Transepithelial chemotaxis of rat peritoneal exudate cells.

Br. J. exp. Path. (1983) 64, 644

TRANSEPITHELIAL CHEMOTAXIS OF RAT PERITONEAL EXUDATE CELLS C. W. EVANS*, J. E. TAYLOR*, J. D. WALKER* AND N. L. SIMMONSt From the Departments of *Anatomy and Experimental Pathology, and tPhysiology and Pharmacology, University of St Andrews, St Andrews, Fife KY16 9TS, Scotland Received for publication March 28, 1983

Summary.- The migration of peritoneal exudate (PE) cells into plain Millipore filters mounted in Boyden chambers occurs under random, chemokinetic and chemotactic conditions. Significant migration of such cells in vivo, however, involves both transendothelial and transepithelial penetration and occurs predominantly under pathological conditions where chemotactic agents are presumed to be present in gradient form. When Madin-Darby canine kidney (MDCK) epithelial cells are grown as a confluent monolayer on the Millipore filter of a Boyden chamber, transepithelial migration is seen only under chemotactic conditions thus modelling in vivo behaviour more effectively. The MDCK cell line exists as 2 variant strains which model different regions of the mammalian nephron. Strain I MDCK cells share features of the distal and collecting tubules and have relatively high junctional resistance (> 1k Q cm2). Strain II MDCK cells model the proximal segment of the nephron and have relatively low junctional resistance (c. 70 Q cm2). We have found that PE cells penetrate the less resistant strain II MDCK monolayer at a faster rate (as assessed by leading front migration) than they penetrate the tighter strain I monolayer. We have also utilized the electrophysiological features of MDCK monolayers to monitor transepithelial penetration. Our electrophysiological data indicate that rat PE cells penetrate MDCK monolayers of either strain by a transjunctional route with consequent reversible dissolution of the junctional complex. This extracellular path of PE cell migration was confirmed by ultrastructural observations. The extent of junctional dissolution and the delay in re-establishment of monolayer integrity (as assessed by electrophysiological means) are related to the concentration of PE cells added to the MDCK monolayer. Brief treatment (10 min) of the MDCK monolayer with the cation chelating agent EDTA also disrupts monolayer integrity, although its re-establishment is significantly faster than when disruption occurs by PE cell transmigration. Our results show a clear parallel between PE cell migration across an MDCK monolayer and changes in its electrophysiological parameters and thus suggest that transepithelial chemotaxis may be directly assessed by electrophysiological means. The use of Boyden chambers modified by the incorporation of epithelial monolayers may prove useful in in vitro studies of inflammation and could be adapted for studies of other pathological processes, such as metastasis, where considerable cell invasion is involved.

In vitro studies have shown that certain chemical substances modify the locomotory behaviour of randomly moving leucocytes. If the leucocytes, in response to the chemical stimulus, continue to move at random but display altered speed or turning then the active substance is considered to have a chemokinetic effect. If

leucocytes display nonthe moving random, directional locomotory behaviour then the active substance is considered to have exerted a chemotactic effect (Keller et al., 1977). The relative contributions of chemotaxis and chemokinesis to leucocyte accumulation at inflammatory sites in vivo remain uncertain because of currently

TRANSEPITHELIAL CHEMOTAXIS

unsurmountable technical difficulties. Consequently, the majority of studies on the locomotory behavioural properties of leucocytes have been performed under in vitro conditions using either the Boyden chamber technique (Boyden, 1962) or the under agarose method (Cutler, 1974; Nelson, Quie and Simmons, 1975). Both of these techniques have considerable limitations as models of in vivo conditions, although attempts have been made to improve their relevance. We have adopted a modified Boyden chamber technique in which Madin-Darby canine kidney (MDCK) epithelial cells are grown on the surface of a Millipore filter separating the 2 compartments of the Boyden chamber (Cramer, Milks and Ojakian, 1980; Walker, Evans and Simmons, 1981). When MDCK cells are grown to form a confluent monolayer upon a permeable substrate they display morphological and functional characteristics identical to a natural epithelium, including vectorial transport of salt and water (Simmons, 1982). In this way, leucocytes which emigrate from the upper (apical) chamber into the Millipore filter in response to a gradient of chemoattractant emanating from the lower (basal) chamber must cross a living cellular barrier as they would do in most circumstances in vivo. The majority of cases of leucocyte emigration in vivo, however, involve transendothelial rather than transepithelial extravasation. In some conditions, such as acute pyelonephritis caused by ascending infection from the lower urinary tract, transepithelial migration must be involved as well. In such examples, responding leucocytes leave small blood vessels in the vicinity of the inflammatory focus, traverse the connective tissue matrix and then penetrate the epithelial boundary to be ultimately expelled in the urine. The modification of the Boyden chamber used in this study models the last 2 steps in this process by incorporating collagen into the matrix of the Millipore filter and by growing a confluent epithelial monolayer on its surface. We now report on the effects of the

645

synthetic chemoattractant f-met-leu-phe (FMLP) on transepithelial migration of rat peritoneal exudate (PE) cells. MATERIALS AND METHODS

Cells. Peritoneal exudate (PE) cells were obtained from male Wistar rats 4 h after the i.p. injection of 10 ml of 1% oyster glycogen (Type II, Sigma). The exudate typically contained 62% polymorphonuclear neutrophil leucocytes, 34% macrophages and 4% lymphocytes. The cells were washed once and resuspended at appropriate concentration in Dulbecco's modification of Eagle's medium (DMEM) containing 20 mm HEPES buffer and 10% calf serum

(Flow Labs). Madin-Darby canine kidney (MDCK) cells (Strains I and II) were maintained as stock cultures in DMEM supplemented with 2 mM glutamine, non-essential amino acids, 50 iu ml-1 streptomycin sulphate and 10% foetal calf serum (all Flow Labs) essentially as described before (Simmons, 1981). Cell suspensions from stock cultures were prepared following treatment with Ca2+ and Mg2+ free Dulbecco's phosphate buffered saline (CMF) containing 0-1% trypsin (Difco 1:250) and 1 mm EDTA, washed once, and resuspended in growth medium. Appropriate concentrations of MDCK cells (max. 3 x 106 per filter) were then seeded on to 25 mm diameter Millipore filters (0-22 or 3 0 ,um pore size) clamped in mini-Marbrook chambers (Hendley Eng. Co., Essex) and grown to confluence under standard conditions. Locomotory behaviour. The locomotory behaviour of rat PE cells was assessed in modified Boyden chambers each of which consisted of a Millipore filter clamped into a mini-Marbrook chamber that sat in 35 mm diameter Petri dish. The top compartment of the mini-Marbrook chamber contained the PE cells (107 in 0-8 ml) and the bottom compartment and Petri dish contained the medium (4-6 ml). Chemotaxis was studied in the presence of a gradient of the chemoattractant n-formyl-l-methionyl-l-leucyl1-phenylalanine (FMLP, Miles Labs) by incorporating it into the lower compartment at appropriate concentration. The chemokinetic behaviour of PE cells was studied in the presence of an absolute concentration of FMLP (i.e. the same concentration of FMLP in both upper and lower compartments). Random locomotion was assessed in the complete absence of FMLP. Experiments were carried out with or without a confluent monolayer of MDCK cells grown on the upper surface of the Millipore filter. In some experiments, the Millipore filter was impregnated with rat tail or calf skin collagen (2-3 mg ml-1) and gelled in situ (Bornstein, 1958). The extent of locomotion into

646

C. W. EVANS, J. E. TAYLOR, J. D. WALKER AND N. L. SIMMONS

the Millipore filter was determined from alcohol fixed preparations stained with Mayer's haemalum by using the leading front method (Zigmond and Hirsch, 1973). Electrophysiology. The integrity of the MDCK cell monolayers grown on Millipore filters was monitored by electrophysiological means. The 2 strains of MDCK cells maintained in our laboratory possess epithelial characteristics, when grown in monolayer culture on Millipore filters, similar to the 2 extremes of transepithelial resistance found in the mammalian nephron (Simmons, 1982). Differences in transepithelial resistances in natural and MDCK epithelia reflect differences between the permeability of the paracellular (tight junctional) pathway (Barker and Simmons, 1981; Simmons, 1982). The high transepithelial resistance (> 1K Q cm2) of strain 1 MDCK epithelia, similar to that found in distal and collecting tubule segments, allowed direct assessment of epithelial integrity by measurement of the potential difference between the upper and lower compartments in response to an external current passed across the cell layer. Epithelial resistance was then determined from Ohm's law. Strain II MDCK epithelial layers possess a low transepithelial resistance (c. 70 Q cm2) similar to the mammalian proximal tubule. Since this value of resistance approaches that of an equivalent volume of physiological saline, resistance measurements are not sufficiently accurate to directly assess changes in the integrity of the epithelium. However, like proximal tubule epithelium, strain II MDCK layers possess a cation selective paracellular pathway (Richardson, Scalera and Simmons, 1981; Barker and Simmons, 1981). Accordingly, epithelial integrity was measured by the magnitude of dilution potentials elicited across the epithelium. Briefly, replacement of the basal medium NaCl with isosmotic choline chloride elicits a diffusion potential (basal solution electropositive) the magnitude of which is given by the Nernzt equation: RT NaCla, ,Y= -F- (tN a-tcl) lnNCl where y is the observed potential difference, R, T and F have their standard meanings, NaCla and NaClb refer to the NaCl concentrations in the apical and basal solutions, and tN a and tci are the transference numbers for Na+ and Cl-. The ratio of transference numbers (tN a/tci) correlates with the morphological criteria indicating monolayer confluency (Barker and Simmons, 1981). Electron microscopy. Millipore filters for transmission electron microscopy were fixed in

4% paraformaldehyde/5% glutaraldehyde in 0-08M Na cacodylate buffer, post-fixed in 1%

OS04, and embedded in LR white acrylic resin

(London Resin Co.). Ultrathin sections were double stained with 2% uranyl acetate and 0-4% lead citrate (Venable and Coggeshall, 1965) and examined in a Zeiss EM 9S-2 electron microscope. Scanning electron microscopy was carried out on a Cambridge Stereoscan 600 following alcohol fixation, critical point drying and gold sputter coating of the Millipore filter and cells. RESULTS

The effects of FMLP on rat PE cell chemotaxis across plain Millipore filters are shown in Fig. 1. The peak chemotactic response was seen at 10-8 FMLP. Concentrations of FMLP above 10-7M have an inhibitory effect on locomotion relative to random movement. Concentrations between 10-8 and 10-14M FMLP exert a chemotatic effect which declines logarithmically from the peak value. In a series of preliminary experimeints designed to test the dose-dependent effects of FMLP on transepithelial chemotaxis, we measured the extent of migration at 90 min of rat PE cells through confluent MDCK strain II monolayers seeded at 3 x 106 cells per filter 3 days earlier. As seen in the Table, the peak chemotactic effect under these conditions was found at 10-7M FMLP although higher concentrations elicited a significant response. In subsequent experiments the concentration of FMLP used varied between 10-6 and 10-8M. The Table also shows that no penetration of MDCK strain II monolayers occurred in the absence of FMLP (random conditions), in absolute concentrations of FMLP (chemokinesis) or in the presence of a negative gradient of FMLP (negative chemotaxis). The higher concentration of FMLP needed to elicit chemotaxis under these conditions compared to a plain Millipore filter is consistent with the greater diffusion restriction to FMLP afforded by the epithelial cell barrier. The effect of transepithelial migration of rat PE cells on the integrity of confluent MDCK strain II monolayers is shown in Fig. 2. Epithelial integrity, as indicated by the ratio of transference numbers,

647


TABLE.-Migration of PE cells across Strain II monolayersa Concentration FMLP (M) Positive gradientc Zero gradientd Negative gradiente

Ob

10-8

0

ND 0 0

5x

10-8

42-6+9-9 0

ND

10-7 65*9+8*8

54-5+4-9

10-6 51*1+7*1

0

0

ND

0

5x

0 0

10-7

3 x 106 MDCK strain TI cells were seeded on collagen impregnated, 3 ,um pore size Millipore filteirs and left for 3 days to reach confluence. b Random locomotion (absence of FMLP, PE cells in apical compartment of Boyden chamber). c Chemotaxis (FMLP in basal compartment, PE cells in apical compartment). d Chemokinesis (FMLP in basal and apical compartments, PE cells in apical compartment). e Negative chemotaxis (FMLP and PE cells in apical compartment). ND, not done. Results represent leading front migration ([Lm) ± s.d., 3 filters per group. a

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.

t }SM,S,,* E L'. pmmu

FIG.

1.-Effects of varying concentrations of

FMLP

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rat PE cell chemotaxis into

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extent of mi incubation filter.

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Shaded

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the

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PE cells per migration

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at each

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concentration.

chemotactic

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Under

con-

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when 10-7m FMLP was

basal

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and basal

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monolayer and

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was

Our

change in by electro-

no

observed

I 30 time (min)

45-

60

FIG. 2.-Electrophysiological assessment of PE cell penetration of confluent MDCK strain II monolayers grown on Millipore filters (0-22 ,um pore size). The ratio of transference numbers tNa/tcl corresponds with monolayer integrity (see text). Penetration of the monolayer occurs under chemotactic conditions (A, positive gradient of 10-7M FMLP) but not under chemokinetic conditions (*, absolute concentration of 10-7M FMLP). Points shown are mean values + s.e. mean for at least 3 separate experiments.

electrophysio- tration of MDCK strain I monolayers by rat PE cells upon changes in transepithelial resistance. As before, 10-7M FMLP which does not promote transepithelial migration under chemokinetic conditions, had no effect upon transepithelial resistance. Under chemotactic conditions, however, where transepithelial migration occurs, a significant drop in epithelial integrity was

logical recordings (Fig. 2) thus parallel our visual observations (Table). In addition, no changes in monolayer integrity were detected when PE cells were added under random conditions or when FMLP alone (no PE cells) was added to the system (not shown). Fig. 3 shows the effects of the pene-


648

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FIG. 3. Transepithelial penetration of MDCK strain I monolayers by PE cells as assessed by resistance changes (see text). Disruption occurs under chemotactic (A) but not under chemokinetic (*) conditions. Other conditions as in Fig. 2.

observed. Again, no significant changes in monolayer integrity were detected by electrophysiological means under random conditions or in the presence of FMLP alone (not shown). In both epithelial MDCK strains therefore, transepithelial chemotaxis was associated with a significant decrease in monolayer integrity. Since both monolayer resistance and paracellular permselectivity are measures of the junctional (non-cellular) pathway (Barker and Simmons, 1981; Simmons, 1982) it is likely that the route for transepithelial chemotaxis by PE cells does not involve cellular penetration, but rather transjunctional migration (see below). Because a decrease in junctional integrity was observed, it is also evident that during transepithelial migration by PE cells the MDCK monolayer is incapable of maintaining its barrier properties in contradiction to the data of Cramer et al. (1980). The clear parallels between transepithelial chemotaxis and changes in electrophysical parameters suggest that chemotaxis may be directly monitored by electrophysiological means, although the precise temporal relationship between these 2 properties needs to be evaluated further. The rates of penetration of epithelial monolayers composed of either Strain I or Strain II MDCK cells were assessed histologically by the leading front method and are compared in Fig. 4. The rate of

2

xs

-,

30

I

0

1

~ I Il 2 3-1 time hr)

4

Fi. 4. Rates of PE cell chemotatic migration through Strains I and II MDCK monolayers grown to confluence on Millipore filters (3 ,um pore size) impregnated with rat tail collagen (3 mg. ml-1). The extent of migration in response to 10-6M FMLP was assessed by the leading front method after incubation with 1 07 PE cells for various time intervals. Points shown are mean values for 3 experiments. Strain I MDCK (0), Strain II (0).

transepithelial penetration by rat PE cells was found to be greater for Strain II monolayers than for Strain I. This increased rate of PE cell migration corresponds to the lower electrical resistance and higher permeability of Strain II compared to Strain I epithelial monolayers. Comparison of the data in Fig. 2 and 3, however, shows that the relative differences in the decline of epithelial integrity of Strain I and Strain II monolayers as assessed electrophysiologically were apparent only over the initial 30 min. This variation may reflect the different time scales involved, with initial but slower junctional disruption ultimately being reflected in a decreased rate of migration. An exact correspondence between the rate of monolayer penetration by PE cells and changes in transepithelial electrophysiological para-

649

TRANSEPITHELIAL CHEMOTAXIS 1.0'

0

0

8 0.5

z L C

10

\

30

20

70 80 time (min)

90

100

110

120

FIG. 5.-Disruption and recovery of MDCK Strain I monolayer resistance following treatment with CMF-EDTA. After 10 min incubation, CMF-EDTA was replaced with normal medium. Points shown are from 4 experiments normalized to take into account variations in starting resistance. Monolayers were grown to confluence on 0-22 ,um pore size Millipore filters.

meters has not been established since electrophysiological changes are dependent upon the form of PE cell penetration (few multicellular foci or multiple single cell sites) and the recovery of epithelial integrity at each separate migration locus. The effects of cell junction disruption by CMF containing 1mM EDTA (CMFEDTA) upon the electrical resistance of Strain I MDCK epithelial monolayers are shown in Fig. 5. Transepithelial resistance dropped rapidly to 38% of its starting value within 10 min of monolayer incubation with CMF-EDTA. Following replacement of CMF-EDTA with normal medium, transepithelial resistance was seen to be re-established to 75% of its starting value by 80 min. Similar data have been obtained for low-resistance MDCK epithelia by Cereijido and coworkers (Cereijido, Meza and MartinezPalomo, 1981).

1.0r-

._

U) n

0.5

l

N

E 0

z

OL_

I 0

I

20

I

I

40

I

I

60

time (hr) FIG. 6.-Recovery of transepithelial resistance by MDCK Strain I monolayers following chemotactic penetration by PE cells in response to 10-6M FMLP. Confluenent MDCK monolayers on Millipore filters (3 Fm pore size, impregnated with calf skin collagen at 2 mg ml-1) were seeded with rat PE cells at 3 different inocula: 0.33 x 107 (-) 0.67 x 107 (A) and 107 per filter (-). After 90 min both PE cells and FMLP were replaced with normal medium and the re-establishment of monolayer resistance was followed. Points shown are normalized results from 3 single filters.

650


FI G. 7.-Penetration through an MDCK strain I monolayer (grown on a 0.22 ;m pore size Millipore filter) by a polymorphonuclear neiutrophil leucocyte (PMNL) obtained from a rat peritoneal cell exudate((107cells per filter). Note that the MDCK cells appear to have re-established contact on the apical side of the migrating cell. Bar= 5 ,um.

Fig. 6 shows the rate of recovery of Strain I MDCK monolayer integrity (monitored by changes in electrical resistance) following chemotactic penetration by rat PE cells at 3 different inocula concentrations. The initial decrease in electrical resistance was dependent upon inoculum concentration up to 0-67 x 107 cells/ filter, after which no further decrement was observed. The rate of recovery of monolayer resistance was also dependent upon PE cell inoculum concentration. It should be noted that recovery under these conditions was considerably slower than that observed after CMF-EDTA treatment (Fig. 5) possibly indicating the need for local cell repair and junctional protein synthesis following emigration of PE cells. In several experiments morphological correlation of rat PE cell chemotaxis across MDCK epithelia was sought by scanning and transmission electron micro-

scopy. Fig. 7 shows transepithelial migration by a polymorphonuclear neutrophil leucocyte. This demonstrates unequivocally that PE cell migration occurs via a paracellular (junctional) route and not via an intracytoplasmic route and directly confirms our electrophysiological data. Fig. 8 shows the presence of PE cell foci on the surface of the epithelial monolayer. Fractures of such foci, as shown in Fig. 9 confirmed monolayer disruption and showed rat PE cell penetration of the Millipore filter. Areas of the MDCK monolayer not associated with PE foci development did not display significant numbers of adherent PE cells (Figs 8 and 9). DISCUSSION

Strong circumstantial evidence that chemotaxis might operate in vivo during the extravasation of leucocytes from small


FIG. 8. Foci (F) of rat PE cells formed during chemotactic penetration of an MDCK Strain I monolayer in response to 10-6M FMLP. The MDCK cells were grown to confluence on a 3 [Lm pore size Millipore filter and pre-treated with CMF-EDTA for 10 min prior to addition of PE cells (107 per filter). Similar but less abundant foci were found on preparations not treated with CMF-EDTA. Bar= 100 ,um. FIG. 9.- Fracture through a small PE cell focus formed as in Fig. 8. Note the disruption of monolayer integrity and PE cell penetration of the Millipore filter (arrows). Bar = 20 ,um.

651

652


blood vessels and their accumulation at inflammatory foci has been provided from in vitro studies using the Boyden chamber technique (reviewed in Wilkinson, 1982). However, the use of theBoyden chamber technique as a model for in vivo events has obvious limitations. The Millipore filter through which the cells migrate, for example, is an inadequate model for connective tissue and no cellular barrier to migration is provided. Thus, in the standard Boyden chamber migration of inflammatory cells proceeds under random, chemokinetic or chemotactic conditions. When an epithelial barrier is grown on the Millipore filter, however, emigration occurs only under chemotactic and not chemokinetic or random conditions. This finding is intuitively correct, since leucocytes are normally confined to blood vessels and extravasate specifically under in vivo conditions (Allison, Smith and Wood, 1955). The question immediately arises as to whether an epithelial barrier adequately models other cellular barriers which leucocytes must penetrate in vivo such as the blood vessel endothelium. Recent evidence suggests that leucocytes spontaneously cross endothelial monolayers in vitro (De Bono, 1976; Beesley et at., 1978) but since this is not known to occur in vivo it seems reasonable to argue that in vitro culture of endothelial cells perturbs an essential feature of their integrity. The use of a kidney epithelial barrier as described in this report may have specific relevance to certain pathological conditions. Chemotactic activity has been demonstrated in the urine of patients suffering from acute or chronic cystitis, and chronic pyelonephritis (Tsujimura et al., 1980). Leucocyte infiltration of the urinary tract was evident in these cases indicating that transepithelial migration had occurred. Transepithelial migration also occurs in many invasive tumours. Bladder metastases are often derived from tumour fragments carried in the urine from primary growths in the renal pelvis and ureter. Development of the metastases is associated with tumour cell invasion of

the bladder epithelium from the apical epithelial surface (Willis, 1973). The replacement of rat PE cells in the modified Boyden chamber vith invasive tumour cells may provide a means for exploring further the phenomenon of metastasis. Although we have measured transepithelial migration in the apical to basal direction (as would happen in bladder metastasis) leucocyte migration in the reverse direction would be expected in other pathological conditions such as pyelonephritis. Cramer et al. (1980), however, have shown that similar events occur whether migration is in the apical to basal or reverse direction. Thus, epithelial cell polarity does not determine the direction of leucocyte migration (Cramer et al., 1980). Our results show that greater concentrations of the chemoattractant FMLP are required for transepithelial migration than for migration through plain Millipore filters. We attribute this difference to the effects of the tight junction seal on FMLP permeation through the MDCK monolayer. Collagen impregnated into the filter to model the connective tissue matrix had no observable effect on transepithelial migration. It remains uncertain, however, as to whether the PE cells digested the collagen in advance of movement or whether they merely pushed it aside as they moved on the Millipore filter substrate. Studies are currently in progress to examine this aspect further. Penetration of the MDCK monolayers occurs via an intercellular route involving disruption of the junctional complex (tight junctions, gap junctions and desmosomes). The major barrier to migration is the tight junction, since if this barrier was penetrated and the cells were held up at desmosomes, for example, then an electrophysiological change would have been detected without observable migration into the Millipore filter. Such examples were never observed. Rat PE cells penetrate strain II MDCK cells faster than Strain I cells which correlates with significant differences in the electrophysiological properties of the 2 strains


(Simmons, 1982). Thus, the greater the resistance of the monolayer, the tighter the junctional seals and the longer the time necessary for penetration. Maintenance of both endothelial and epithelial cell layer integrity is essential in vivo after leucocyte migration if appropriate physiological function is to be sustained. Our ultrastructural observations show that epithelial monolayers need not be significantly damaged during PE cell migration and that epithelial cells may rapidly re-associate (Fig. 7). The sealing properties of tight junctions, however, develop progressively as shown by our experiments involving replacement of migrating PE cells and FMLP with fresh medium. Increasing numbers of rat PE cells show more extensive changes in monolayer penetration which take longer to return to normal. These results parallel clinical observations during the development of an extensive inflammatory response. The integrity of the epithelial monolayer can also be diminished by cation chelation which leads to disruption of the junctional complex (Cereijido et al., 1978; 1981). Substantial recovery of monolayer integrity occurs within 80 min of such treatment, whereas monolayer recovery after PMN penetration is considerably delayed. We attribute this difference in recovery rates to the more severe effects of PE cell products on junctional disruption and to the presence of residual PE cells, some of which may have only partially penetrated the epithelial monolayer. Following treatment of MDCK Strain I monolayers with PE cells under chemotactic conditions with or without prior junctional disruption with CMF-EDTA, it was apparent that monolayer penetration occurred in discrete foci. Such foci were not so obvious when Strain II MDCK monolayers were used. The presence of foci, however, implies that not all junctions are disrupted following such treatment. The surface of the epithelial monolayer was generally non-adherent for PE cells. Whether there was any change in PE cell/epithelium adhesion at foci of

653

penetration remains to be elucidated, although our ultrastructural observations showed no evidence of close contacts between these cells. We conclude that rat PE cells penetrate MDCK epithelial monolayers via an intercellular route with reversible dissolution of membrane junctions. The effects of transepithelial migration can be followed by monitoring tight junction integrity by electrophysiological methods. In an earlier report, Cramer and her colleagues failed to detect resistance changes during transepithelial migration (Cramer et al., 1980). This was probably due to a combination of the low electrical resistance of these MDCK monolayers (c. 181 Q cm2) and low assay sensitivity. We have found that high resistance MDCK monolayers (> 1 kQ cm2) give unequivocal results by direct resistance measurements whereas low resistance monolayer (c. 70 Q cm2) require indirect assessment of epithelial integrity using an assay based on the cation selectivity of the intercellular pathway.

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