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Interactions of neutrophil granulocytes (PMNs) and endothelium in vitro
MICROVASCULAR
RESEARCH
Interactions
13, 107-112 (1977)
of Neutrophil Granulocytes and Endothelium In Vitro
(PMNs)
J. M. LACKIE’ AND D. DE BONO Strangeways Research Laboratory, Cambridge, England, and Department of Surgery, University of Cambridge, England Received May 3, 1976 Rabbit peritoneal polymorphonuclear leucocytes (PMNs) adhere readily to serum-coated glass and to the surfaces of pig aortic endothelial cells grown in vitro, though adhesion to pig fibroblast surfaces is much weaker. Using time-lapse cinephotomicrography it has been shown that PMNs will move freely over the surfaces of endothelial cells and that they show no contact paralysis when they collide with the leading lamella of moving endothelial cells.
One of the earliest observable events in the acute inflammatory reaction is the adhesion of polymorphonuclear leucocytes (PMNs) to the endothelium of capillary vessels. This margination process has been described in vivo (Clark et al., 1936; Atherton and Born, 1972) but relatively little is known about the adhesive interaction which is presumed to take place, nor is much known of the cellular activities involved in the subsequent phase of emigration (diapedesis). In this paper we present in vitro observations which suggest that endothelial cells may provide a particularly good substrate for PMN adhesion and that there is no inhibition of locomotion of PMNs following their contact
with
endothelial
cells.
METHODS Cells PMNs were obtained from New Zealand white rabbits following the intraperitoneal injection of 500 ml of sterile physiological saline containing 0.1 ‘A oyster glycogen (Sigma) and its removal after 4 hr. At 4 hr, this exudate contains almost pure neutro-
phi1 granulocytes (Cohn and Morse, 1959). PMNs were stored at 4” in the exudate fluid for up to 48 hr until required, under which conditions they remain viable as judged by dye exclusion and their behaviour in a variety of systems. Immediately before use, the cells were washed with divalent-cation-free Hank’s Hepes (CMF) and with CMF plus 1 mM EDTA, filtered through lens tissue to remove large clumps of cells, and then suspended in Medium 199 + 10% calf serum at a concentration of approximately 2 x IO6 cells/ml (Lackie, 1974). 1 Present address and address to which all communications Biology, The University, Glasgow Gil 6NU, Scotland. Copyright 0 1977 by Academic Press, Inc. 107 All rights of reproduction in any form reserved. Printed
in Great
Britain
should be sent: Department
of GelI
ISSN 0026-22862
108
LACKIE
AND
DE BONO
Endothelial cells were obtained from pig aortas by treatment with 0.2 % Worthington collagenase (Type 1) in phosphate-buffered saline and were cultured in Waymouth’s medium with 20% calf serum (de Bono, 1974). Pig fibroblasts were obtained by incubating minced pig lung or oesophagus for 1 hr at 37” with 0.2 % collagenase, passing the cell suspension through a strainer, washing, and plating out in Waymouth’s medium. These cells were judged to be fibroblasts on the basis of their morphology under light and electron microscopy, their growth as multilayers, and their production of collagen. The behaviour of these cells was similar to the behaviour of chick heart fibroblasts in an identical assay. For adhesion experiments, endothelial cells which had been grown for 47 days (to confluence) after removal from aortas were suspended using trypsinEDTA (0.5 % Difco-trypsin in 0.5 mM EDTA-CMF) or collagenase (0.2 x, in complete medium) and plated onto 13-mm-diameter coverslips at a density sufficient to give confluent monolayers within 24 hr. For filming, cells were plated sparsely onto 32 x 32mm coverslips. Adhesion Experiments Adhesion experiments were carried out in Linbro tissue culture plates with confluent endothelium, confluent fibroblasts, or glass as the collecting substrate (Armstrong and Lackie, 1975). A suspension of PMNs was added to each well, the PMNs were allowed to settle and adhere at 37” for 30 min, and the coverslips were then removed, washed by dipping four times through an air-medium interface, and fixed in buffered formolsaline. Coverslips were then stained with Giemsa, dehydrated in isopropyl alcohol, cleared in xylene, and mounted. Using a 10 x 10 eyepiece grid, the PMNs on 10 microscope fields per coverslip were counted and the mean number of PMNs per unit area was calculated for each coverslip. Only confluent areas of endothelium or fibroblasts were scored. PMNs can be distinguished readily from endothelial cells or fibroblasts. Time-Lapse Filming A suspension of PMNs was placed over a sparsely seeded coverslip of endothelial cells in which the cells covered approximately half the area available, and the PMNs were allowed to settle and adhere until a sufficient number of PMNs remained on the coverslip when it was inverted (approximately 50 PMNs per field). The coverslip with adherent PMNs was inverted over a filming chamber consisting of a 16-mm-diameter hole in a 0.75-mm-thick stainless steel slide with a coverslip sealed on the lower surface. A lo-set lapse interval was used and the best film for analysis proved to be that taken using Nomarski optics on a Zeiss microscope with a x 16 objective. Two sequences of film in which the contrast and focus were most suitable for analysis were examined frame by frame using an L & W stop-action projector. The time each PMN spent on cellular or noncellular substrate and the results of collision with endothelial cells or fibroblasts were recorded. The dorsal surface of the spread cells is taken as the surface remote from the glass substrate, though the filming was in fact done with the cells inverted, i.e., dorsal side down. Strong overlapping was recorded if more than half the PMN overlapped the border of the spread fibroblast or endothelial cell and slight overlapping was recorded if the PMN had more than half its area in contact with glass. Underlapping was defined as movement of the PMN between the glass and the ventral surface of the spread cell.
PMNS
AND
ENDOTHELIUM
IN
109
VITRO
RESULTS Adhesion Experiments
As can be seen from Table 1, endothelial cells are a good substrate for the adhesion of PMNs, rather more of which adhere to the cellular substrate than adhere to serumcoated glass, though the difference is small and unlikely to be of biological significance. In contrast, fibroblasts are a relatively poor substrate (Table 2; see also, Armstrong -,and TABLE
1
OF RABBIT PMNs TO PIG ENDOTHELIAL MONOLAYERS USING TRYPSIN-EDTA) AFTER 30 MIN AT 37””
ADHESION
Experiment 1
2 3 4 5 6 7 8 9 10
Mean number of PMNs per lOO+m square on glass 19.0 10.2 8.5 8.7 6.6 9.0 8.8 7.1 8.4 25.8
(2) (3) (3) (2) (1) (2) (2) (3) (6) (6)
(SUBCULTURED
Mean number of PMNs per lOO+m square on endothelial monolayer 19.4 (2) 9.0 (3) 12.5 (3) 13.6 (2) 8.4 (1) 6.1 (2) 6.8 (2) 12.4 (3) 14.6 (6) 26.6 (6)
n Each experiment was carried out on a different batch of endothelium. Differences between experiments reflect differences in the number of PMNs added and are not significant (Armstrong and Lackie, 1975). The figure in parentheses is the number of replicate coverslips. Mean difference between paired samples (+ standard error of mean) = 2.37 f 0.61 [t (29) = 3.888, P < 0.002] i.e., significantly more adhere to endothelium. The mean difference was calculated using a paired-sample t test; coverslips were paired within each experiment because of the way in which the assay was set up, though only the mean value for each experiment is shown in this table.
Lackie, 1975). The high collection efficiency on endothelium was reliably obtained when the endothelial cells were subcultured using trypsin-EDTA to detach the cells, but a much lower collection was observed when endothelial cells were detached using Worthington collagenase in complete medium. The latter procedure requires a much longer treatment with enzyme, and despite the fact that the endothelial cells have had at least 24 hr to recover they seem more to resemble fibroblasts in their adhesiveness (Table 3). Contact Interactions
When a PMN moving across a serum-coated surface contacted a spread fibroblast, there appeared to be no contact paralysis (Armstrong and Lackie, 1975) since the PMN overlapped the fibroblast and, in a small proportion of cases, moved onto the dorsal surface of the fibroblast. Though extensive overlapping was exhibited, only a few PMNs
110
LACKIE AND DE BONO TABLE ADHESION
OF
PMNs TO SERUM-COATED FIBROBLAST
2
GLASS, ENDOTHELIAL MONOLAYERS’
MONOLAYERS,
AND
Number adhering per 100~grn square on : Experiment
Glass
Endothelial cells
Fibroblasts
1. PMNs 2. PMNs 3. Lymphocytes
25.8 (6) 8.4 (6) 0 (6)
26.6 (6) 14.6 (6) 21.6 (6)
5.1(6) 5.1 (6) 1.5 (6)
a Experiment 3 is included for comparison (from de Bono, 1976). The figure in parentheses is the number of replicates within each experiment. TABLE
3
OF PMNs TO ENDOTHELKJM WHICH WAS SUBCULTURED USING COLLAGENASE IN COMPLETE MEDIUM
ADHESION
Mean number adhering per 100~pm square on: Experiment 1 2 3 4
Glass 20.3 39.8 32.4 20.8
(6) (6) (3) (6)
Endothelial monolayer 8.7 15.4 2.5 1.9
(6) (2) (3) (2)
relinquished their adhesion to the glassand moved onto the fibroblast, a result that might have been predicted since the relative adhesivenessof the two substrateswould tend to favour PMNs remaining on glass.A few of the PMNs were apparently firmly attached by tail fibres and seemedunable to move away from their anchorage point, though they were free to swivel around. Several clear examples of underlapping were observed and in all casesthe PMN was able to move extensively below the spread fibroblast. Contact with an endothelial cell was much more likely to lead to a complete overlapping of the PMN onto the dorsal surface of the spread cell. No underlapping was observed, though the significance of this is hard to estimate since no deliberate search for underlapping events was carried out. Since complete overlapping occurs readily, the area of the field covered by endothelium was accessibleto PMNs, which therefore spent a smaller proportion of their time on glass-the ratio of time spent on glassand on endothelium being in almost exact proportion to the relative areas. This contrasts with the situation with fibroblasts, in which PMNs were much more limited in their substrate, sincethe probability of them completely overlapping waslow and they spent much more of their time on the serum-coatedglasssubstrate. These results are shown in Table 4. The PMNs contacting endothelium were followed for an average of 300
PMNS
AND
ENDOTHELIUM
IN
TABLE
ANALYSIS OF CONTACTS BETWEEN PMNs
Frames Frames
Total
number
a Underlapping b Fibroblasts: and six never c Endothelium: anchored and sis. * The areas t Less than
4
AND SPREAD FIBROBLASTS
OR ENDOTHELIAL
CELLS
Fibroblast/Glass Substrak? (% of total)
Endothelium/Glass SubstrateC (% of total)
7010 (75) 2369t (25)
3917 (49) 4078 (51)
74 (82) 11 (12) 5 (6)
67 (46) 36 (25) 43 (29)
spent on glass* spent in contact*
Number of collisions leading Slight overlapping Strong overlapping Complete overlapping
111
VfZXO
to:
90
of collisions” has not been considered Area of glass in field = 42 contacted fibroblasts. These Area of glass in field = five never made contact with
146
a contact event for the purposes of this analysis. ‘A. Forty cells were followed, of which six remained anchored 12 were excluded from the analysis. 52 %. Forty-nine cells were followed, of which 10 remained an endothelial cell. These 15 were excluded from the analy-
of glass and spread cells have been equalised. 3 % of the time was occupied in complete overlapping.
300 frames each, a real-time equivalent of approximately 1 hr. In fact, the actual film was considerably longer since somePMNs leave the field and others enter. DISCUSSION It haspreviously been argued (Armstrong and Lackie, 1975; Lackie and Armstrong, 1975) that the ability of PMNs to invade tissuesmay be a consequenceof their ability to move freely following contact, that is, their failure to show contact paralysis of locomotion. Therefore, it is not surprising that PMNs should move freely upon the surfaces of endothelial cells, sincethis is the primary site of adhesion and invasion in vivo. The differences
observed
between
fibroblasts
and
endothelial
cells
in their
adhesiveness
for
PMNs are reflected in the relative easewith which PMNs move from glassto the cellular substrate and suggestthat the relative adhesiveness of the substrate may play a role in the process of diapedesis. Though the PMNs used are from a different species,it is unlikely that this is too serious a problem since the adhesion of PMNs to fibroblasts from several speciesseemsto be similar and their adhesion to human umbilical cord vein endothelium does not seemto differ from that shown to pig aortic endothelium (results not shown). A further criticism is that PMNs do not normally adhere to aortic endothelium but rather to capillary endothelium; whether endothelial cells differ in the two sitesis hard to judge, but the plasticity of endothelium in culture suggeststhat apparent morphological differences in vivo may not be significant, and the flow rates in the major arteries may well produce shear stresseswhich exceed the adhesive interactions, thus normally preventing adhesion to arterial endothelium. In vitro studiessuch as we have described provide an imperfect model for the in vivo situation but do show
112
LACKIE AND DE BONO
that endothelial cells are more suitable for PMN adhesion than are fibroblasts and that PMNs are not contact inhibited for locomotion by endothelial cells. ACKNOWLEDGMENTS This work was supported by the Eastwood Memorial Foundation (D. de Bono).
(J. M. Lackie) and by the MRC
REFERENCES ARMSTRONG,
J. M. (1975). Studies on intercellular invasion in vitro using rabbit granulocytes (PMNs). I. Role of contact inhibition of locomotion. J. Cell
P. B., AND LACKIE,
peritoneal neutrophil
Biol. 65,439%462. ATHERTON, A., AND BORN,
G. V. R. (1972). Quantitative investigations of the adhesiveness of circulating polymorphonuclear leucocytes to blood vessel walls. J. Physiol. 222,447-474. CLARK, E. R., CLARK, E. L., AND REX, R. 0. (1936). Observations on polymorphonuclear leucocytes in the living animal. Amer. J. Anat. 59,123-173. COHN, Z. A., AND MORSE, S. I. (1959). Interactions between rabbit polymorphonuclear leucocytes and staphylococci. J. Exp. Med. 110,419. DE BONO, D. (1974). Effects of cytotoxic sera on endothelium in vitro. Nature (London) 252, 83-84. DE BONO, D. (1976), in preparation. LACKIE, J. M. (1974). The aggregation of rabbit polymorphonuclear leucocytes: Effects of antimitotic agents, cyclic nucleotides, and methyl xanthines. J. CeN Sci. 16, 167-180. LACKIE, J. M., AND ARMSTRONG, P. B. (1975). Studies on intercellular invasion in oitro using rabbit peritoneal neutrophil granulocytes. II. Adhesive intreactions between cells. J. Cell Sci. 19, 645-652.
RESEARCH
Interactions
13, 107-112 (1977)
of Neutrophil Granulocytes and Endothelium In Vitro
(PMNs)
J. M. LACKIE’ AND D. DE BONO Strangeways Research Laboratory, Cambridge, England, and Department of Surgery, University of Cambridge, England Received May 3, 1976 Rabbit peritoneal polymorphonuclear leucocytes (PMNs) adhere readily to serum-coated glass and to the surfaces of pig aortic endothelial cells grown in vitro, though adhesion to pig fibroblast surfaces is much weaker. Using time-lapse cinephotomicrography it has been shown that PMNs will move freely over the surfaces of endothelial cells and that they show no contact paralysis when they collide with the leading lamella of moving endothelial cells.
One of the earliest observable events in the acute inflammatory reaction is the adhesion of polymorphonuclear leucocytes (PMNs) to the endothelium of capillary vessels. This margination process has been described in vivo (Clark et al., 1936; Atherton and Born, 1972) but relatively little is known about the adhesive interaction which is presumed to take place, nor is much known of the cellular activities involved in the subsequent phase of emigration (diapedesis). In this paper we present in vitro observations which suggest that endothelial cells may provide a particularly good substrate for PMN adhesion and that there is no inhibition of locomotion of PMNs following their contact
with
endothelial
cells.
METHODS Cells PMNs were obtained from New Zealand white rabbits following the intraperitoneal injection of 500 ml of sterile physiological saline containing 0.1 ‘A oyster glycogen (Sigma) and its removal after 4 hr. At 4 hr, this exudate contains almost pure neutro-
phi1 granulocytes (Cohn and Morse, 1959). PMNs were stored at 4” in the exudate fluid for up to 48 hr until required, under which conditions they remain viable as judged by dye exclusion and their behaviour in a variety of systems. Immediately before use, the cells were washed with divalent-cation-free Hank’s Hepes (CMF) and with CMF plus 1 mM EDTA, filtered through lens tissue to remove large clumps of cells, and then suspended in Medium 199 + 10% calf serum at a concentration of approximately 2 x IO6 cells/ml (Lackie, 1974). 1 Present address and address to which all communications Biology, The University, Glasgow Gil 6NU, Scotland. Copyright 0 1977 by Academic Press, Inc. 107 All rights of reproduction in any form reserved. Printed
in Great
Britain
should be sent: Department
of GelI
ISSN 0026-22862
108
LACKIE
AND
DE BONO
Endothelial cells were obtained from pig aortas by treatment with 0.2 % Worthington collagenase (Type 1) in phosphate-buffered saline and were cultured in Waymouth’s medium with 20% calf serum (de Bono, 1974). Pig fibroblasts were obtained by incubating minced pig lung or oesophagus for 1 hr at 37” with 0.2 % collagenase, passing the cell suspension through a strainer, washing, and plating out in Waymouth’s medium. These cells were judged to be fibroblasts on the basis of their morphology under light and electron microscopy, their growth as multilayers, and their production of collagen. The behaviour of these cells was similar to the behaviour of chick heart fibroblasts in an identical assay. For adhesion experiments, endothelial cells which had been grown for 47 days (to confluence) after removal from aortas were suspended using trypsinEDTA (0.5 % Difco-trypsin in 0.5 mM EDTA-CMF) or collagenase (0.2 x, in complete medium) and plated onto 13-mm-diameter coverslips at a density sufficient to give confluent monolayers within 24 hr. For filming, cells were plated sparsely onto 32 x 32mm coverslips. Adhesion Experiments Adhesion experiments were carried out in Linbro tissue culture plates with confluent endothelium, confluent fibroblasts, or glass as the collecting substrate (Armstrong and Lackie, 1975). A suspension of PMNs was added to each well, the PMNs were allowed to settle and adhere at 37” for 30 min, and the coverslips were then removed, washed by dipping four times through an air-medium interface, and fixed in buffered formolsaline. Coverslips were then stained with Giemsa, dehydrated in isopropyl alcohol, cleared in xylene, and mounted. Using a 10 x 10 eyepiece grid, the PMNs on 10 microscope fields per coverslip were counted and the mean number of PMNs per unit area was calculated for each coverslip. Only confluent areas of endothelium or fibroblasts were scored. PMNs can be distinguished readily from endothelial cells or fibroblasts. Time-Lapse Filming A suspension of PMNs was placed over a sparsely seeded coverslip of endothelial cells in which the cells covered approximately half the area available, and the PMNs were allowed to settle and adhere until a sufficient number of PMNs remained on the coverslip when it was inverted (approximately 50 PMNs per field). The coverslip with adherent PMNs was inverted over a filming chamber consisting of a 16-mm-diameter hole in a 0.75-mm-thick stainless steel slide with a coverslip sealed on the lower surface. A lo-set lapse interval was used and the best film for analysis proved to be that taken using Nomarski optics on a Zeiss microscope with a x 16 objective. Two sequences of film in which the contrast and focus were most suitable for analysis were examined frame by frame using an L & W stop-action projector. The time each PMN spent on cellular or noncellular substrate and the results of collision with endothelial cells or fibroblasts were recorded. The dorsal surface of the spread cells is taken as the surface remote from the glass substrate, though the filming was in fact done with the cells inverted, i.e., dorsal side down. Strong overlapping was recorded if more than half the PMN overlapped the border of the spread fibroblast or endothelial cell and slight overlapping was recorded if the PMN had more than half its area in contact with glass. Underlapping was defined as movement of the PMN between the glass and the ventral surface of the spread cell.
PMNS
AND
ENDOTHELIUM
IN
109
VITRO
RESULTS Adhesion Experiments
As can be seen from Table 1, endothelial cells are a good substrate for the adhesion of PMNs, rather more of which adhere to the cellular substrate than adhere to serumcoated glass, though the difference is small and unlikely to be of biological significance. In contrast, fibroblasts are a relatively poor substrate (Table 2; see also, Armstrong -,and TABLE
1
OF RABBIT PMNs TO PIG ENDOTHELIAL MONOLAYERS USING TRYPSIN-EDTA) AFTER 30 MIN AT 37””
ADHESION
Experiment 1
2 3 4 5 6 7 8 9 10
Mean number of PMNs per lOO+m square on glass 19.0 10.2 8.5 8.7 6.6 9.0 8.8 7.1 8.4 25.8
(2) (3) (3) (2) (1) (2) (2) (3) (6) (6)
(SUBCULTURED
Mean number of PMNs per lOO+m square on endothelial monolayer 19.4 (2) 9.0 (3) 12.5 (3) 13.6 (2) 8.4 (1) 6.1 (2) 6.8 (2) 12.4 (3) 14.6 (6) 26.6 (6)
n Each experiment was carried out on a different batch of endothelium. Differences between experiments reflect differences in the number of PMNs added and are not significant (Armstrong and Lackie, 1975). The figure in parentheses is the number of replicate coverslips. Mean difference between paired samples (+ standard error of mean) = 2.37 f 0.61 [t (29) = 3.888, P < 0.002] i.e., significantly more adhere to endothelium. The mean difference was calculated using a paired-sample t test; coverslips were paired within each experiment because of the way in which the assay was set up, though only the mean value for each experiment is shown in this table.
Lackie, 1975). The high collection efficiency on endothelium was reliably obtained when the endothelial cells were subcultured using trypsin-EDTA to detach the cells, but a much lower collection was observed when endothelial cells were detached using Worthington collagenase in complete medium. The latter procedure requires a much longer treatment with enzyme, and despite the fact that the endothelial cells have had at least 24 hr to recover they seem more to resemble fibroblasts in their adhesiveness (Table 3). Contact Interactions
When a PMN moving across a serum-coated surface contacted a spread fibroblast, there appeared to be no contact paralysis (Armstrong and Lackie, 1975) since the PMN overlapped the fibroblast and, in a small proportion of cases, moved onto the dorsal surface of the fibroblast. Though extensive overlapping was exhibited, only a few PMNs
110
LACKIE AND DE BONO TABLE ADHESION
OF
PMNs TO SERUM-COATED FIBROBLAST
2
GLASS, ENDOTHELIAL MONOLAYERS’
MONOLAYERS,
AND
Number adhering per 100~grn square on : Experiment
Glass
Endothelial cells
Fibroblasts
1. PMNs 2. PMNs 3. Lymphocytes
25.8 (6) 8.4 (6) 0 (6)
26.6 (6) 14.6 (6) 21.6 (6)
5.1(6) 5.1 (6) 1.5 (6)
a Experiment 3 is included for comparison (from de Bono, 1976). The figure in parentheses is the number of replicates within each experiment. TABLE
3
OF PMNs TO ENDOTHELKJM WHICH WAS SUBCULTURED USING COLLAGENASE IN COMPLETE MEDIUM
ADHESION
Mean number adhering per 100~pm square on: Experiment 1 2 3 4
Glass 20.3 39.8 32.4 20.8
(6) (6) (3) (6)
Endothelial monolayer 8.7 15.4 2.5 1.9
(6) (2) (3) (2)
relinquished their adhesion to the glassand moved onto the fibroblast, a result that might have been predicted since the relative adhesivenessof the two substrateswould tend to favour PMNs remaining on glass.A few of the PMNs were apparently firmly attached by tail fibres and seemedunable to move away from their anchorage point, though they were free to swivel around. Several clear examples of underlapping were observed and in all casesthe PMN was able to move extensively below the spread fibroblast. Contact with an endothelial cell was much more likely to lead to a complete overlapping of the PMN onto the dorsal surface of the spread cell. No underlapping was observed, though the significance of this is hard to estimate since no deliberate search for underlapping events was carried out. Since complete overlapping occurs readily, the area of the field covered by endothelium was accessibleto PMNs, which therefore spent a smaller proportion of their time on glass-the ratio of time spent on glassand on endothelium being in almost exact proportion to the relative areas. This contrasts with the situation with fibroblasts, in which PMNs were much more limited in their substrate, sincethe probability of them completely overlapping waslow and they spent much more of their time on the serum-coatedglasssubstrate. These results are shown in Table 4. The PMNs contacting endothelium were followed for an average of 300
PMNS
AND
ENDOTHELIUM
IN
TABLE
ANALYSIS OF CONTACTS BETWEEN PMNs
Frames Frames
Total
number
a Underlapping b Fibroblasts: and six never c Endothelium: anchored and sis. * The areas t Less than
4
AND SPREAD FIBROBLASTS
OR ENDOTHELIAL
CELLS
Fibroblast/Glass Substrak? (% of total)
Endothelium/Glass SubstrateC (% of total)
7010 (75) 2369t (25)
3917 (49) 4078 (51)
74 (82) 11 (12) 5 (6)
67 (46) 36 (25) 43 (29)
spent on glass* spent in contact*
Number of collisions leading Slight overlapping Strong overlapping Complete overlapping
111
VfZXO
to:
90
of collisions” has not been considered Area of glass in field = 42 contacted fibroblasts. These Area of glass in field = five never made contact with
146
a contact event for the purposes of this analysis. ‘A. Forty cells were followed, of which six remained anchored 12 were excluded from the analysis. 52 %. Forty-nine cells were followed, of which 10 remained an endothelial cell. These 15 were excluded from the analy-
of glass and spread cells have been equalised. 3 % of the time was occupied in complete overlapping.
300 frames each, a real-time equivalent of approximately 1 hr. In fact, the actual film was considerably longer since somePMNs leave the field and others enter. DISCUSSION It haspreviously been argued (Armstrong and Lackie, 1975; Lackie and Armstrong, 1975) that the ability of PMNs to invade tissuesmay be a consequenceof their ability to move freely following contact, that is, their failure to show contact paralysis of locomotion. Therefore, it is not surprising that PMNs should move freely upon the surfaces of endothelial cells, sincethis is the primary site of adhesion and invasion in vivo. The differences
observed
between
fibroblasts
and
endothelial
cells
in their
adhesiveness
for
PMNs are reflected in the relative easewith which PMNs move from glassto the cellular substrate and suggestthat the relative adhesiveness of the substrate may play a role in the process of diapedesis. Though the PMNs used are from a different species,it is unlikely that this is too serious a problem since the adhesion of PMNs to fibroblasts from several speciesseemsto be similar and their adhesion to human umbilical cord vein endothelium does not seemto differ from that shown to pig aortic endothelium (results not shown). A further criticism is that PMNs do not normally adhere to aortic endothelium but rather to capillary endothelium; whether endothelial cells differ in the two sitesis hard to judge, but the plasticity of endothelium in culture suggeststhat apparent morphological differences in vivo may not be significant, and the flow rates in the major arteries may well produce shear stresseswhich exceed the adhesive interactions, thus normally preventing adhesion to arterial endothelium. In vitro studiessuch as we have described provide an imperfect model for the in vivo situation but do show
112
LACKIE AND DE BONO
that endothelial cells are more suitable for PMN adhesion than are fibroblasts and that PMNs are not contact inhibited for locomotion by endothelial cells. ACKNOWLEDGMENTS This work was supported by the Eastwood Memorial Foundation (D. de Bono).
(J. M. Lackie) and by the MRC
REFERENCES ARMSTRONG,
J. M. (1975). Studies on intercellular invasion in vitro using rabbit granulocytes (PMNs). I. Role of contact inhibition of locomotion. J. Cell
P. B., AND LACKIE,
peritoneal neutrophil
Biol. 65,439%462. ATHERTON, A., AND BORN,
G. V. R. (1972). Quantitative investigations of the adhesiveness of circulating polymorphonuclear leucocytes to blood vessel walls. J. Physiol. 222,447-474. CLARK, E. R., CLARK, E. L., AND REX, R. 0. (1936). Observations on polymorphonuclear leucocytes in the living animal. Amer. J. Anat. 59,123-173. COHN, Z. A., AND MORSE, S. I. (1959). Interactions between rabbit polymorphonuclear leucocytes and staphylococci. J. Exp. Med. 110,419. DE BONO, D. (1974). Effects of cytotoxic sera on endothelium in vitro. Nature (London) 252, 83-84. DE BONO, D. (1976), in preparation. LACKIE, J. M. (1974). The aggregation of rabbit polymorphonuclear leucocytes: Effects of antimitotic agents, cyclic nucleotides, and methyl xanthines. J. CeN Sci. 16, 167-180. LACKIE, J. M., AND ARMSTRONG, P. B. (1975). Studies on intercellular invasion in oitro using rabbit peritoneal neutrophil granulocytes. II. Adhesive intreactions between cells. J. Cell Sci. 19, 645-652.