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Concanavalin-A agglutination of human pulmonary alveolar macrophages from nonsmokers and cigarette smokers: A comparison
Life Sciences Vol . 18, pp . 1177-1184, 1976 Printed in the U .S .A .
Pargamon Preea
CONCANAVALIN-A AGGLUTINATION OF HUMAN PULMONARY ALVEOLAR MACROPHAGES FROM NONSMOKERS AND CIGARETTE SMOKERS : A COMPARISON Glenn A . Warr and R . Russell Martin Departments of Medicine, and Microbiology A Immunology Baylor College of Medicine, Houston, Texas 77030 (Received in final form April 12, 1976)
Incubation of pulmonary alveolar macrophages (PAMs) from nonsmokers in phosphate buffered saline (PBS) leads to minimal autoagglutination of these cells, while smokers PAMs incubated in medium remained individually dispersed in suspension . Incubation with the lectin concanavalin A (Con-A) induced agglutination of PAMs from cigarette smokers, while PAMs from nonsmokers were not further agglutinated . After trypsin treatment, both nonsmoker and smoker PAMs agglutinated in the p~~ence of Con-A . Both PAM populations bound the same amount of I-Con-A . Low temperatures (4°C), a-methylmannose, and pre-fixation of smoker PAMs with Candida glutaraldehyde prevented Con-A induced cell agglutination . bound to PAMs by Con-A were similarly distributed on the surfaces of nonsmoker and smoker cells . Cigarette smoking has been associated with morphological changes in-pulmonary alveolar macrophages (PAMs), the most striking being increased cytoplasmic inclusions (1,2), and alterations in a number of functional characteristics (3-5) . lie have reported changes in surface topography and a reduction in membrane complement receptors of PAMs from cigarette smokers (G .A . WARR, R .R . MARTIN, and L .O . GENTRY, Bulletin of the XXIIIrd Conference of the International Union Against Tuberculosis, 1975, in press) . Fibroblast cell lines which are transformed by oncogenic viruses have changes in cellular cyclic AMP levels, surface topography, and in agglutinability induced by the plant lectin, Con-A (5) . The association of agglutina tion induced by Con-A and transformation of cell lines has also been reported by others (7-11) . The present study examines binding of Con-A and lectin-induced cell agglutination, using PAMs from young healthy nonsmokers and cigarette smokers . Methods Pre orat ion of pu l monary alveolar macrophages : Using a fiberoptic bronchoscope as previously described (3), PAMs were obtained by saline lavage from 26 healthy volunteers, ranging in age from 21 to 27 years . Lavaged cells (79 to 87x of which were PAMs) were washed twice in cold (4°C) Dulbecco's, calcium and magnesium free, phosphate buffered saline, pH 7 .4 (PBS, Grand Island Biological Co ., Grand Is1and, N . Y .), and resuspended in PBS at a final cell concentration of 4 x 106 /~nl . 1177
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Çonçanavalin-A induçed agglutination_ assay : The method of Inbar and Sachs (11) was followed to quantitate cell agglutination . The PAMs suspensions, 0 .5 ml, were pi petted into 35 mm plastic petri dishes and mixed with 0 .5 ml of Con-A (Grade IV, Sigma Chem . Co ., St . Louis, Mo .) at concentrations from 0 to 1000 u9/ml PBS . The cells were incubated at room temperature (22-24°C) and agglutination examined microscopically at 5 minute intervals for 30 minutes . As a control, a-methylmannose at a final concentration of 0 .15 M was incubated with PAMs in the presence of 300 ug Con-A to inhibit the lectin-induced agglutination . Membrane stabilized PAMs were prepared in cold (4°C) PBS buffered 1% glutaraldehyde for 1 hour, w~shed in PBS three times and resuspended at a cell concentration of 4 x 10 /ml PBS . To study the effects of low temperature on Con-A induced agglutination, pre-cooled reagents and PAMs were mixed and maintained at 4°C . Con-A induced agglutination was assayed as above . Trypsinized PAMs were prepared by incubating the cells in the presence of 0 .01% trypsin solution (Sigma Chem . Co ., St . Louis, Mo .) for 5 minutes at 22°C, washed three times, and resuspended in PBS . Quantitation of Conca naval i n-A binding to PAMs : Iodination of Con-A ( 125 I_Con-A~ was prepared by the iodine monochloride method (12), with carrier-free Na 12 I (New England Nuclear Corp ., Boston, Mass .) . The 1251-Con-A was dialyzed overnight at 4°C against 100 volurt~s of PBS . Final 1251-Con-A concentration was determingd by the method of Lowry et al . (13), using cold Con-A as a standard . The 12 I-Con-A had a specific Virtually all radioactivity was precipitable by activity of 32,000 cpm/ug . 10% trichloroacetic acid . To quantitate 125I_Ca -~ binding to PAMs, 10 6 PAMs were incubated with varying concentrations of R 2 I-Con-A, 0-250 u9/ml, at room temperature (22°C) and 4°C for 30 minutes, washed three times with PBS, and resuspended in 1 ml PBS . Controls included incubation with a-methylmannose, cold Con-A, PBS, and 10 mM Iodoacetate . All samples and standard series of 1251-Con-A concentrations were counted in a Nuclear-Chicago Automatiç Gamma Counter . Sample counts were corrected I-Con-A to the plastic surfaces of the counting .for nonspecific binding of 1 vials and for Con-A binding which was not blocked by a-methylmannose . B inding of Candida krusei to PAMs via Con-A binding sites : Candida krusei were grown in tryptic soy broth for 48 hours, treated 24 hours in 0 .5% formaldehyde, washed four times with 0 .15 M NaCI and resuspended in saline at 2 x 108 cells/ml . PAMs, 10 5/ml, suspended in tissue culture medium 199 (TCM, Grand Island Biological Co ., Grand Island, N . Y .) were allowed to attach to the glass surface of tissue culture slides (No . 4804, Lab-Tek Products, Division Miles Lab . Inc ., Naperville, I11 .) for 1 hour at 37°C . The PAMs were washed with TCM and equilibrated to room temperature (22-24°C) . Con-A with final concentrations of 0, 10, 25, and 100 ug/ml TCM were incubated with the PAMs at 22°C for 15 minutes, the PAMs were then washed three times with TCM . Candida krusei diluted to 2 x 107 cells/ml TCM were incubated with the PAMs~r-T5 m ûßs ni at 22°C, washed three times with TCM, fixed with absolute methanol,
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and stained with Wright-Giemsa stain . PAMs were also fixed for 15 minutes with 3% glutaraldehyde following incubation at 37°C, thoroughly washed with TCM, and incubated with Con-A and Distribution of candida binding via Con-A candida as described above . receptors was determined microscopically . Results Smokers PAMs remained individually dispersed while in PBS . The presence of as little as 10 ug Con-A/ml was adequate to induce agglutination of smoker PAMs, and maximal agglutination occurred with Con-A concentration of 100 pg/ml . The rate of cell agglutination varied with each individual ; however, agglutination was complete within the 30 minute incubation period . When a-methylmannose was present in the assay system, agglutination induced by Con-A was blocked (FIG . 1) .
,.
~+
'' +~
`~
~ ~~-
~-~
FIG . 1 Agglutination of pulmonary alveolar macrophages from a smoker with 100 u9 Con-A, (A) in the presence of 0 .15 M a-methylmannose, and (B) in the absence of a-methylmannose . In contrast, nonsmoker PAMs demonstrate a low grade (1+) agglutination while in suspension in PBS without addition of Con-A, and agglutination is only slightly increased even in the resence of 500 u9 Con-A/ml (FIG . 2) . Agglutination remained unchanged (1+~ in the presence of 0 .15 M a-methylmannose whether PAMs were incubated in the absence of Con-A or in medium containing as much as 300 ug Con-A/ml . The effects of low temperature (4°C) on Con-A induced agglutination were also examined in parallel with assays done at 22°C . Both nonsmoker and
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smoker PAMs failed to agglutinate with Con-A after 30 minutes at the low incubation temperature . When returned to 22 °C, Con-A induced agglutintion of smoker PAMs proceeded nornially. The converse was not true, once agglutination had occurred at 22°C, cell dissociation would not occur at 4°C .
e 0 C ir O1 O
Conc .
Con-A
(psa/M~)
FIG . 2 Degree of agglutination of pulmonary alveolar macrophages (PAMs) from nonsmokers and smokers in the presence of varying concentrations of concanavalin A Con-A) . Clumping was scored as follows : No clumping "0" ; 2-4 cells/clump "0 .5" ; 5-10 cells "1+'~ ; 10-50 cells "2+" ; 50-100 cells "3+~~ ; >100 cells/clump "4+", Each bar represents the mean (± SE) agglutination values for 10 subjects in each group . The presence of 0 .15 _M a-methylmannose is represented by a-CH3-M . Brief treatment of nonsmoker PAMs with trypsin increased the spontaneous agglutination in the absence of Con-A from 1+ to 2+ and resulted in a 3+ to 4+ agglutination in the presence of 100 ug Con-A/ml at 22°C . Trypsinization of smoker PAMs resulted in a 1+ agglutination of some cell samples in PBS, but did not alter their agglutinability in the presence of Con-A . Pre-fixation of all PAM populations with glutaraldehyde prevented Con-A induced agglutination under all assay conditions . Using the radio-labelled (1251) Con-A, no significant difference was found in the amount of Con-A bound at 22°C to 10 6 PAMs from either nonsmokers or smokers (FIG . 3) . Approximately 80~ as much Con-A was bound at 4°C, with equivalent binding to both nonsmoker and smoker PAMs .
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Con-A Induced Agglutiaatioa of Sumaa PAMs
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10~
e ..
..0 e
0
m e
0 N W N
v n= 3 n= 3
nonamokara o-~ amokara ~ -
1G1~--300 PO
12SI Con-A 1 ncubatad ~ 10 0 PAwI FI6 . 3
Quantitation of 1251 Con-A binding to h~n pulmonary alveolar macrophages at 22°C in the presence of I-Con-A concentrations ranging from 1 to 100 ug/ml . Based on a molecular weight of 110,000 for Con-A at pH 7 .4 (14), both nonsmoker and smoker PAMs bound a mean of 4 .3 x 10~ molecules of Con-A per PAM when incubated with 100 u9 Con-A/ml/106 PAMs t 22°C . The presence of 0 .15 M Iodoacetate did not reduce the amount of 1 ~ 5 I-Con-A associated with either nonsmoker or smoker PAMs and did not affect Con-A induced agglutination. In addition to agglutinating mammalian cells, Con-A can induce agglutination of certain gram-positive microorganisms (15) . Using Candida krusei as a marker for Con-A bound to the membrane of PAMs, we were una~to ~dnônstrate any differences 1n Con-A distribution on nonsmoker or smoker PAMs . Under these incubation conditions, candida did not bind to PAMs in the absence of Con-A, and the binding of candida could be inhibited by a-methylmannose . When PAMs were prefixed with glutaraldehyde, some of the cells bound small numbers of candida even in the absence of Con-A . When PAMs from nonsmokers or suwkers (either prefixed with glutaraldehyde, or with terminal fixation)were incubated for 15 minutes at 22°C with Con-A in concentrations ranging from 10 to 100 ug/ml, gg + 0 .5% of the PAMs bound candida to their surface . No differences were detectedin the distribution of binding of candida on the cell membranes . Under all incubation conditions, the candida localized to the spreading veil of membrane surrounding the cell body (FIG .4) . There was a decrease in the number of candlda bound from a range of 13-36 candida/PAM at 25 ug Con-A/ml or higher concentrations,to 7-21 candida/PAM when 10 ug Con-A/ml was present . This indicates a relationship between the amount of PAM-associated Con-A and candida binding .
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FIG . 4 Binding of candida to pulmonary alveolar macrophages from a nonsmoker, incubated with 25 ug Con-A/ml . No binding was observed in the absence of Con-A . Original magnification 400x diameters . Discussion It has been proposed that cell agglutination induced by Con-A depends on the cell having mobile Con-A receptors which can move over short distances on the membrane surface . Proper alignment of receptors on adjacent cells results in a Con-A bridge and hence cell agglutination (16) . Structurally, the ability of Con-A to promote agglutination of cells has been associated with the presence of abundant membraneous microvilli on cells adhering to glass surfaces . Willingham and Paston (6) reported Con-A agglutinability developed as cells became transformed, or following trypsinization, both of which lead to increased microvilli formation . Treatment with dibutyryl cyclic AMP or with pharmacological agents known to increase cellular c-AMP levels also reduced surface microvilli and cell agglutination, perhaps by altering the organization of the intracellular microtubular-microfibrillar systems (17 ) . Recently, we have reported alterations in the surface morphology of PAMs associated with cigarette smoking (G .A . WARR, R .R . MARTIN, and L .O . GENTRY, Bulletin of the XXIIIrd Conference of the International Union Against Tuber culosis, 1975, in press) . When PAMs from nonsmokers were fixed in suspension and examined by scanning electron microscopy, their surfaces had highly undulating membranes similar to PAMs recovered from various laboratory animals (18) . However, PAMs from cigarette smokers had few undulations of the membrane
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and the surface topography ranged from smooth to cobblestone in appearance, with occasional short club-like projections . These differences in surface topography would explain the presence of spontaneous clumping of nonsmoker PAMs in suspension, since the more redundant surfaces would promote cell-to-cell interaction . Smoker PAMs, lacking the extensive undulating membrane area, would have less interaction of cell surfaces and would tend to remain individually dispersed . However, after exposure to Con-A, smoker PAMs agglutinate while nonsmoker PAMs do not . Trypsin treatment of nonsmoker PAMs renders them agglutinable by Con-A, an effect which has been attributed to increased microvilli formation (6,10) . If the extent of microvilli formation were the critical determinant for Con-A agglutinability, however, one would expect that nonsmoker PAMs would be more agglutinable than PAMs from smokers . In fact, the previous studies of the surface morphology of agglutinable and non-agglutinable cell lines have described cells which have settled on flat surface (6,17) . When PAMs are allowed to settle on glass before fixation for scanning electron microscopy, extensive spreading and loss of ruffled membrane occurs (G .A . WARR and R .R .MARTIN, . unpublished observations) . Many smoker PAMs, however, maintain a raised central cell body iJith protruding vesicle-like blebs, with radiating membranous spikes, or microvilli, extending to the margin of the spreading cell . Under these conditions, smoker PAMs more closely resemble the agglutinable cell lines described by others (6,17) . As in other cell systems (6-11), the Con-A induced agglutination is blocked by a-methylmannose, low temperatures, and membrane stabilization . Since both PAM populations bound equivalent numbers of Con-A molecules, the differences in agglutination with nonsmoker and smoker PAMs are not the result of differing amounts of membrane-bound lectin . Inhibition of agglutination at low temperatures also is not due to a lack of Con-A binding since, even though a slight decrease in binding was observed at 4°C, at least 3 times as many Con-A molecules are bound to the PAMs at this temperature as are required to induce agglutination at 22°C . Similar binding of candida to nonsmoker and smoker PAMs via Con-A receptors indicate that there are no gross differences, such as capping, in the distribution of Con-A receptors on the cell surfaces . Redistribution of Con-A binding sites could occur over limited areas of the membrane surface without affecting the binding of the relatively large candida particles . To detect short-range rearrangement of Con-A receptors requires more sensitive methods, such as electron microscopy (19,20) . The significance of changes in surface topography and agglutinability of macrophages in the presence of Con-A is uncertain . At the present, these differences remain phenomena which cannot be related in any concrete fashion with cellular impairment . We are presently examining the association of cigarette smoking with alterations in a nurt~ber of functional characteristics of pulmonary macrophages . Future studies should be directed toward defining the relationship between alterations in the surface of macrophages and the intracellular structures which are important to the functions of these phagocytic cells in host defense . Acknowledgements We thank Mrs . Margaret Reisberg for assistance in preparation of 125ICon-A, and Mrs . Phyllis Faulkner for typing the manuscript . This work was supported in part by a grant ACS-IM-53 from the American Cancer Society, and NIH grants AI-12048 'and AI-00446 . Dr . Martin is a
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Con-A Induced Agglutination of H~maa PAMs
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recipient of a Research Career Development Award AI-70335 from the National Institutes of Health . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 .
S.A . PRATT, T.N . FINLEY, M.H . SMITH, and A .J . LADMAN, Anat . Rec . 163 497-508 (1969) . R.R . MARTIN, Amer . Rev. ~Res . Dis . 107 596-601 (1973) . G.A . WARR, and. R~üARTIN, In~èct-T~~~m~munit~ 8 222-227 (1973) G.A . WARR, and R.R . MARTIN, Inert. Imnunit 9169-771 (1974) H. YEAGER, Proc . Soc . ~Ex B~oT.-ife -250 (1969) . M.C . WILLING(, a~ I . PASfiA~ Proc . fiat. Acad . Sci . USA 72 1263-1267 (1975) . C. BOREK, M . GROB, and M.M . BURGER, ~Ex tl . Cell Res . 77 207-215 (1973) . H . BEN-BASSAT, M. INBAR, and L. SACHS, ~Viro~ ~85d-859 (1970) U. MINTZ, and L . SACHS, Proc . Nat. Acad . Sci . US1~72 2428-2432 (1975) . M. INBAR, H . BEN-BASSAT,~ L~ACR~proc. Nat. mad . Sçi . USA _68 2748-2751 (1971) . M. INBAR, and L . SACHS, Proc . Nat . Acad . Sci . USA 63 1418-1425 (1969) . R .W . HELMKAMP, M.A . CONT~¬ftAS,and ü~ BALS, Int .~_. Appl . Radiat . Isot . 18 737-741 (1967) . ~.FI . LOWRY, N .J . ROSEBROUGH, A.L . FARR, and R .J . RANDALL, _J . Biol . Chem . 193 265-275 (1951) . ~C. WANG, R.A . CUNNINGHAM, and G .M . EDELMAN, Proc . Nat . Acad . Sçi . USA 68 1130-1134 (1971) . ~.M . ALLEN, G .M .W . COOK, and A .R . POOLE, E~x tl . Cell Res .68 466-471 (1971). U . RUTISHAUSER, and L . SACHS, Proc . Nat . cA aâ. Sc~US~7~2456-2460 (1974) . T .T . PUCK, C .A . WALDREN, and A .W . HSIE, Proc . Nat . Acad . Sçi . USA _69 1943-1947 (1972) . E.S . LEAKS, M.J . WRIGHT, and Q .N . MYRYIK, _J . Reticulcendothel . Soc . 17 370-379 (1975) . S.B . SMITH, and J .P . REVEL, Devel . Biol . 27 434-441 (1972) . J .H .M . TEMMINK, J .G . COLLARD, L~SPifS, a~ E, RODS, Exptl . Cell Res . 92 307-322 (1975) .
Pargamon Preea
CONCANAVALIN-A AGGLUTINATION OF HUMAN PULMONARY ALVEOLAR MACROPHAGES FROM NONSMOKERS AND CIGARETTE SMOKERS : A COMPARISON Glenn A . Warr and R . Russell Martin Departments of Medicine, and Microbiology A Immunology Baylor College of Medicine, Houston, Texas 77030 (Received in final form April 12, 1976)
Incubation of pulmonary alveolar macrophages (PAMs) from nonsmokers in phosphate buffered saline (PBS) leads to minimal autoagglutination of these cells, while smokers PAMs incubated in medium remained individually dispersed in suspension . Incubation with the lectin concanavalin A (Con-A) induced agglutination of PAMs from cigarette smokers, while PAMs from nonsmokers were not further agglutinated . After trypsin treatment, both nonsmoker and smoker PAMs agglutinated in the p~~ence of Con-A . Both PAM populations bound the same amount of I-Con-A . Low temperatures (4°C), a-methylmannose, and pre-fixation of smoker PAMs with Candida glutaraldehyde prevented Con-A induced cell agglutination . bound to PAMs by Con-A were similarly distributed on the surfaces of nonsmoker and smoker cells . Cigarette smoking has been associated with morphological changes in-pulmonary alveolar macrophages (PAMs), the most striking being increased cytoplasmic inclusions (1,2), and alterations in a number of functional characteristics (3-5) . lie have reported changes in surface topography and a reduction in membrane complement receptors of PAMs from cigarette smokers (G .A . WARR, R .R . MARTIN, and L .O . GENTRY, Bulletin of the XXIIIrd Conference of the International Union Against Tuberculosis, 1975, in press) . Fibroblast cell lines which are transformed by oncogenic viruses have changes in cellular cyclic AMP levels, surface topography, and in agglutinability induced by the plant lectin, Con-A (5) . The association of agglutina tion induced by Con-A and transformation of cell lines has also been reported by others (7-11) . The present study examines binding of Con-A and lectin-induced cell agglutination, using PAMs from young healthy nonsmokers and cigarette smokers . Methods Pre orat ion of pu l monary alveolar macrophages : Using a fiberoptic bronchoscope as previously described (3), PAMs were obtained by saline lavage from 26 healthy volunteers, ranging in age from 21 to 27 years . Lavaged cells (79 to 87x of which were PAMs) were washed twice in cold (4°C) Dulbecco's, calcium and magnesium free, phosphate buffered saline, pH 7 .4 (PBS, Grand Island Biological Co ., Grand Is1and, N . Y .), and resuspended in PBS at a final cell concentration of 4 x 106 /~nl . 1177
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Çonçanavalin-A induçed agglutination_ assay : The method of Inbar and Sachs (11) was followed to quantitate cell agglutination . The PAMs suspensions, 0 .5 ml, were pi petted into 35 mm plastic petri dishes and mixed with 0 .5 ml of Con-A (Grade IV, Sigma Chem . Co ., St . Louis, Mo .) at concentrations from 0 to 1000 u9/ml PBS . The cells were incubated at room temperature (22-24°C) and agglutination examined microscopically at 5 minute intervals for 30 minutes . As a control, a-methylmannose at a final concentration of 0 .15 M was incubated with PAMs in the presence of 300 ug Con-A to inhibit the lectin-induced agglutination . Membrane stabilized PAMs were prepared in cold (4°C) PBS buffered 1% glutaraldehyde for 1 hour, w~shed in PBS three times and resuspended at a cell concentration of 4 x 10 /ml PBS . To study the effects of low temperature on Con-A induced agglutination, pre-cooled reagents and PAMs were mixed and maintained at 4°C . Con-A induced agglutination was assayed as above . Trypsinized PAMs were prepared by incubating the cells in the presence of 0 .01% trypsin solution (Sigma Chem . Co ., St . Louis, Mo .) for 5 minutes at 22°C, washed three times, and resuspended in PBS . Quantitation of Conca naval i n-A binding to PAMs : Iodination of Con-A ( 125 I_Con-A~ was prepared by the iodine monochloride method (12), with carrier-free Na 12 I (New England Nuclear Corp ., Boston, Mass .) . The 1251-Con-A was dialyzed overnight at 4°C against 100 volurt~s of PBS . Final 1251-Con-A concentration was determingd by the method of Lowry et al . (13), using cold Con-A as a standard . The 12 I-Con-A had a specific Virtually all radioactivity was precipitable by activity of 32,000 cpm/ug . 10% trichloroacetic acid . To quantitate 125I_Ca -~ binding to PAMs, 10 6 PAMs were incubated with varying concentrations of R 2 I-Con-A, 0-250 u9/ml, at room temperature (22°C) and 4°C for 30 minutes, washed three times with PBS, and resuspended in 1 ml PBS . Controls included incubation with a-methylmannose, cold Con-A, PBS, and 10 mM Iodoacetate . All samples and standard series of 1251-Con-A concentrations were counted in a Nuclear-Chicago Automatiç Gamma Counter . Sample counts were corrected I-Con-A to the plastic surfaces of the counting .for nonspecific binding of 1 vials and for Con-A binding which was not blocked by a-methylmannose . B inding of Candida krusei to PAMs via Con-A binding sites : Candida krusei were grown in tryptic soy broth for 48 hours, treated 24 hours in 0 .5% formaldehyde, washed four times with 0 .15 M NaCI and resuspended in saline at 2 x 108 cells/ml . PAMs, 10 5/ml, suspended in tissue culture medium 199 (TCM, Grand Island Biological Co ., Grand Island, N . Y .) were allowed to attach to the glass surface of tissue culture slides (No . 4804, Lab-Tek Products, Division Miles Lab . Inc ., Naperville, I11 .) for 1 hour at 37°C . The PAMs were washed with TCM and equilibrated to room temperature (22-24°C) . Con-A with final concentrations of 0, 10, 25, and 100 ug/ml TCM were incubated with the PAMs at 22°C for 15 minutes, the PAMs were then washed three times with TCM . Candida krusei diluted to 2 x 107 cells/ml TCM were incubated with the PAMs~r-T5 m ûßs ni at 22°C, washed three times with TCM, fixed with absolute methanol,
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and stained with Wright-Giemsa stain . PAMs were also fixed for 15 minutes with 3% glutaraldehyde following incubation at 37°C, thoroughly washed with TCM, and incubated with Con-A and Distribution of candida binding via Con-A candida as described above . receptors was determined microscopically . Results Smokers PAMs remained individually dispersed while in PBS . The presence of as little as 10 ug Con-A/ml was adequate to induce agglutination of smoker PAMs, and maximal agglutination occurred with Con-A concentration of 100 pg/ml . The rate of cell agglutination varied with each individual ; however, agglutination was complete within the 30 minute incubation period . When a-methylmannose was present in the assay system, agglutination induced by Con-A was blocked (FIG . 1) .
,.
~+
'' +~
`~
~ ~~-
~-~
FIG . 1 Agglutination of pulmonary alveolar macrophages from a smoker with 100 u9 Con-A, (A) in the presence of 0 .15 M a-methylmannose, and (B) in the absence of a-methylmannose . In contrast, nonsmoker PAMs demonstrate a low grade (1+) agglutination while in suspension in PBS without addition of Con-A, and agglutination is only slightly increased even in the resence of 500 u9 Con-A/ml (FIG . 2) . Agglutination remained unchanged (1+~ in the presence of 0 .15 M a-methylmannose whether PAMs were incubated in the absence of Con-A or in medium containing as much as 300 ug Con-A/ml . The effects of low temperature (4°C) on Con-A induced agglutination were also examined in parallel with assays done at 22°C . Both nonsmoker and
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smoker PAMs failed to agglutinate with Con-A after 30 minutes at the low incubation temperature . When returned to 22 °C, Con-A induced agglutintion of smoker PAMs proceeded nornially. The converse was not true, once agglutination had occurred at 22°C, cell dissociation would not occur at 4°C .
e 0 C ir O1 O
Conc .
Con-A
(psa/M~)
FIG . 2 Degree of agglutination of pulmonary alveolar macrophages (PAMs) from nonsmokers and smokers in the presence of varying concentrations of concanavalin A Con-A) . Clumping was scored as follows : No clumping "0" ; 2-4 cells/clump "0 .5" ; 5-10 cells "1+'~ ; 10-50 cells "2+" ; 50-100 cells "3+~~ ; >100 cells/clump "4+", Each bar represents the mean (± SE) agglutination values for 10 subjects in each group . The presence of 0 .15 _M a-methylmannose is represented by a-CH3-M . Brief treatment of nonsmoker PAMs with trypsin increased the spontaneous agglutination in the absence of Con-A from 1+ to 2+ and resulted in a 3+ to 4+ agglutination in the presence of 100 ug Con-A/ml at 22°C . Trypsinization of smoker PAMs resulted in a 1+ agglutination of some cell samples in PBS, but did not alter their agglutinability in the presence of Con-A . Pre-fixation of all PAM populations with glutaraldehyde prevented Con-A induced agglutination under all assay conditions . Using the radio-labelled (1251) Con-A, no significant difference was found in the amount of Con-A bound at 22°C to 10 6 PAMs from either nonsmokers or smokers (FIG . 3) . Approximately 80~ as much Con-A was bound at 4°C, with equivalent binding to both nonsmoker and smoker PAMs .
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10~
e ..
..0 e
0
m e
0 N W N
v n= 3 n= 3
nonamokara o-~ amokara ~ -
1G1~--300 PO
12SI Con-A 1 ncubatad ~ 10 0 PAwI FI6 . 3
Quantitation of 1251 Con-A binding to h~n pulmonary alveolar macrophages at 22°C in the presence of I-Con-A concentrations ranging from 1 to 100 ug/ml . Based on a molecular weight of 110,000 for Con-A at pH 7 .4 (14), both nonsmoker and smoker PAMs bound a mean of 4 .3 x 10~ molecules of Con-A per PAM when incubated with 100 u9 Con-A/ml/106 PAMs t 22°C . The presence of 0 .15 M Iodoacetate did not reduce the amount of 1 ~ 5 I-Con-A associated with either nonsmoker or smoker PAMs and did not affect Con-A induced agglutination. In addition to agglutinating mammalian cells, Con-A can induce agglutination of certain gram-positive microorganisms (15) . Using Candida krusei as a marker for Con-A bound to the membrane of PAMs, we were una~to ~dnônstrate any differences 1n Con-A distribution on nonsmoker or smoker PAMs . Under these incubation conditions, candida did not bind to PAMs in the absence of Con-A, and the binding of candida could be inhibited by a-methylmannose . When PAMs were prefixed with glutaraldehyde, some of the cells bound small numbers of candida even in the absence of Con-A . When PAMs from nonsmokers or suwkers (either prefixed with glutaraldehyde, or with terminal fixation)were incubated for 15 minutes at 22°C with Con-A in concentrations ranging from 10 to 100 ug/ml, gg + 0 .5% of the PAMs bound candida to their surface . No differences were detectedin the distribution of binding of candida on the cell membranes . Under all incubation conditions, the candida localized to the spreading veil of membrane surrounding the cell body (FIG .4) . There was a decrease in the number of candlda bound from a range of 13-36 candida/PAM at 25 ug Con-A/ml or higher concentrations,to 7-21 candida/PAM when 10 ug Con-A/ml was present . This indicates a relationship between the amount of PAM-associated Con-A and candida binding .
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FIG . 4 Binding of candida to pulmonary alveolar macrophages from a nonsmoker, incubated with 25 ug Con-A/ml . No binding was observed in the absence of Con-A . Original magnification 400x diameters . Discussion It has been proposed that cell agglutination induced by Con-A depends on the cell having mobile Con-A receptors which can move over short distances on the membrane surface . Proper alignment of receptors on adjacent cells results in a Con-A bridge and hence cell agglutination (16) . Structurally, the ability of Con-A to promote agglutination of cells has been associated with the presence of abundant membraneous microvilli on cells adhering to glass surfaces . Willingham and Paston (6) reported Con-A agglutinability developed as cells became transformed, or following trypsinization, both of which lead to increased microvilli formation . Treatment with dibutyryl cyclic AMP or with pharmacological agents known to increase cellular c-AMP levels also reduced surface microvilli and cell agglutination, perhaps by altering the organization of the intracellular microtubular-microfibrillar systems (17 ) . Recently, we have reported alterations in the surface morphology of PAMs associated with cigarette smoking (G .A . WARR, R .R . MARTIN, and L .O . GENTRY, Bulletin of the XXIIIrd Conference of the International Union Against Tuber culosis, 1975, in press) . When PAMs from nonsmokers were fixed in suspension and examined by scanning electron microscopy, their surfaces had highly undulating membranes similar to PAMs recovered from various laboratory animals (18) . However, PAMs from cigarette smokers had few undulations of the membrane
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and the surface topography ranged from smooth to cobblestone in appearance, with occasional short club-like projections . These differences in surface topography would explain the presence of spontaneous clumping of nonsmoker PAMs in suspension, since the more redundant surfaces would promote cell-to-cell interaction . Smoker PAMs, lacking the extensive undulating membrane area, would have less interaction of cell surfaces and would tend to remain individually dispersed . However, after exposure to Con-A, smoker PAMs agglutinate while nonsmoker PAMs do not . Trypsin treatment of nonsmoker PAMs renders them agglutinable by Con-A, an effect which has been attributed to increased microvilli formation (6,10) . If the extent of microvilli formation were the critical determinant for Con-A agglutinability, however, one would expect that nonsmoker PAMs would be more agglutinable than PAMs from smokers . In fact, the previous studies of the surface morphology of agglutinable and non-agglutinable cell lines have described cells which have settled on flat surface (6,17) . When PAMs are allowed to settle on glass before fixation for scanning electron microscopy, extensive spreading and loss of ruffled membrane occurs (G .A . WARR and R .R .MARTIN, . unpublished observations) . Many smoker PAMs, however, maintain a raised central cell body iJith protruding vesicle-like blebs, with radiating membranous spikes, or microvilli, extending to the margin of the spreading cell . Under these conditions, smoker PAMs more closely resemble the agglutinable cell lines described by others (6,17) . As in other cell systems (6-11), the Con-A induced agglutination is blocked by a-methylmannose, low temperatures, and membrane stabilization . Since both PAM populations bound equivalent numbers of Con-A molecules, the differences in agglutination with nonsmoker and smoker PAMs are not the result of differing amounts of membrane-bound lectin . Inhibition of agglutination at low temperatures also is not due to a lack of Con-A binding since, even though a slight decrease in binding was observed at 4°C, at least 3 times as many Con-A molecules are bound to the PAMs at this temperature as are required to induce agglutination at 22°C . Similar binding of candida to nonsmoker and smoker PAMs via Con-A receptors indicate that there are no gross differences, such as capping, in the distribution of Con-A receptors on the cell surfaces . Redistribution of Con-A binding sites could occur over limited areas of the membrane surface without affecting the binding of the relatively large candida particles . To detect short-range rearrangement of Con-A receptors requires more sensitive methods, such as electron microscopy (19,20) . The significance of changes in surface topography and agglutinability of macrophages in the presence of Con-A is uncertain . At the present, these differences remain phenomena which cannot be related in any concrete fashion with cellular impairment . We are presently examining the association of cigarette smoking with alterations in a nurt~ber of functional characteristics of pulmonary macrophages . Future studies should be directed toward defining the relationship between alterations in the surface of macrophages and the intracellular structures which are important to the functions of these phagocytic cells in host defense . Acknowledgements We thank Mrs . Margaret Reisberg for assistance in preparation of 125ICon-A, and Mrs . Phyllis Faulkner for typing the manuscript . This work was supported in part by a grant ACS-IM-53 from the American Cancer Society, and NIH grants AI-12048 'and AI-00446 . Dr . Martin is a
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recipient of a Research Career Development Award AI-70335 from the National Institutes of Health . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 .
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