M.: Elevated alpha-l-antitr)'psin serum levels in lung cancer patients. Cancer, 34:280-281, 1974. 9 3. Lieberman, J.: t]eterozygous and homozygous alphar antitrypsin deficiency in patients with pulmonary emphysema. New Eng. J. Med., 281:279-284, 1969. 4. Caplin, M., and Festenstein, F.: Relation between lung cancer, chronic bronchitis and airways obstruction. Brit. Med.J.,3:678-680, 1975. 5. Cohen, A. B.: huerrelationships between tim human alveolar macrophage and alpha-l-antitrypsin. J. Clin. Invest., 52:2793-2799, 1973. 6. Tuttle, S. C., and Jones, R. K.: Fluorescent antibody studies of alpha-l-antitrypsin in adult human lung. Amer. J. Clin. Path., 64:477--482, 1975. 7. Cooper, H. C., and Gupta, P. K.: Hepatic t3topathoIogy in alpha-l-antitrypsin deficiency. Amer. J. Clin. Path., 62 : 118-122, 1974. 8. Frost, J. K., Gupta, P. K., Erozau, Y. S., C~rter, D.,
MEDICAL INTELLIGENCE Hollander, D. It., Levin, M. L., and Ball, W. C., Jr.: Pulmonary c)tologlc alterations in toxic environmental inhalation, ttuman Path., 4:521-536, 1973. 9. Walker, K. R., and Fulhner, C. D.: Observations of eosinophilic extracytoplasmic processes in pulmonarT macrophages. Acta Cytol.,15:363-364, 1971. 10. Saccomanno, G., Saundcrs, R. P., Ellis, tt., Archer, V. E., Wood, B. G., and Beckler, P. A.: Concentration of carcinoma or atypical ceils in sputum. Acta Cytol., 7:305-310, 1963.
JUXTAPOSITION OF THE ATRIAL APPENDAGES
PAULJ. BOOR,M.D.,* AND PATRICK R. McALLISTER, P n . D . t
Abstract
Juxtaposition of the atrial appendages is a rare congenital cardiac anomaly ahnost universally associated with severe conotruncal abnormalities, especially transposition of the aorta. This presentation describes a case of right sided juxtaposition of the atrial appendages without concomitant cdnotruncal malformations. This finding contradicts previous hypotheses concerning the genesis of jlLxtaposition. Juxtaposition o f the atrial a p p e n d a g e s is a rare cardiac anomaly in which both atrial appendages lie to one side o f the great arteries. This malformation may be diagnosed clinically b)' cardiac catbeterization and in tile majority o f cases is associated with other severe conotruncal cardiac anomalies, especially transposition o f the aorta. 15 Because o f this association, most authors consider juxtaposition to be a consequence o f the m o r e severe associated cardiac abnormalities. As an exception to this concept, this communication describes a case o f right sided juxtaposition of the atrial a p p e n d a g e s without severe conotruncal malformation. CASE H I S T O R Y A 930 gin. infant boy was delivered by cesarean section to a l~rimagravida I.atin American female in severe eclampsia. T h e patient was i m m a t u r e (estimated gestational age: 28 weeks), fed poorly, and was noted d u r i n g the second day o f life to have a g r a d e 3/6 systolic m u r m u r consistent with a patefit ductns *Assistant Professor of Pathology, University of Texas Medical Branch. Staff Pathologist, University of Texas Medical Branch Hospitals. Galveston, Texas. "LMedical Student, University of Texas Medical Branch, Galveston, Texas.
arteriosus. He was transferred to tile John Seal)" Hospital where surgical r e p a i r o f the patent ductus arteriosus was p e r f o r m e d at 15 days of age. Following surgery, the patient did well. However, a persistent softer bigh pitched systolic m u r m u r raised the diagnostic possibility o f ventricular septal defect, At 19 days o f age the patient suffered an episode p r e s u m e d to be sepsis and developed Enterobacter cloacae meningitis, which r e s p o n d e d to therapy. Subsequentl)' cardiac catheterization confirmed the diagnosis o f ventricular septal defect. T h e patient became severely hypotensive immediately following the procedure, develo p e d necrotizing enterocolitis and sepsis, and died at the age o f two months.
PATHOLOGY At autopsy the patient weighed 1000 gill. and showed no external congenital abnormalities. T h e normal orientation of the abdominal organs, atria, and inferior and s u p e r i o r venae cavae was indicative o f a solitus type o f visceroatrial situs. Tile surgical ligation o f the ductus arteriosus was intact. During the in situ examination o f tile thorax, both atrial a p p e n d a g e s were f o u n d to tile right o f the great vessels. ~ a c h a p p e n d a g e retained its characteristic m0r,'Ohology; i.e., the right atrial a p p e n d a g e was ~0rgad based, flat, and cuboidal, whereas the left atrial a p p e n dage was thin, finger-like, and curved. T h e left a p p e n d a g e bent toward the left a n d thus was overlying tile aorta (Fig. I). Dissection o f the h e a r t confirmed tile connection o f each atrial a p p e n d a g e with its respective atrium. T i l e foramen ovale was minimally patent (0.2 cm. in diameter). Tile right ventricle was moderately h)'pertrophic and a p p r o x i m a t e d the left ventricle in size. A small ventricular septal defect (1.5 ram. in diameter) was located just b e l o w the tricuspid valve in the muscular tissue o f the septal band (Fig. 2). T h e p u l m 0 n a r y artery and aorta were o f normal size and orientatiou. Tile left atrinm and ventricle showed no abnormalities o t h e r than the ventrictflar septal defect beneath the aortic vah'e. All cardiac valves were norlnal. Other significant pathologic findings that presumably led to the patient's demise included riglit middle and lower Iobc h e m o r r h a g i c p n e u m o n i a and severe necrotizing ileocolitis. Postmortem cuhures o f lung and meninges grew Enlerobacter cloacae. Neuropathologic changes included unusually severe nutritional d a m a g e in the hrain, presumably related to the patient's long p e r i o d o f intravenous nourishment.
347