Phalloidin prevents by proinflammatory HIROSHI
ASAKO,
leukocyte emigration induced stimuli in rat mesentery
ROBERT
E. WOLF,
D. NEIL
GRANGER,
AND RONALD
J. KORTHUIS
Departments of Physiology and Medicine, Center for Excellence for Arthritis and Rheumatology, Louisiana State University Medical Center, School of Medicine in Shreveport, Shreveport, Louisiana 71130 Asako, Hiroshi, Robert E. Wolf, D. Neil Granger, and Ronald J. Korthuis. Phalloidin prevents leukocyte emigration induced by proinflammatory stimuli in rat mesentery. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1637-H1642, 1992.-The objective of this study was to determine whether phalloidin, a potent microfilament stabilizer, can modify inflammatory mediator-induced leukocyte adhesion and extravasation in postcapillary venules of the rat mesentery. To address this issue, the rat mesentery was prepared for in vivo microscopic observation. Venules with initial diameters ranging between 25 and 35 pm were selected for study. Erythrocyte velocity, vessel diameter, leukocyte rolling velocity, and the number of adherent (stationary for 30 s) and emigrated leukocytes were initially determined during superfusion of the mesentery with phosphate-buffered saline. After these variables were recorded during the control period, either 100 nM platelet-activating factor (PAF), 20 nM leukotriene B4 (LTB*), or 1 PM N-formylmethionyl-leucyl-phenylalanine (FMLP) was added to the superfusate. Repeat measurements were obtained between 50 and 60 min after initial exposure to the inflammatory mediator. In some experiments, rats were given phalloidin (25 or 500 pg/kg iv) 30 min before superfusion with the inflammatory mediators. Superfusion of the mesentery with either PAF, LTB4, or FMLP enhanced leukocyte adherence and emigration and reduced leukocyte rolling velocity. Pretreatment with the low dose of phalloidin effectively prevented leukocyte emigration but had no effect on the increased leukocyte adherence elicited by the three inflammatory mediators. However, when administered at the higher dose, phalloidin prevented both leukocyte adherence and emigration. Neither dose of phalloidin altered the upregulation of neutrophil membrane CD1 l/CD18 glycoprotein adherence complex induced by PAF or LTB,. These results are consistent with the concept that PAF, LTB4, and FMLP increase leukocyte extravasation by a process that may involve alterations in the endothelial cell cytoskeleton. cytoskeleton; microfilaments; F-actin; leukocyte adherence; leukocyte rolling; postcapillary venules; platelet-activating factor; leukotriene B,; formyl-methionyl-leucyl-phenylalanine; inflammation ACCUMULATION of polymorphonuclear leukocytes in the interstitial space is one of the major hallmarks of an acute inflammatory response (12, 16, 30, 33). Although the process whereby these adherent leukocytes breach the microvascular barrier to enter the interstitial space is not well understood, there is a growing body of evidence which suggests that reorganization of actin microfilaments in the endothelial cell cytoskeleton may play a key role in modulating leukocyte diapedesis. For example, leukocytes emigrate through interendothelial cell junctions, and the integrity of these junctions is regulated by cytoplasmic microfilaments (1,4,6,7,9,12, 13, 20, 23-27, 29). In addition, coincident and rather remarkable changes in endothelial ceil topography and cytoskeletal organization occur as leukocytes emigrate across postcapillary venules (23). Perhaps the strongest
THE
0363-6135/92
$2.00
Copyright
support for this concept is derived from the observation that inflammatory mediator-induced leukocyte emigration across cultured endothelial cells or into dermabrasion chambers in vivo is prevented by prior treatment with phalloidin (9,25), an agent that promotes junctional integrity by stabilizing existing filamentous actin and by inducing actin filament formation from actin monomers and oligomers (7, 35). Before a leukocyte can emigrate into the tissues in response to a chemotactic signal, these cells must first adhere to postcapillary venular endothelium (12, 14-16, 30,33). Thus it is possible that phalloidin acts indirectly to reduce leukocyte diapedesis by preventing the adhesion of these phagocytic cells to microvascular endothelium. The aim of the present study was to further explore the role of the endothelial cell cytoskeleton in the process of leukocyte emigration by examining this process in vivo using intravital microscopic approaches. In these studies, leukocyte adherence and emigration were elicited by superfusing the rat mesentery with platelet-activating factor (PAF), leukotriene B4 (LTB4), or N-formyl-methionyl-leucyl-phenylalanine (FMLP) in control animals and in rats pretreated with phalloidin. Our results indicate that phalloidin attenuates leukocyte emigration without exerting a significant influence on leukocyte adhesion to venular endothelium. MATERIALS AND METHODS Surgical procedure. Male Sprague-Dawley rats weighing between 200 and 250 g were fasted for 24 h before the experiments. After the animals were anesthetized with pentobarbital sodium (65 mg/kg body wt), a tracheotomy was performed to facilitate respiration during the experiment. The right carotid artery and left jugular vein were cannulated for monitoring systemic arterial pressure and for intravenous administration of drugs (phalloidin and supplemental doses of anesthetic), respectively. After a midline abdominal incision was made, the right and left renal arteries and veins were ligated to prevent the renal excretion of phalloidin. Intravital microscopy. After the animal was placed in a laterally recumbent position on an adjustable Plexiglas microscope stage, a segment of midjejunum was exteriorized through the abdominal incision, and the mesentery was prepared for microscopic observation as described previously (2, 14). The mesentery was draped over an optically clear cover slip that allowed for observation of a 2-cm2 segment of tissue. All other exposed tissue was covered with saline-soaked gauze and Saran Wrap to minimize evaporative water loss. The temperature of the pedestal was maintained at 37°C with a constant temperature water circulator (model 80, Fisher Scientific). Rectal and mesenteric temperatures were continuously monitored using an electrothermometer. The mesentery was then superfused with warmed bicarbonate-buffered saline (pH 7.4) that was bubbled with a mixture of 5% CO,-95% N,. An inverted microscope (Nikon Optiphoto, Japan) with a
0 1992 The
American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 15, 2019.
H1637
H1638
PHALLOIDIN
ATTENUATES
x40 objective lens (Nikon Optiphoto, Japan) and a ~10 eyepiece was used to observe the mesenteric microcirculation. The mesentery was transilluminated with a 12 V-100 W direct current-stabilized light source. A video camera (VK-C150, Hitachi, Japan) mounted on the microscope projected the image onto a color monitor (PVM-2030, Sony, Japan), and the images were recorded using a video cassette recorder (NV8950 Panasonic, Japan). A video time-date generator (WJ810 Panasonic) projected the time, date, and a stopwatch function onto the monitor. Single unbranched venules with diameters ranging between 25 and 35 pm and a length X50 pm were selected for study. Venular diameter (D,) was measured either on- or off-line using a video image-shearing monitor (IPM, LaMesa, CA). The number of adherent leukocytes was determined off-line during playback of video-taped images. A leukocyte was considered to be adherent to venular endothelium if it remained stationary for a period 230 s (14). Adherent cells were expressed as the number per 100~pm length of venule. The number of emigrated leukocytes was also determined off-line during playback of videotaped images. Any interstitial leukocytes present in the mesentery at the onset of the experiment were subtracted from the total number of leukocytes that accumulated during the course of the experiment. Leukocyte emigration was expressed as the number per microscopic field (1.7 x lo-” mm2 or 150 ,ccrnlength of venule). Rolling leukocytes were defined as those white blood cells that moved at a velocity less than that of erythrocytes in the same stream. Leukocyte rolling velocity was determined from the time required for a leukocyte to traverse a given distance along the length of a venule. Centerline red blood cell velocity ( VItHC) was measured using an optical Doppler velocimeter (Microcirculation Research Institute, Texas A&M University) that was calibrated against a rotating glass disk coated with red blood cells. Venular blood flow was calculated from the product of mean red blood cell velocity [ Vlxlean = centerline cross-sectional area, asvelocity t 1.6 (S)] and microvascular suming cylindrical geometry. Venular wall shear rate (y) was calculated based on Newtonian definition: y = 8( V,,,,/D,) (18) . Flow cytometry studies. Flow cytometry was used to quantitate surface expression of CD18 on rat polymorphonuclear leukocytes. The analysis was performed using whole blood samples to minimize artifactual upregulation of CD18 associated with ex vivo manipulation (34). A 200-~1 aliquot of heparinized whole blood obtained from Sprague-Dawley rats was mixed with 200 ~1 of phosphate-buffered saline (PBS) in polypropylene test tubes and incubated either alone or after gentle mixing with either phalloidin alone (0.5 or 10 pg/ml final concentration, which approximates the initial blood concentration achieved in the in vivo experiments) or with one of the three proinflammatory mediators [PAF (100 nM), LTB, (20 nM), or FMLP (1 PM)] in the presence and absence of phalloidin (at either 0.5 or 10 pg/ml) for 30 min at 37°C. After incubation, 500 ~1 of a solution containing 7 pg/ml of a monoclonal antibody (WT3) directed against CD18 (3.9 pg/ml final concentration) (32) were added to the samples, and the test tubes were placed at 4°C for 20 min. The samples were then washed twice with PBS and centrifuged at 1,000 rpm for 3 min. Then 200 ~1 of a loo-fold dilution of fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin (7.5 pg/ml final concentration, Zymed Laboratories, San Francisco, CA) were added, and the mixture was incubated for 20 min at 4°C. The samples were then washed twice with PBS and centrifuged at 1,000 rpm for 3 min. The red cells were then lysed by addition of 1 ml of 25-fold dilution of Immuno-lyze reagent (Coulter Immunology, Hialeah, FL) to each tube followed by vigorous vortexing, after which the samples were allowed to sit for 90 min. Then 250 ~1 of fixative (Coulter Immunology, Hialeah, FL) was added to
LEUKOCYTE
EMIGRATION
each tube. After the samples were vigorously vortexed, the samples were washed twice with 3 ml of PBS at 1,000 rpm for 3 min. Then 200 ~1 of PBS was added to each tube. Analysis was performed on an EPICS 753 flow cytometer/sorter (Coulter Electronics, Hialeah, FL) for the simultaneous accumulation of immunofluorescence in addition to forward angle and 90” light scatter signals. Dead cells and debris were excluded by forward angle and 90” light scatter gating or, in some experiments, by the exclusion of dead cells that incorporate propidium iodide (21). Controls included cells stained with the secondary reagent alone or cells stained with an irrelevant-isotype-matched control monoclonal antibodies. Twenty-five thousand cells were analyzed in each experiment, and each experiment was repeated four times. Experimental protocols. After all parameters measured online (arterial pressure, erythrocyte velocity, and venular diameter) were in a steady state, images from the mesenteric preparation were recorded on videotape for 10 min for subsequent analysis of leukocyte adherence, emigration, and rolling velocity. Immediately thereafter, the mesentery was superfused for 60 min with either 100 nM PAF (Sigma Chemical, St. Louis, MO), 20 nM LTB, (Calbiochem, LaJolla, CA), or 1 PM FMLP (Sigma Chemical) dissolved in bicarbonate-buffered saline (n = 4-6 in each group). Previous reports indicate that these doses elicit significant leukocyte adherence and emigration (2, 5). Video recording and measurements of all parameters were repeated during the final 10 min of the 60-min period of inflammatory mediator superfusion. In additional experiments, animals received an intravenous injection of phalloidin (dissolved in saline, stock solution 1 mg/ml, Sigma Chemical) at doses of either 25 or 500 pg/kg 30 min before the addition of an inflammatory mediator to the superfusate. The lower dose of phalloidin produces an initial blood concentration of 0.4-0.6 PM, a dose similar to that which has been used in earlier work from our laboratory and by others (1,9,20,25). Video recordings and measurement of all variables were then obtained according to the protocol outlined above (n = 4-6 in each group). Statistical analysis. All values are reported as means t SE. The data were initially analyzed by using a one-way analysis of variance. To determine which groups were statistically different, multiple comparisons were analyzed using Scheffe’s post hoc test (31). Statistical significance was defined as P < 0.05.
RESULTS Figure 1 summarizes the effect of PAF, LTB4, and FMLP on leukocyte adherence in rat mesenteric venules. Under baseline conditions, there were 4.5 t 0.4 adherent leukocytes per 100 pm length of venule. Superfusion of the mesentery with either 100 nM PAF, 20 nM LTB4, or 1 PM FMLP was associated with a significant increase in the number of adherent leukocytes to 29.5 t 4.7, 39.4 t 6.5, and 63.0 t 5.9 per 100 pm venule length, respectively. The increased leukocyte adherence induced by either PAF, LTB4, or FMLP was not affected by treatment with phalloidin at 25 pg/kg (32.0 t 2.6, 33.6 t 6.7, 57.7 t 4.1 per IOO-pm length of venule, respectively) but was significantly attenuated by treatment with 500 pg/kg phalloidin (8.3 t 2.0 or 21.0 t 1.4 or 22.4 t 5.1 per loo-pm length of venule, respectively). The effect of PAF, LTB4, and FMLP on the number of emigrated leukocytes per microscopic field is illustrated in Fig. 2. Superfusion with either PAF, LTB*, or FMLP significantly increased the number of emigrated leukocytes from 3.1 t 0.5 under control condi-
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 15, 2019.
PHALLOIDIN
ATTENUATES
LEUKOCYTE
x
I
.--b-J 0 ?a Oi *El 3’ aE --la. *g C-
50
0 a, >
e60-
40
c
50-
l
-
. u
=z s
4o
H1639
EMIGRATION
as +A0 0 Y 3
30
20
10
3 0
PAF
LTB,
Fig. 1. Effects of phalloidin on inflammatory mediator-induced leukocyte adherence in rat mesenteric venules. PAF, platelet-activating factor; LTB4, leukotriene B,; FMLP, N-formyl-methionyl-leucyl-phenylalanine; IM, inflammatory mediator. Solid bars, control; open bars, IM; hatched bars, IM + phalloidin (low); cross-hatched bars, IM + phalloidin (high). * P < 0.05 relative to control; ** P < 0.05 relative to corresponding IM. 60
I/) al -w x
50
0
40
0 24 33;
b
-0L 2: o.-
L CP
E
W
30
20
10
-
0 .
PAF Fig. 2. Effects cyte emigration and symbols.
LTB,
of phalloidin on inflammatory in rat mesenteric venules.
PAF
FMLP
FMLP mediator-induced leukoSee Fig. 1 for abbreviations
tions to 19.3 t 2.8, 13.0 t 2.8, and 34.4 t 6.2 per microscopic field, respectively. The increased number of emigrated leukocytes induced by either PAF, LTB4, or FMLP was significantly attenuated by treatment with phalloidin at 25 pg/kg (7.4 t 1.3, 4.2 t 1.0, 14.0 t 4.5, leukocytes/field, respectively). Administration of 500 pg/kg phalloidin did not produce a further reduction in the leukocyte emigration induced by these proinflammatory mediators (4.8 t 0.8, 3.6 t 1.1, 4.8 t 1.4 leukocytes/field, respectively). Figure 3 summarizes the response of leukocyte rolling velocity in postcapillary venules to either PAF, LTB*, or FMLP. Leukocyte rolling velocity in rat mesenteric venules under baseline conditions was 44.1 t 1.0 pm/s, a value similar to that previously reported for cat mesenteric venules (14). Leukocyte rolling velocity was significantly reduced by superfusion of the mesentery with PAF (25.9 -+ 2.5 pm/s), LTB4 (26.5 + 3.8 pm/s), or FMLP (19.2 t 1.0 urn/s). The PAF-. LTBn-. or FMLP-induced
Fig. 3. Effects rolling velocity and symbols.
LTB,
of phalloidin on IM-induced in rat mesenteric venules.See
FMLP alterations in leukocyte Fig. 1 for abbreviations
reduction in leukocyte rolling velocity was significantly attenuated by 500 pg/kg phalloidin (37.4 t 4.3,38.2 t 3.9, 27.2 t 2.0 pm/s, respectively) but not by 25 pg/kg phalloidin (23.2 t 1.5, 21.3 t 0.7, 23.2 t 2.0 pm/s, respectively). Red blood cell velocity, venular diameter, and venular wall shear rate were not significantly altered by superfusion with either PAF or LTB4 or FMLP in the presence or absence of phalloidin (25 or 500 pg/kg) (Table 1). Flow cytometric analysis of CD 1 l/CD 18 expression in isolated rat neutrophils revealed that basal expression of this adhesive glycoprotein was not altered by either the low or high dose of phalloidin. Although a significant increase in fluorescence intensity was detected by flow cytometry in response to PAF and FMLP (6.4 t 2.1 and 22.2 t 3.5% increase relative to unstimulated cells), LTB4 did not appear to increase the surface expression of CDll/CD18 (4.9 t 2.8% increase relative to unstimulated cells). This latter observation Table 1. Effects of platelet-activating factor, leukotriene Bq, and formyl-methionyl-leucyl-phenylalanine in absence and presence of phalloidin on mean red blood cell velocity, venular diameter, and wall shear rate RBC
Control PAF PAF + PH(L) PAF + PH(H) Control LTB, LTB, + PH(L) LTB4 + PH(H) Control FMLP FMLP + PH(L) FMLP + PH(H)
Velocity, mm/s
2.3t0.3 1.7kO.3 1.5t0.3 1 J3kO.9 3.6t0.4 2.1t0.2 2.9tl.O 2.8t0.5 2.8t0.3 3.1t0.7 2.0t0.2 1.6kO.5
Diameter, Pm
28.6k0.7 27.8kl.O 29.4t1.7 29.3t1.7 29.2kO.7 32.1kO.9 34.6k2.5 27.81k0.6 29.6k0.7 28.8tl.l 33.0t0.9 29.8k0.8
Wall
Shear s-l
Rate,
408t56 301t62 256t38 325k175 603k62 330t35* 443t142 497tlOl 476t62 533t115 300t35 264t88
Values are means t SE. PAF, platelet-activating factor; PH(L), phalloidin at low (25 pg/kg) dose; PH(H), phalloidin at high (500 pg/kg) leukotriene B,; FMLP, N-formyl-methionyl-leucyldose; LTB4, phenylalanine; RBC, red blood cell. *Values were statistically different from control.
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 15, 2019.
H1640
PHALLOIDIN
ATTENUATES
LEUKOCYTE
EMIGRATION
is consistent with the fact that rat neutrophils are reported to be less responsive to LTB4 than granulocytes from other species (10, 11) and suggests that LTB4-induced leukocyte adhesion and emigration may be related to an effect on the endothelium. In any event, neither the low nor the high dose of phalloidin modified the increase in fluorescence intensity induced by PAF (5.1 t 2.0 and 6.4 t 1.4%, respectively) or FMLP (24.3 t 3.7 and 23.7 t 3.1%, respectively), indicating that the bicyclic heptapeptide does not influence the upregulation and activation of CD18 on rat neutrophils.
reperfusion alters neutrophil sequestration and increases microvascular permeability through a direct effect on the endothelium that is due in part to alterations in the endothelial cell cytoskeleton (20). These observations suggest that cytoskeletal alterations induced by proinflammatory stimuli lead to disruption of junctional complexes which, in turn, facilitates the penetration of the microvascular barrier by the emigrating leukocyte. Based on the aforementioned observations, it has been suggested that reorganization of microfilamentous cytoskeletal elements within the endothelium modulates the integrity of interendothelial cell junctions thereby DISCUSSION allowing leukocyte emigration to proceed (9, 20, 25). The extravascular accumulation of polymorphonuclear However, the fact that leukocytes must first adhere to leukocytes is a characteristic feature of an acute inflamthe endothelium before they can emigrate into the tissues matory response (5, 12, 14, 30, 33). Although it is well (12,14, 16,29,30) suggestsanother potential explanation known that leukocytes emigrate through interendothelial for the ability of phalloidin to attenuate leukocyte diacell junctions, our understanding of the mechanisms pedesis. That is, it is possible that phalloidin may act whereby leukocytes penetrate this barrier to gain access indirectly to reduce leukocyte diapedesis by preventing to the interstitial space remains unclear. The purpose of leukocyte adhesion to microvascular endothelium. This this study was to determine whether alterations in the notion is based on the possibility that the cytoskeleton endothelial cell cytoskeleton play an important role in may play a role in either the assembly, expression, or modulating the extravascular accumulation of leukocytes surface distribution of endothelial cell adhesion receptors induced by proinflammatory stimuli. To address this is[e.g., intercellular adhesion molecule- 1, granule memsue, leukocyte emigration was induced by superfusing the brane protein-140 (GMP-140), and endothelial leukocyte rat mesentery with three well-known proinflammatory adhesion molecule-l]. Thus an important aim of the stimuli (PAF, LTB4, or FMLP) in the presence and abpresent study was to determine whether phalloidin inhibsence of phalloidin, a bicyclic heptapeptide that acts to its leukocyte adhesion to the endothelium. Our results prevent the disassembly of microfilamentous cytoskeletal suggest that such an explanation is unlikely for experielements in endothelial cells (7, 35). Our results indicate ments in which phalloidin was administered at 25 pg/kg, that stabilization of F-actin with phalloidin completely prevented the increase in leukocyte emigration induced since this dose did not attenuate leukocyte adhesion but abolished emigration. However, such a scenario may exby PAF, LTB4, or FMLP. The notion that leukocytes breach the endothelial cell plain the reduction in leukocyte adherence and extravabarrier by mechanisms that involve alterations in the sation noted in experiments where the dose of this binumber and distribution of actin filaments within the cyclic heptapeptide was 20-fold higher. Because the endothelial cell cytoskeleton is supported by several re- mobilization of the endothelial cell adhesion molecule GMP-140 from Weibel-Palade bodies may involve cyports already in the literature. For example, microfilatoskeletal alterations, and because the expression of ment-rich endothelial cell lamellopodia extend luminally and envelope leukocytes as they emigrate in response to GMP-140 appears to support leukocyte rolling (22), this may also explain why the high dose of phalloidin attenLTB4 (23). In addition, agents that modify junctional uated the reduction in leukocyte rolling velocity induced integrity via alterations in the disposition of microfilaby PAF, LTB*, and FMLP. mentous cytoskeletal elements within the endothelium An assumption inherent to our conclusion that the influence neutrophil emigration across endothelial cell monolayers (9). For example, administration of agents endothelial cell cytoskeleton plays an active role in modulating leukocyte emigration in response to PAF, LTB*, that disrupt interendothelial cell junctions (e.g., histamine or cytochalasin B) tend to promote diapedesis. On and FMLP is that phalloidin acts to stabilize F-actin only in endothelial cells and exerts little effect on leukocytes. the other hand, agents that stabilize cytoskeletal microfilThis is an important consideration, since most of the aments and maintain junctional integrity (e.g., phalloidin, norepinephrine, or serotonin) significantly reduce stimulated responses of neutrophils (e.g., adherence, secretion, superoxide and leukotriene production, and moleukocyte emigration across endothelial cell monolayers. tility) appear to involve cytoskeletal alterations (12, 17, Stabilization of microfilaments within the endothelial 19, 28). However, phalloidin does not gain access to the cell cytoskeleton with phalloidin prevents LTB4 or zymointracellular compartment of leukocytes unless the cell San-activated plasma-induced accumulation of leukocytes in skin dermabrasion chambers (25). More recently, we membrane is first permeabilized and thus would not be have demonstrated that phalloidin attenuates postis- expected to interfere with leukocyte responses that depend on the cytoskeleton (19). Indeed, phalloidin does chemic neutrophil infiltration and increased microvascunot attenuate neutrophil adherence to nonbiological surlar permeability in skeletal muscle (20). Because phalloidin also attenuated the loss of cell-to-cell contact in faces (e.g., plastic) (20), endothelial cell monolayers (9), monolayers exposed to anoxia-reoxygenation and pre- or postcapillary venules (present study). This agent also vented the increase in microvascular permeability in- does not influence neutrophil superoxide production (20) duced by cytochalasin D, we concluded that ischemia- or locomotion (25). Moreover, the results of the present
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 15, 2019.
PHALLOIDIN
ATTENUATES
study indicate that phalloidin does not alter the increased surface expression of CD1 l/CD18 on neutrophils activated by PAF or FMLP. On the other hand, antamanide (which also acts to stabilize F-actin but differs from phalloidin in that it can readily gain access to the intracellular compartment of neutrophils due to its much greater lipid solubility) does alter neutrophil function (20). More relevant to-the present study isthe fact that phalloidin does not attenuate the migration of neutrophils across nonbiological membranes (13). In this study, it was shown that FMLP added to the lower well in a Boyden apparatus induces neutrophil migration from the upper to lower chamber. Inclusion of phalloidin in the upper and lower wells failed to attenuate neutrophil migration induced by FMLP. In contrast to the lack of effect in neutrophils, phalloidin enters the endothelial cell cytoplasm by pinocytosis without permeabilization of the cell membrane (1, 3, 7, 20, 25, 26) and prevents the concurrent reorganization of microfilamentous cytoskeletal elements, disruption of junctional integrity, and alterations in cell shape in endothelial cells exposed to proinflammatory stimuli (1, 9, 13, 20,26). The results of the present study also indicate that phalloidin at doses that effectively abolish leukocyte diapedesis do not influence the adherence of these phagocytic cells to venular endothelium. These considerations suggest that the ability of phalloidin to block leukocyte emigration in vivo may relate to stabilization of endothelial cell cytoskeletal elements rather than to effects on neutrophilic function. In summary, the most significant finding of this study is that phalloidin effectively prevented leukocyte emigration induced by PAF, LTB4, and FMLP at doses that had no effect on the increased leukocyte adherence elicited by these well-known inflammatory mediators. Because phalloidin does not appear to alter neutrophilic function, our data suggest that PAF, LTB4, and FMLP increase leukocyte extravasation by a process that involves alterations in the endothelial cell cytoskeleton. This study was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grant DK-33594 and the National Heart, Lung, and Blood Institute Grant HL-36069. R. J. Korthuis is the recipient of an Established Investigatorship from the American Heart Association. Address for reprint requests: R. J. Korthuis, Dept. of Physiology, LSU Medical Center, 1501 Kings Highway, PO Box 33932, Shreveport, LA 71130-3932. Received
2 March
1992; accepted
in final
form
8 July
1992.
REFERENCES 1. Alexander, J. S., H. B. Hechtman, and D. Shepro. Phalloidin enhances endothelial barrier function and reduces inflammatory permeability in vitro. Microuasc. Res. 35: 308-315, 1988. 2. Asako, H., P. Kubes, B. A. Baethge, R. E. Wolf, and D. N. Granger. Colchicine and methotrexate reduce leukocyte adherence and emigration in rat mesenteric venules. Inflammation 16: 45-56, 1992. 3. Barak, L. S., R. R. Rogers, and W. W. Webb. In vivo staining of cytoskeletal actin by autointernalization of nontoxic concentrations of nitrobenzoxadiazole-phallicidin. J. Cell Biol. 89: 368372, 1981. 4. Bentzel, C. J., B. Hainau, S. Ho, S. W. Hui, A. Edelman, T. Aganostopoulos, and E. G. Benedetti. Cytoplasmic regulation of tight-junction permeability: Effect of plant cytokines. Am. J. Phvsiol. 239 (Cell Phvsiol. 8): C75-C89, 1980.
LEUKOCYTE
EMIGRATION
H1641
J., P. Hedqvist, and K.-E. Arfors. Increase in vascular 5. Bjork, permeability induced by leukotriene B, and the role of polymorphonuclear leukocytes. Inflammation 6: 189-200, 1982. 6. Bussolino, F., G. Cammussi, M. Aglietta, P. Braquet, A. Bosia, G. Pescarmona, F. Sanavio, N., and P. C. Marchisio. Human endothelial cells are target for platelet-activating factor. I. Platelet-activating factor induces changes in cytoskelton structures. J. Immunol. 139: 2439-2446, 1987. J. A. Effects of cytochalasin and phalloidin on actin. J. 7. Cooper, Cell Biol. 105: 1473-1478, 1987. 8. Davis, M. J. Determination of volumetric flow in capillary tubes using an optical Doppler velocimeter. Microuasc. Res. 34: 223-230, 1987. 9. Doukas, J., D. Shepro, and H. B. Hechtman. Vasoactive amines directly modify endothelial cells to affect polymorphonuclear leukocyte diapedesis in vitro. Blood 69: 1563-1569, 1987. 10. Ford-Hutchinson, A. W., B. Brunet, P. Savard, and S. Charleson. Leukotriene Bq, polymorphonuclear leukocytes, and inflammatory exudates in the rat. Prostaglandins 28: 13-27, 1984. 11. Foster, S. J., M. E. McCormick, A. Howarth, and D. Aked. Leukocyte recruitment in the subcutaneous sponge implant model of acute inflammation in the rat is not mediated by leukotriene Bq. Biochem. Pharmacol. 35: 1709-1717, 1986. 12. Gallin, J. I., I. M. Goldstein, and R. Snyderman. Inflammation: Basic Principles and Clinical Correlates, edited by J. I. Gallin, I. M. Goldstein, and R. Snyderman. New York: Raven, 1988. G., R. Welbourne, J. M. Klausner, S. Alexander, 13. Goldman, L. Kobzik, C. R. Valeri, D. Shepro, and H. B. Hechtman. Attenuation of acid aspiration edema with phalloidin. Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3): L378-L383, 1990. 14. Granger, D. N., J. N. Benoit, M. Suzuki, and M. B. Grisham. Leukocyte adherence to venular endothelium during ischemia-reperfusion. Am. J. Physiol. 257 (Gastrointest. Liver Physiol. 20): G683-G688, 1989. 15. Granger, D. N., B. J. Zimmerman, E. Sekizuka, and M. B. Grisham. Intestinal microvascular exchange in the rat during luminal perfusion with formyl-methionyl-leucyl-phenylalanine. Gastroenterology 94: 673-681, 1988. 16. Harlan, J. M. Leukoycte-endothelial cell interactions. Blood 65: 513-525, 1985. 17. Henson, P. M., J. E. Henson, C. Fittschen, G. Kimani, D. L. Bratton, and D. W. H. Riches. Phagocytic cells: degranulation and secretion. In: Inflammation: Basic Principles and Clinical Correlates, edited by J. I. Gallin, I. M. Goldstein, and R. Snyderman. New York: Raven, 1988, chapt. 22, p. 363-390. 18. House, S. D., and H. Lipowsky. Leukocyte-endothelium adhesion: Microdynamics in mesentery of the cat. Microvasc. Res. 34: 363-379, 1987., 19. Howard, T. H., and W. H. Meyer. Chemotactic peptide modulation of actin assembly and locomotion in neutrophils. J. Cell Biol. 98: 1265-1271, 1984. 20. Korthuis, R. J., D. L. Carden, P. R. Kvietys, D. Shepro, and J. Fuseler. Phalloidin attenuates postischemic neutrophil infiltration and increased microvascular permeability. J. Appl. Physiol. 71: 1261-1269, 1991. 21. Krishnan, A. Rapid flow cytometric analysis of mammalian cell cycle by propidium iodide staining. J. Cell Biol. 66: 188-192, 1975. M. B., and T. A. Springer. Leukocytes roll on a 22. Lawrence, selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65: 859-873, 1991. 23. Lewis, R. E., and H. J. Granger. Diapedesis and the permeability of venous microvessels to protain macromolecules: the impact of leukotriene B, (LTB4). Microuasc. Res. 35: 27-47, 1988. 24. Montesano, R. D., G. Gabbiani, A. Perreld, and L. Orci. In vivo assembly of tight junctions in fetal rat liver. J. Cell Biol. 67: 310-319, 1975. 25. Paterson, I. S., J. M. Klausner, G. Goldman, R. Welbourn, J. S. Alexander, D. Shepro, and H. B. Hechtman. The endothelial cell cytoskelton modulates extravascular polymorphonuclear leukocyte accumulations in vivo. Microuasc. Res. 38: 49-56, 1989. 26. Phillips, P. G., H. Lum, A. B. Malik, and M. Tsan. Phallicidin prevents thrombin-induced increases in endothelial cell permeability to albumin. Am. J. Physiol. 257 (Cell Physiol. 26): C562C567, 1989.
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 15, 2019.
H1642
PHALLOIDIN
ATTENUATES
Histamine type I receptor D., and J. L. Gallin. occupancy increase endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers. J. Cell Biol. 103: 2379-2387, 1986. 28. Shalit, M., G. A. Dabiri, and F. S. Southwick. Plateletactivating factor both stimulates and “primes human polymorphonuclear leukocyte actin filament assembly. Blood 70: 1921-1927, 1987. 29. Shasby, D. M., S. S. Shasby, J. M. Sullivan, and M. J. Peach. Role of endothelial cell cytoskelton in control of endothelial permeability. Circ. Res. 51: 657-661, 1982. 27. Rotrosen,
30. Springer, R. Rothlein.
T.
A., D. Leukocyte
C. Anderson, A. Adhesion Molecules.
S. Rosenthal,
and
New York: Springer-
Verlag, 1989. 31. Steele, R. G. D., and J. H. Torie. of Statistics. A BiometricaL Approach.
1980.
Principles
and Procedures
New York: McGraw-Hill,
LEUKOCYTE
EMIGRATION
32. Tamatani, T., M. Kotani, and M. Miyasaka. Characterization of the rat leukocyte integrin, CDll/CD18, by the use of LFA- 1 subunit-specific monoclonal antibodies. Eur. J. Immunol. 21:627-633, 1991. 33 Tonnenson, M. G. Neutrophil-endothelial cell interactions: ’ mechanisms of neutrophil adherence to vascular endothelium. J. Invest. Dermatol. 93: 53s-58s, 1989. Welbourn, R., G. Goldman, L. Kobzik, 34* Valeri, D. Shepro, and H. B. Hechtman.
I. Paterson,
C. R.
Neutrophil adherence receptors (CDl8) in ischemia: dissociation between quantitative cell surface expression and diapedesis mediated by leukotriene B+ J. Immunol. 145: 1906-1911, 1990. 35. Wieland, T., and H. Faulstich. Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms. CRC Crit. Rev. Biochem. 5: 185-260, 1978.
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 15, 2019.
ASAKO,
leukocyte emigration induced stimuli in rat mesentery
ROBERT
E. WOLF,
D. NEIL
GRANGER,
AND RONALD
J. KORTHUIS
Departments of Physiology and Medicine, Center for Excellence for Arthritis and Rheumatology, Louisiana State University Medical Center, School of Medicine in Shreveport, Shreveport, Louisiana 71130 Asako, Hiroshi, Robert E. Wolf, D. Neil Granger, and Ronald J. Korthuis. Phalloidin prevents leukocyte emigration induced by proinflammatory stimuli in rat mesentery. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1637-H1642, 1992.-The objective of this study was to determine whether phalloidin, a potent microfilament stabilizer, can modify inflammatory mediator-induced leukocyte adhesion and extravasation in postcapillary venules of the rat mesentery. To address this issue, the rat mesentery was prepared for in vivo microscopic observation. Venules with initial diameters ranging between 25 and 35 pm were selected for study. Erythrocyte velocity, vessel diameter, leukocyte rolling velocity, and the number of adherent (stationary for 30 s) and emigrated leukocytes were initially determined during superfusion of the mesentery with phosphate-buffered saline. After these variables were recorded during the control period, either 100 nM platelet-activating factor (PAF), 20 nM leukotriene B4 (LTB*), or 1 PM N-formylmethionyl-leucyl-phenylalanine (FMLP) was added to the superfusate. Repeat measurements were obtained between 50 and 60 min after initial exposure to the inflammatory mediator. In some experiments, rats were given phalloidin (25 or 500 pg/kg iv) 30 min before superfusion with the inflammatory mediators. Superfusion of the mesentery with either PAF, LTB4, or FMLP enhanced leukocyte adherence and emigration and reduced leukocyte rolling velocity. Pretreatment with the low dose of phalloidin effectively prevented leukocyte emigration but had no effect on the increased leukocyte adherence elicited by the three inflammatory mediators. However, when administered at the higher dose, phalloidin prevented both leukocyte adherence and emigration. Neither dose of phalloidin altered the upregulation of neutrophil membrane CD1 l/CD18 glycoprotein adherence complex induced by PAF or LTB,. These results are consistent with the concept that PAF, LTB4, and FMLP increase leukocyte extravasation by a process that may involve alterations in the endothelial cell cytoskeleton. cytoskeleton; microfilaments; F-actin; leukocyte adherence; leukocyte rolling; postcapillary venules; platelet-activating factor; leukotriene B,; formyl-methionyl-leucyl-phenylalanine; inflammation ACCUMULATION of polymorphonuclear leukocytes in the interstitial space is one of the major hallmarks of an acute inflammatory response (12, 16, 30, 33). Although the process whereby these adherent leukocytes breach the microvascular barrier to enter the interstitial space is not well understood, there is a growing body of evidence which suggests that reorganization of actin microfilaments in the endothelial cell cytoskeleton may play a key role in modulating leukocyte diapedesis. For example, leukocytes emigrate through interendothelial cell junctions, and the integrity of these junctions is regulated by cytoplasmic microfilaments (1,4,6,7,9,12, 13, 20, 23-27, 29). In addition, coincident and rather remarkable changes in endothelial ceil topography and cytoskeletal organization occur as leukocytes emigrate across postcapillary venules (23). Perhaps the strongest
THE
0363-6135/92
$2.00
Copyright
support for this concept is derived from the observation that inflammatory mediator-induced leukocyte emigration across cultured endothelial cells or into dermabrasion chambers in vivo is prevented by prior treatment with phalloidin (9,25), an agent that promotes junctional integrity by stabilizing existing filamentous actin and by inducing actin filament formation from actin monomers and oligomers (7, 35). Before a leukocyte can emigrate into the tissues in response to a chemotactic signal, these cells must first adhere to postcapillary venular endothelium (12, 14-16, 30,33). Thus it is possible that phalloidin acts indirectly to reduce leukocyte diapedesis by preventing the adhesion of these phagocytic cells to microvascular endothelium. The aim of the present study was to further explore the role of the endothelial cell cytoskeleton in the process of leukocyte emigration by examining this process in vivo using intravital microscopic approaches. In these studies, leukocyte adherence and emigration were elicited by superfusing the rat mesentery with platelet-activating factor (PAF), leukotriene B4 (LTB4), or N-formyl-methionyl-leucyl-phenylalanine (FMLP) in control animals and in rats pretreated with phalloidin. Our results indicate that phalloidin attenuates leukocyte emigration without exerting a significant influence on leukocyte adhesion to venular endothelium. MATERIALS AND METHODS Surgical procedure. Male Sprague-Dawley rats weighing between 200 and 250 g were fasted for 24 h before the experiments. After the animals were anesthetized with pentobarbital sodium (65 mg/kg body wt), a tracheotomy was performed to facilitate respiration during the experiment. The right carotid artery and left jugular vein were cannulated for monitoring systemic arterial pressure and for intravenous administration of drugs (phalloidin and supplemental doses of anesthetic), respectively. After a midline abdominal incision was made, the right and left renal arteries and veins were ligated to prevent the renal excretion of phalloidin. Intravital microscopy. After the animal was placed in a laterally recumbent position on an adjustable Plexiglas microscope stage, a segment of midjejunum was exteriorized through the abdominal incision, and the mesentery was prepared for microscopic observation as described previously (2, 14). The mesentery was draped over an optically clear cover slip that allowed for observation of a 2-cm2 segment of tissue. All other exposed tissue was covered with saline-soaked gauze and Saran Wrap to minimize evaporative water loss. The temperature of the pedestal was maintained at 37°C with a constant temperature water circulator (model 80, Fisher Scientific). Rectal and mesenteric temperatures were continuously monitored using an electrothermometer. The mesentery was then superfused with warmed bicarbonate-buffered saline (pH 7.4) that was bubbled with a mixture of 5% CO,-95% N,. An inverted microscope (Nikon Optiphoto, Japan) with a
0 1992 The
American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 15, 2019.
H1637
H1638
PHALLOIDIN
ATTENUATES
x40 objective lens (Nikon Optiphoto, Japan) and a ~10 eyepiece was used to observe the mesenteric microcirculation. The mesentery was transilluminated with a 12 V-100 W direct current-stabilized light source. A video camera (VK-C150, Hitachi, Japan) mounted on the microscope projected the image onto a color monitor (PVM-2030, Sony, Japan), and the images were recorded using a video cassette recorder (NV8950 Panasonic, Japan). A video time-date generator (WJ810 Panasonic) projected the time, date, and a stopwatch function onto the monitor. Single unbranched venules with diameters ranging between 25 and 35 pm and a length X50 pm were selected for study. Venular diameter (D,) was measured either on- or off-line using a video image-shearing monitor (IPM, LaMesa, CA). The number of adherent leukocytes was determined off-line during playback of video-taped images. A leukocyte was considered to be adherent to venular endothelium if it remained stationary for a period 230 s (14). Adherent cells were expressed as the number per 100~pm length of venule. The number of emigrated leukocytes was also determined off-line during playback of videotaped images. Any interstitial leukocytes present in the mesentery at the onset of the experiment were subtracted from the total number of leukocytes that accumulated during the course of the experiment. Leukocyte emigration was expressed as the number per microscopic field (1.7 x lo-” mm2 or 150 ,ccrnlength of venule). Rolling leukocytes were defined as those white blood cells that moved at a velocity less than that of erythrocytes in the same stream. Leukocyte rolling velocity was determined from the time required for a leukocyte to traverse a given distance along the length of a venule. Centerline red blood cell velocity ( VItHC) was measured using an optical Doppler velocimeter (Microcirculation Research Institute, Texas A&M University) that was calibrated against a rotating glass disk coated with red blood cells. Venular blood flow was calculated from the product of mean red blood cell velocity [ Vlxlean = centerline cross-sectional area, asvelocity t 1.6 (S)] and microvascular suming cylindrical geometry. Venular wall shear rate (y) was calculated based on Newtonian definition: y = 8( V,,,,/D,) (18) . Flow cytometry studies. Flow cytometry was used to quantitate surface expression of CD18 on rat polymorphonuclear leukocytes. The analysis was performed using whole blood samples to minimize artifactual upregulation of CD18 associated with ex vivo manipulation (34). A 200-~1 aliquot of heparinized whole blood obtained from Sprague-Dawley rats was mixed with 200 ~1 of phosphate-buffered saline (PBS) in polypropylene test tubes and incubated either alone or after gentle mixing with either phalloidin alone (0.5 or 10 pg/ml final concentration, which approximates the initial blood concentration achieved in the in vivo experiments) or with one of the three proinflammatory mediators [PAF (100 nM), LTB, (20 nM), or FMLP (1 PM)] in the presence and absence of phalloidin (at either 0.5 or 10 pg/ml) for 30 min at 37°C. After incubation, 500 ~1 of a solution containing 7 pg/ml of a monoclonal antibody (WT3) directed against CD18 (3.9 pg/ml final concentration) (32) were added to the samples, and the test tubes were placed at 4°C for 20 min. The samples were then washed twice with PBS and centrifuged at 1,000 rpm for 3 min. Then 200 ~1 of a loo-fold dilution of fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin (7.5 pg/ml final concentration, Zymed Laboratories, San Francisco, CA) were added, and the mixture was incubated for 20 min at 4°C. The samples were then washed twice with PBS and centrifuged at 1,000 rpm for 3 min. The red cells were then lysed by addition of 1 ml of 25-fold dilution of Immuno-lyze reagent (Coulter Immunology, Hialeah, FL) to each tube followed by vigorous vortexing, after which the samples were allowed to sit for 90 min. Then 250 ~1 of fixative (Coulter Immunology, Hialeah, FL) was added to
LEUKOCYTE
EMIGRATION
each tube. After the samples were vigorously vortexed, the samples were washed twice with 3 ml of PBS at 1,000 rpm for 3 min. Then 200 ~1 of PBS was added to each tube. Analysis was performed on an EPICS 753 flow cytometer/sorter (Coulter Electronics, Hialeah, FL) for the simultaneous accumulation of immunofluorescence in addition to forward angle and 90” light scatter signals. Dead cells and debris were excluded by forward angle and 90” light scatter gating or, in some experiments, by the exclusion of dead cells that incorporate propidium iodide (21). Controls included cells stained with the secondary reagent alone or cells stained with an irrelevant-isotype-matched control monoclonal antibodies. Twenty-five thousand cells were analyzed in each experiment, and each experiment was repeated four times. Experimental protocols. After all parameters measured online (arterial pressure, erythrocyte velocity, and venular diameter) were in a steady state, images from the mesenteric preparation were recorded on videotape for 10 min for subsequent analysis of leukocyte adherence, emigration, and rolling velocity. Immediately thereafter, the mesentery was superfused for 60 min with either 100 nM PAF (Sigma Chemical, St. Louis, MO), 20 nM LTB, (Calbiochem, LaJolla, CA), or 1 PM FMLP (Sigma Chemical) dissolved in bicarbonate-buffered saline (n = 4-6 in each group). Previous reports indicate that these doses elicit significant leukocyte adherence and emigration (2, 5). Video recording and measurements of all parameters were repeated during the final 10 min of the 60-min period of inflammatory mediator superfusion. In additional experiments, animals received an intravenous injection of phalloidin (dissolved in saline, stock solution 1 mg/ml, Sigma Chemical) at doses of either 25 or 500 pg/kg 30 min before the addition of an inflammatory mediator to the superfusate. The lower dose of phalloidin produces an initial blood concentration of 0.4-0.6 PM, a dose similar to that which has been used in earlier work from our laboratory and by others (1,9,20,25). Video recordings and measurement of all variables were then obtained according to the protocol outlined above (n = 4-6 in each group). Statistical analysis. All values are reported as means t SE. The data were initially analyzed by using a one-way analysis of variance. To determine which groups were statistically different, multiple comparisons were analyzed using Scheffe’s post hoc test (31). Statistical significance was defined as P < 0.05.
RESULTS Figure 1 summarizes the effect of PAF, LTB4, and FMLP on leukocyte adherence in rat mesenteric venules. Under baseline conditions, there were 4.5 t 0.4 adherent leukocytes per 100 pm length of venule. Superfusion of the mesentery with either 100 nM PAF, 20 nM LTB4, or 1 PM FMLP was associated with a significant increase in the number of adherent leukocytes to 29.5 t 4.7, 39.4 t 6.5, and 63.0 t 5.9 per 100 pm venule length, respectively. The increased leukocyte adherence induced by either PAF, LTB4, or FMLP was not affected by treatment with phalloidin at 25 pg/kg (32.0 t 2.6, 33.6 t 6.7, 57.7 t 4.1 per IOO-pm length of venule, respectively) but was significantly attenuated by treatment with 500 pg/kg phalloidin (8.3 t 2.0 or 21.0 t 1.4 or 22.4 t 5.1 per loo-pm length of venule, respectively). The effect of PAF, LTB4, and FMLP on the number of emigrated leukocytes per microscopic field is illustrated in Fig. 2. Superfusion with either PAF, LTB*, or FMLP significantly increased the number of emigrated leukocytes from 3.1 t 0.5 under control condi-
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 15, 2019.
PHALLOIDIN
ATTENUATES
LEUKOCYTE
x
I
.--b-J 0 ?a Oi *El 3’ aE --la. *g C-
50
0 a, >
e60-
40
c
50-
l
-
. u
=z s
4o
H1639
EMIGRATION
as +A0 0 Y 3
30
20
10
3 0
PAF
LTB,
Fig. 1. Effects of phalloidin on inflammatory mediator-induced leukocyte adherence in rat mesenteric venules. PAF, platelet-activating factor; LTB4, leukotriene B,; FMLP, N-formyl-methionyl-leucyl-phenylalanine; IM, inflammatory mediator. Solid bars, control; open bars, IM; hatched bars, IM + phalloidin (low); cross-hatched bars, IM + phalloidin (high). * P < 0.05 relative to control; ** P < 0.05 relative to corresponding IM. 60
I/) al -w x
50
0
40
0 24 33;
b
-0L 2: o.-
L CP
E
W
30
20
10
-
0 .
PAF Fig. 2. Effects cyte emigration and symbols.
LTB,
of phalloidin on inflammatory in rat mesenteric venules.
PAF
FMLP
FMLP mediator-induced leukoSee Fig. 1 for abbreviations
tions to 19.3 t 2.8, 13.0 t 2.8, and 34.4 t 6.2 per microscopic field, respectively. The increased number of emigrated leukocytes induced by either PAF, LTB4, or FMLP was significantly attenuated by treatment with phalloidin at 25 pg/kg (7.4 t 1.3, 4.2 t 1.0, 14.0 t 4.5, leukocytes/field, respectively). Administration of 500 pg/kg phalloidin did not produce a further reduction in the leukocyte emigration induced by these proinflammatory mediators (4.8 t 0.8, 3.6 t 1.1, 4.8 t 1.4 leukocytes/field, respectively). Figure 3 summarizes the response of leukocyte rolling velocity in postcapillary venules to either PAF, LTB*, or FMLP. Leukocyte rolling velocity in rat mesenteric venules under baseline conditions was 44.1 t 1.0 pm/s, a value similar to that previously reported for cat mesenteric venules (14). Leukocyte rolling velocity was significantly reduced by superfusion of the mesentery with PAF (25.9 -+ 2.5 pm/s), LTB4 (26.5 + 3.8 pm/s), or FMLP (19.2 t 1.0 urn/s). The PAF-. LTBn-. or FMLP-induced
Fig. 3. Effects rolling velocity and symbols.
LTB,
of phalloidin on IM-induced in rat mesenteric venules.See
FMLP alterations in leukocyte Fig. 1 for abbreviations
reduction in leukocyte rolling velocity was significantly attenuated by 500 pg/kg phalloidin (37.4 t 4.3,38.2 t 3.9, 27.2 t 2.0 pm/s, respectively) but not by 25 pg/kg phalloidin (23.2 t 1.5, 21.3 t 0.7, 23.2 t 2.0 pm/s, respectively). Red blood cell velocity, venular diameter, and venular wall shear rate were not significantly altered by superfusion with either PAF or LTB4 or FMLP in the presence or absence of phalloidin (25 or 500 pg/kg) (Table 1). Flow cytometric analysis of CD 1 l/CD 18 expression in isolated rat neutrophils revealed that basal expression of this adhesive glycoprotein was not altered by either the low or high dose of phalloidin. Although a significant increase in fluorescence intensity was detected by flow cytometry in response to PAF and FMLP (6.4 t 2.1 and 22.2 t 3.5% increase relative to unstimulated cells), LTB4 did not appear to increase the surface expression of CDll/CD18 (4.9 t 2.8% increase relative to unstimulated cells). This latter observation Table 1. Effects of platelet-activating factor, leukotriene Bq, and formyl-methionyl-leucyl-phenylalanine in absence and presence of phalloidin on mean red blood cell velocity, venular diameter, and wall shear rate RBC
Control PAF PAF + PH(L) PAF + PH(H) Control LTB, LTB, + PH(L) LTB4 + PH(H) Control FMLP FMLP + PH(L) FMLP + PH(H)
Velocity, mm/s
2.3t0.3 1.7kO.3 1.5t0.3 1 J3kO.9 3.6t0.4 2.1t0.2 2.9tl.O 2.8t0.5 2.8t0.3 3.1t0.7 2.0t0.2 1.6kO.5
Diameter, Pm
28.6k0.7 27.8kl.O 29.4t1.7 29.3t1.7 29.2kO.7 32.1kO.9 34.6k2.5 27.81k0.6 29.6k0.7 28.8tl.l 33.0t0.9 29.8k0.8
Wall
Shear s-l
Rate,
408t56 301t62 256t38 325k175 603k62 330t35* 443t142 497tlOl 476t62 533t115 300t35 264t88
Values are means t SE. PAF, platelet-activating factor; PH(L), phalloidin at low (25 pg/kg) dose; PH(H), phalloidin at high (500 pg/kg) leukotriene B,; FMLP, N-formyl-methionyl-leucyldose; LTB4, phenylalanine; RBC, red blood cell. *Values were statistically different from control.
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 15, 2019.
H1640
PHALLOIDIN
ATTENUATES
LEUKOCYTE
EMIGRATION
is consistent with the fact that rat neutrophils are reported to be less responsive to LTB4 than granulocytes from other species (10, 11) and suggests that LTB4-induced leukocyte adhesion and emigration may be related to an effect on the endothelium. In any event, neither the low nor the high dose of phalloidin modified the increase in fluorescence intensity induced by PAF (5.1 t 2.0 and 6.4 t 1.4%, respectively) or FMLP (24.3 t 3.7 and 23.7 t 3.1%, respectively), indicating that the bicyclic heptapeptide does not influence the upregulation and activation of CD18 on rat neutrophils.
reperfusion alters neutrophil sequestration and increases microvascular permeability through a direct effect on the endothelium that is due in part to alterations in the endothelial cell cytoskeleton (20). These observations suggest that cytoskeletal alterations induced by proinflammatory stimuli lead to disruption of junctional complexes which, in turn, facilitates the penetration of the microvascular barrier by the emigrating leukocyte. Based on the aforementioned observations, it has been suggested that reorganization of microfilamentous cytoskeletal elements within the endothelium modulates the integrity of interendothelial cell junctions thereby DISCUSSION allowing leukocyte emigration to proceed (9, 20, 25). The extravascular accumulation of polymorphonuclear However, the fact that leukocytes must first adhere to leukocytes is a characteristic feature of an acute inflamthe endothelium before they can emigrate into the tissues matory response (5, 12, 14, 30, 33). Although it is well (12,14, 16,29,30) suggestsanother potential explanation known that leukocytes emigrate through interendothelial for the ability of phalloidin to attenuate leukocyte diacell junctions, our understanding of the mechanisms pedesis. That is, it is possible that phalloidin may act whereby leukocytes penetrate this barrier to gain access indirectly to reduce leukocyte diapedesis by preventing to the interstitial space remains unclear. The purpose of leukocyte adhesion to microvascular endothelium. This this study was to determine whether alterations in the notion is based on the possibility that the cytoskeleton endothelial cell cytoskeleton play an important role in may play a role in either the assembly, expression, or modulating the extravascular accumulation of leukocytes surface distribution of endothelial cell adhesion receptors induced by proinflammatory stimuli. To address this is[e.g., intercellular adhesion molecule- 1, granule memsue, leukocyte emigration was induced by superfusing the brane protein-140 (GMP-140), and endothelial leukocyte rat mesentery with three well-known proinflammatory adhesion molecule-l]. Thus an important aim of the stimuli (PAF, LTB4, or FMLP) in the presence and abpresent study was to determine whether phalloidin inhibsence of phalloidin, a bicyclic heptapeptide that acts to its leukocyte adhesion to the endothelium. Our results prevent the disassembly of microfilamentous cytoskeletal suggest that such an explanation is unlikely for experielements in endothelial cells (7, 35). Our results indicate ments in which phalloidin was administered at 25 pg/kg, that stabilization of F-actin with phalloidin completely prevented the increase in leukocyte emigration induced since this dose did not attenuate leukocyte adhesion but abolished emigration. However, such a scenario may exby PAF, LTB4, or FMLP. The notion that leukocytes breach the endothelial cell plain the reduction in leukocyte adherence and extravabarrier by mechanisms that involve alterations in the sation noted in experiments where the dose of this binumber and distribution of actin filaments within the cyclic heptapeptide was 20-fold higher. Because the endothelial cell cytoskeleton is supported by several re- mobilization of the endothelial cell adhesion molecule GMP-140 from Weibel-Palade bodies may involve cyports already in the literature. For example, microfilatoskeletal alterations, and because the expression of ment-rich endothelial cell lamellopodia extend luminally and envelope leukocytes as they emigrate in response to GMP-140 appears to support leukocyte rolling (22), this may also explain why the high dose of phalloidin attenLTB4 (23). In addition, agents that modify junctional uated the reduction in leukocyte rolling velocity induced integrity via alterations in the disposition of microfilaby PAF, LTB*, and FMLP. mentous cytoskeletal elements within the endothelium An assumption inherent to our conclusion that the influence neutrophil emigration across endothelial cell monolayers (9). For example, administration of agents endothelial cell cytoskeleton plays an active role in modulating leukocyte emigration in response to PAF, LTB*, that disrupt interendothelial cell junctions (e.g., histamine or cytochalasin B) tend to promote diapedesis. On and FMLP is that phalloidin acts to stabilize F-actin only in endothelial cells and exerts little effect on leukocytes. the other hand, agents that stabilize cytoskeletal microfilThis is an important consideration, since most of the aments and maintain junctional integrity (e.g., phalloidin, norepinephrine, or serotonin) significantly reduce stimulated responses of neutrophils (e.g., adherence, secretion, superoxide and leukotriene production, and moleukocyte emigration across endothelial cell monolayers. tility) appear to involve cytoskeletal alterations (12, 17, Stabilization of microfilaments within the endothelial 19, 28). However, phalloidin does not gain access to the cell cytoskeleton with phalloidin prevents LTB4 or zymointracellular compartment of leukocytes unless the cell San-activated plasma-induced accumulation of leukocytes in skin dermabrasion chambers (25). More recently, we membrane is first permeabilized and thus would not be have demonstrated that phalloidin attenuates postis- expected to interfere with leukocyte responses that depend on the cytoskeleton (19). Indeed, phalloidin does chemic neutrophil infiltration and increased microvascunot attenuate neutrophil adherence to nonbiological surlar permeability in skeletal muscle (20). Because phalloidin also attenuated the loss of cell-to-cell contact in faces (e.g., plastic) (20), endothelial cell monolayers (9), monolayers exposed to anoxia-reoxygenation and pre- or postcapillary venules (present study). This agent also vented the increase in microvascular permeability in- does not influence neutrophil superoxide production (20) duced by cytochalasin D, we concluded that ischemia- or locomotion (25). Moreover, the results of the present
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 15, 2019.
PHALLOIDIN
ATTENUATES
study indicate that phalloidin does not alter the increased surface expression of CD1 l/CD18 on neutrophils activated by PAF or FMLP. On the other hand, antamanide (which also acts to stabilize F-actin but differs from phalloidin in that it can readily gain access to the intracellular compartment of neutrophils due to its much greater lipid solubility) does alter neutrophil function (20). More relevant to-the present study isthe fact that phalloidin does not attenuate the migration of neutrophils across nonbiological membranes (13). In this study, it was shown that FMLP added to the lower well in a Boyden apparatus induces neutrophil migration from the upper to lower chamber. Inclusion of phalloidin in the upper and lower wells failed to attenuate neutrophil migration induced by FMLP. In contrast to the lack of effect in neutrophils, phalloidin enters the endothelial cell cytoplasm by pinocytosis without permeabilization of the cell membrane (1, 3, 7, 20, 25, 26) and prevents the concurrent reorganization of microfilamentous cytoskeletal elements, disruption of junctional integrity, and alterations in cell shape in endothelial cells exposed to proinflammatory stimuli (1, 9, 13, 20,26). The results of the present study also indicate that phalloidin at doses that effectively abolish leukocyte diapedesis do not influence the adherence of these phagocytic cells to venular endothelium. These considerations suggest that the ability of phalloidin to block leukocyte emigration in vivo may relate to stabilization of endothelial cell cytoskeletal elements rather than to effects on neutrophilic function. In summary, the most significant finding of this study is that phalloidin effectively prevented leukocyte emigration induced by PAF, LTB4, and FMLP at doses that had no effect on the increased leukocyte adherence elicited by these well-known inflammatory mediators. Because phalloidin does not appear to alter neutrophilic function, our data suggest that PAF, LTB4, and FMLP increase leukocyte extravasation by a process that involves alterations in the endothelial cell cytoskeleton. This study was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grant DK-33594 and the National Heart, Lung, and Blood Institute Grant HL-36069. R. J. Korthuis is the recipient of an Established Investigatorship from the American Heart Association. Address for reprint requests: R. J. Korthuis, Dept. of Physiology, LSU Medical Center, 1501 Kings Highway, PO Box 33932, Shreveport, LA 71130-3932. Received
2 March
1992; accepted
in final
form
8 July
1992.
REFERENCES 1. Alexander, J. S., H. B. Hechtman, and D. Shepro. Phalloidin enhances endothelial barrier function and reduces inflammatory permeability in vitro. Microuasc. Res. 35: 308-315, 1988. 2. Asako, H., P. Kubes, B. A. Baethge, R. E. Wolf, and D. N. Granger. Colchicine and methotrexate reduce leukocyte adherence and emigration in rat mesenteric venules. Inflammation 16: 45-56, 1992. 3. Barak, L. S., R. R. Rogers, and W. W. Webb. In vivo staining of cytoskeletal actin by autointernalization of nontoxic concentrations of nitrobenzoxadiazole-phallicidin. J. Cell Biol. 89: 368372, 1981. 4. Bentzel, C. J., B. Hainau, S. Ho, S. W. Hui, A. Edelman, T. Aganostopoulos, and E. G. Benedetti. Cytoplasmic regulation of tight-junction permeability: Effect of plant cytokines. Am. J. Phvsiol. 239 (Cell Phvsiol. 8): C75-C89, 1980.
LEUKOCYTE
EMIGRATION
H1641
J., P. Hedqvist, and K.-E. Arfors. Increase in vascular 5. Bjork, permeability induced by leukotriene B, and the role of polymorphonuclear leukocytes. Inflammation 6: 189-200, 1982. 6. Bussolino, F., G. Cammussi, M. Aglietta, P. Braquet, A. Bosia, G. Pescarmona, F. Sanavio, N., and P. C. Marchisio. Human endothelial cells are target for platelet-activating factor. I. Platelet-activating factor induces changes in cytoskelton structures. J. Immunol. 139: 2439-2446, 1987. J. A. Effects of cytochalasin and phalloidin on actin. J. 7. Cooper, Cell Biol. 105: 1473-1478, 1987. 8. Davis, M. J. Determination of volumetric flow in capillary tubes using an optical Doppler velocimeter. Microuasc. Res. 34: 223-230, 1987. 9. Doukas, J., D. Shepro, and H. B. Hechtman. Vasoactive amines directly modify endothelial cells to affect polymorphonuclear leukocyte diapedesis in vitro. Blood 69: 1563-1569, 1987. 10. Ford-Hutchinson, A. W., B. Brunet, P. Savard, and S. Charleson. Leukotriene Bq, polymorphonuclear leukocytes, and inflammatory exudates in the rat. Prostaglandins 28: 13-27, 1984. 11. Foster, S. J., M. E. McCormick, A. Howarth, and D. Aked. Leukocyte recruitment in the subcutaneous sponge implant model of acute inflammation in the rat is not mediated by leukotriene Bq. Biochem. Pharmacol. 35: 1709-1717, 1986. 12. Gallin, J. I., I. M. Goldstein, and R. Snyderman. Inflammation: Basic Principles and Clinical Correlates, edited by J. I. Gallin, I. M. Goldstein, and R. Snyderman. New York: Raven, 1988. G., R. Welbourne, J. M. Klausner, S. Alexander, 13. Goldman, L. Kobzik, C. R. Valeri, D. Shepro, and H. B. Hechtman. Attenuation of acid aspiration edema with phalloidin. Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3): L378-L383, 1990. 14. Granger, D. N., J. N. Benoit, M. Suzuki, and M. B. Grisham. Leukocyte adherence to venular endothelium during ischemia-reperfusion. Am. J. Physiol. 257 (Gastrointest. Liver Physiol. 20): G683-G688, 1989. 15. Granger, D. N., B. J. Zimmerman, E. Sekizuka, and M. B. Grisham. Intestinal microvascular exchange in the rat during luminal perfusion with formyl-methionyl-leucyl-phenylalanine. Gastroenterology 94: 673-681, 1988. 16. Harlan, J. M. Leukoycte-endothelial cell interactions. Blood 65: 513-525, 1985. 17. Henson, P. M., J. E. Henson, C. Fittschen, G. Kimani, D. L. Bratton, and D. W. H. Riches. Phagocytic cells: degranulation and secretion. In: Inflammation: Basic Principles and Clinical Correlates, edited by J. I. Gallin, I. M. Goldstein, and R. Snyderman. New York: Raven, 1988, chapt. 22, p. 363-390. 18. House, S. D., and H. Lipowsky. Leukocyte-endothelium adhesion: Microdynamics in mesentery of the cat. Microvasc. Res. 34: 363-379, 1987., 19. Howard, T. H., and W. H. Meyer. Chemotactic peptide modulation of actin assembly and locomotion in neutrophils. J. Cell Biol. 98: 1265-1271, 1984. 20. Korthuis, R. J., D. L. Carden, P. R. Kvietys, D. Shepro, and J. Fuseler. Phalloidin attenuates postischemic neutrophil infiltration and increased microvascular permeability. J. Appl. Physiol. 71: 1261-1269, 1991. 21. Krishnan, A. Rapid flow cytometric analysis of mammalian cell cycle by propidium iodide staining. J. Cell Biol. 66: 188-192, 1975. M. B., and T. A. Springer. Leukocytes roll on a 22. Lawrence, selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65: 859-873, 1991. 23. Lewis, R. E., and H. J. Granger. Diapedesis and the permeability of venous microvessels to protain macromolecules: the impact of leukotriene B, (LTB4). Microuasc. Res. 35: 27-47, 1988. 24. Montesano, R. D., G. Gabbiani, A. Perreld, and L. Orci. In vivo assembly of tight junctions in fetal rat liver. J. Cell Biol. 67: 310-319, 1975. 25. Paterson, I. S., J. M. Klausner, G. Goldman, R. Welbourn, J. S. Alexander, D. Shepro, and H. B. Hechtman. The endothelial cell cytoskelton modulates extravascular polymorphonuclear leukocyte accumulations in vivo. Microuasc. Res. 38: 49-56, 1989. 26. Phillips, P. G., H. Lum, A. B. Malik, and M. Tsan. Phallicidin prevents thrombin-induced increases in endothelial cell permeability to albumin. Am. J. Physiol. 257 (Cell Physiol. 26): C562C567, 1989.
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 15, 2019.
H1642
PHALLOIDIN
ATTENUATES
Histamine type I receptor D., and J. L. Gallin. occupancy increase endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers. J. Cell Biol. 103: 2379-2387, 1986. 28. Shalit, M., G. A. Dabiri, and F. S. Southwick. Plateletactivating factor both stimulates and “primes human polymorphonuclear leukocyte actin filament assembly. Blood 70: 1921-1927, 1987. 29. Shasby, D. M., S. S. Shasby, J. M. Sullivan, and M. J. Peach. Role of endothelial cell cytoskelton in control of endothelial permeability. Circ. Res. 51: 657-661, 1982. 27. Rotrosen,
30. Springer, R. Rothlein.
T.
A., D. Leukocyte
C. Anderson, A. Adhesion Molecules.
S. Rosenthal,
and
New York: Springer-
Verlag, 1989. 31. Steele, R. G. D., and J. H. Torie. of Statistics. A BiometricaL Approach.
1980.
Principles
and Procedures
New York: McGraw-Hill,
LEUKOCYTE
EMIGRATION
32. Tamatani, T., M. Kotani, and M. Miyasaka. Characterization of the rat leukocyte integrin, CDll/CD18, by the use of LFA- 1 subunit-specific monoclonal antibodies. Eur. J. Immunol. 21:627-633, 1991. 33 Tonnenson, M. G. Neutrophil-endothelial cell interactions: ’ mechanisms of neutrophil adherence to vascular endothelium. J. Invest. Dermatol. 93: 53s-58s, 1989. Welbourn, R., G. Goldman, L. Kobzik, 34* Valeri, D. Shepro, and H. B. Hechtman.
I. Paterson,
C. R.
Neutrophil adherence receptors (CDl8) in ischemia: dissociation between quantitative cell surface expression and diapedesis mediated by leukotriene B+ J. Immunol. 145: 1906-1911, 1990. 35. Wieland, T., and H. Faulstich. Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms. CRC Crit. Rev. Biochem. 5: 185-260, 1978.
Downloaded from www.physiology.org/journal/ajpheart at Macquarie Univ (137.111.162.020) on February 15, 2019.
