Department of Physiology, University of Alabama College of Medicine, Mobile, Alabama 36688-0002
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ABSTRACT |
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Our studies show that ischemia-reperfusion (I/R) in the isolated rat lung causes retention of lymphocytes, which is associated with increased microvascular permeability, as determined by quantitative measurement of the microvascular filtration coefficient (Kf,c). Immunoneutralization of either CD40 or CD40L, cell surface proteins important in lymphocyte-endothelial cell proinflammatory events, results in significantly lower postischemic Kf,c values. Antagonism of CD40-CD40L signaling also results in attenuation of I/R-elicited macrophage inflammatory protein-2 production. Rat lymphocytes activated ex vivo with phorbol 12-myristate, 13-acetate increased Kf,c in isolated lungs independently of I/R, and this increase was prevented by pretreating lungs with anti-CD40. In addition to lymphocyte involvement via CD40-CD40L interactions, our studies also show that I/R injury is potentiated by antagonism of IL-10 produced locally within the postischemic lung, whereas exogenous, rat recombinant IL-10 provided protection against I/R-induced microvascular damage. Thus acute lymphocyte involvement in lung I/R injury involves CD40-CD40L signaling mechanisms, and these events may be influenced by local IL-10 generation.
inflammation; filtration coefficient; lymphocytes; macrophage inflammatory protein-2
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INTRODUCTION |
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EMERGING DATA FROM many experimental models continue to stress the importance of reactive oxygen species, granulocyte and endothelial cell signaling events, and endothelial cell contractile protein activation in the overall pathogenesis of ischemia and reperfusion (I/R)-induced organ injury. Yet, the complete mechanism producing microvascular dysfunction in response to I/R remains unclear. Overlooked during the search for mechanisms, however, is the fact that the lung harbors a substantial reservoir of marginated lymphocytes (3, 8, 21, 28, 29, 38), and the release of lymphocytes from the acutely reperfused lung correlates with less, rather than more, microvascular injury (27). Recent data from our laboratory and others suggest a role for acute lymphocyte involvement in the initial inflammatory responses leading to I/R-induced microvascular injury (14, 19, 34, 47), although the specific nature of lymphocyte involvement is not known. Although lymphocyte involvement could be related to local cytokine production and inhibition or lymphocyte-lymphocyte cross talk, it is possible that lymphocyte-endothelial cell interactions are vital for the initiation of events leading to postischemic changes in microvascular permeability.
Activated, proinflammatory CD4+ T lymphocytes can
adhere to microvascular endothelium in part by P-selectin mobilization
to endothelial cell surfaces (2, 4, 20, 39, 42, 44, 45),
and acute P-selectin mobilization readily occurs in the postischemic microvasculature (26, 34). It is also
known that a costimulatory molecule for lymphocytes is expressed in the
microcirculation of several organs (32, 43, 46). This
molecule, CD40, is a member of the tumor necrosis factor-
superfamily of receptors (6, 37, 40), and previous studies
have indicated that brief periods of ischemia increase the
levels of CD40 within the cardiac microvasculature (32).
The ligand for CD40, CD40L, is also expressed on activated lymphocytes
(12).
Expression of CD40 in the ischemic microcirculation may prove to be a critical link between P-selectin-mediated lymphocyte retention in the postischemic lung and development of I/R-induced microvascular leak (27). Ligation of CD40 has been shown to stimulate the release of proinflammatory cytokines, matrix metalloproteinases, and neutrophil-activating chemokines, as well as cause expression of endothelial cell adhesion molecules in cultured endothelial cell studies (5, 10, 12, 17, 23, 49). These factors can contribute to increased microvascular permeability associated with various inflammatory conditions, including I/R. Additional support for a CD40 role comes from studies addressing the effects of pulmonary endothelial cell CD40 ligation expressed in situ. Soluble CD40L, when injected in the murine pulmonary circulation, elicits inflammation and edema formation similar in nature to that observed with lung I/R (43). Therefore, during I/R, it is possible that proinflammatory lymphocytes tether to the postischemic lung microvasculature via P-selectin and interact with endothelium-expressed CD40 to affect I/R-induced alterations in microvascular permeability. These events may involve chemokine production to recruit granulocytes, which in turn contribute to the alteration of microcirculatory barrier properties (1, 27).
The present studies test the hypothesis that CD40-CD40L signaling contributes to postischemic inflammation, leading to microvascular injury and edema formation in the lung. Using ELISA and a quantitative measure of microvascular permeability, i.e., the microvascular filtration coefficient (Kf,c), we evaluated the effects of blocking CD40-CD40L signaling on I/R-induced chemokine production and lung damage, respectively. In addition, we determined whether an anti-CD40 antibody could prevent increased permeability elicited by perfusion with phorbol 12-myristate, 13-acetate (PMA)-activated lymphocytes. Additional aspects of the I/R injury process were also tested in regard to the effects of interleukin (IL)-10, which may be released during inflammatory events to modulate the degree of lymphocyte-mediated inflammatory responses (15, 22, 31, 33). Endogenous IL-10 has been shown to limit inflammatory responses in other models of I/R and with endotoxemia (7, 11).
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MATERIALS AND METHODS |
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Materials
Anti-CD40 (pAb L-17) and CD40L (pAb K-19) antibodies as well as goat and rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant rat IL-10 (rIL-10), polyclonal rabbit anti-rIL-10 (neutralizing, azide free), and rat macrophage inflammatory protein (MIP)-2 ELISA kits were purchased from Biosource International (Camarillo, CA). Reagents used for fluorescence-activated cell sorting (FACS) analysis were purchased from PharmIngen (San Diego, CA). All other physiological salt solutions and chemical reagents, including PMA, were purchased from Sigma-Aldrich.Isolated Rat Lung Preparation
Lungs were isolated and perfused according to our previously published methodology (18, 27). Briefly, male CD rats (250-350 g body weight; Charles River Laboratories) were anesthetized with pentobarbital sodium (50 mg/kg body wt ip). A tracheostomy was performed, and lungs were ventilated with a gas mixture composed of 21% O2-5% CO2-74% N2 (Harvard rodent ventilator, model 683) at a rate of 50 breaths/min, a tidal volume of 15 ml/kg body wt, and a positive end-expiratory pressure of 2-3 cmH2O. After median sternotomy, heparin (300 IU) was injected in the right ventricle, and cannulas were placed in the pulmonary artery and left ventricle. The heart, lungs, and mediastinal structures were removed en bloc and suspended from a force-displacement transducer (model FT03; Grass) in a humidified chamber to monitor weight changes. The lungs were perfused at a constant flow rate with Earle's balanced salt solution [in mg/l: 200 CaCl2, 400 KCl, 97.7 MgSO4, 6,800 NaCl, 140 NaH2PO4(H2O), 1,000 glucose, and 10 phenol red] containing 0.21% NaHCO3 and 4% BSA. The first 100 ml of perfusate, which contained the majority of residual blood cells and plasma, were discarded, and an additional 50 ml of perfusate were then used for recirculation during experiments.Pulmonary arterial (Ppa) and pulmonary venous (Ppv) pressures were monitored continuously with pressure transducers (model P23 ID; Gould-Statham) and recorded on a polygraph recorder (model 7E; Grass). Microvascular pressure (Ppc) was estimated using the double-occlusion method, as previously described (18, 22, 30).
The Kf,c was used as an index of microvascular
permeability according to methods previously described (18, 27,
30). Briefly, after an isogravimetric state was achieved in the
lung, i.e., the lung was neither gaining nor losing weight,
Ppv was elevated rapidly by 6-8 cmH2O for
15 min, and the lung weight gain was recorded. The characteristic rapid
weight gain (vascular filling and distension) was followed by a slower
rate of weight gain (capillary filtration). The rate of weight change
(W/
t) during the last 2-min interval was analyzed to
calculate the Kf,c. The
W/
t was
divided by the change measured in Ppc occurring after
venous pressure elevation and then was normalized using the baseline
wet lung weight and expressed as milliliter per minute per
cmH2O per 100 g lung tissue. All lungs in which
Kf,c values were measured were maintained in
zone III conditions, i.e., Ppa > Ppv > alveolar pressure (Palv) throughout
the duration of the experiment.
FACS
Samples for lymphocyte sorting were taken from the right and left ventricles in the in situ lungs and from the venous effluent in the isolated lung groups. Isolated lungs were ventilated and perfused under zone II (Ppa > Palv > Ppv) or zone III (Ppa > Ppv > Palv) conditions. Separation and labeling of the total lymphocyte pool were achieved using three independent sample vials to tag CD4+ and CD8+ cells or B cells or to be used for antibody controls. For each Eppendorf tube, 200 µl of PBS containing 0.3-1.0% albumin and 10 µl monoclonal fluorescent antibody were added to 100 µl of blood or perfusate. CD4+ and CD8+ T cells were tagged with phycoerythrein and FITC, respectively. B cells were labeled with FITC. Tubes were vortexed vigorously for 10 s, and the tubes were then allowed to sit for 20 min at room temperature. PBS-albumin (1 ml) was then added to each tube, and samples were centrifuged at 1,200 revolutions/min (rpm) for 7 min. After centrifugation, the supernatant was removed, and the pellet was washed with PBS-albumin. The pellet was resuspended in 1 ml of Immuno-Lyse solution (Coulter Electronics) diluted 1:25 with PBS-albumin. Samples were left in Immuno-Lyse for 0.5-2.0 min, and 250 ml of fixative (Coulter Electronics) were added. Samples were revortexed and recentrifuged at 2,400 rpm for 3 min. The supernatant was removed again, and the pellet was washed with 1 ml PBS-albumin two times. The pellet was resuspended in a final volume of 0.5 ml of PBS-albumin and stored at 4°C for no longer than 2 h until analyzed.Cells were sorted using a FACSVantage SE cell sorter (Becton-Dickinson, San Jose, CA) located at the University of South Alabama College of Medicine Biopolymer and Cell Sorting Laboratory. Data acquisition and analysis were performed using Cell Quest software (Becton-Dickinson).
MIP-2 Assays
Assays to determine MIP-2 production from isolated lungs were performed according to the ELISA manufacturer's specifications (Cytoscreen Immunoassay Kit, rat MIP-2). Samples for analysis were taken according to the specific protocols as follows.Specific Protocols
After isolation, lungs were placed into one of the following experimental protocols.Time control. Lungs in this group were isolated, ventilated, and perfused without interruption for a period of 180 min. The Kf,c values and perfusate samples for MIP-2 assays and lymphocyte analysis were taken at time points equivalent to those for lungs subjected to I/R as described in the next section.
I/R control and experimental. Lungs in this group were subjected to a 45-min period of global, normothermic ischemia followed by 30 and 90 min of reperfusion. Basal measures of the Kf,c and perfusate samples for MIP-2 assays were taken after lungs achieved an isogravimetric state. Lungs were then subjected to I/R with or without pretreatment with anti-CD40 or CD40L antibodies, goat or rabbit IgG, anti-IL-10 antibody (5 µg/ml), or IL-10 (0.5, 2, and 10 ng/ml doses). All antibodies, control IgG, and IL-10 were allowed to circulate 15 min before inducing ischemia. The Kf,c values and perfusate samples for MIP-2 assays and lymphocyte analysis were collected after 30 min of reperfusion time for all experiments. Kf,c values were measured at 30 and 90 min of reperfusion in the studies using rIL-10.
PMA control and experimental.
Lungs in this group were isolated and perfused in a manner similar to
time control lungs, with the exception that the total duration of
perfusion time was 90 min after the initial permeability measurements.
After basal permeability assessments, lungs were treated with PMA only
(2.57 × 104 M), exogenous lymphocytes (3.0 × 107 lymphocytes, >98% viability as assessed by trypan
blue exclusion), PMA-activated lymphocytes (3.0 × 107
cells challenged with 2.57 × 10
4 M PMA), or
anti-CD40 antibody followed by perfusion with PMA-activated lymphocytes. Isolated lymphocytes were not treated with the anti-CD40 antibody. Rat lymphocytes were harvested using methods previously described by our laboratory (30). Briefly, blood was
collected from healthy rat donors in heparinized syringes and separated into its distinct cellular fractions by cell density centrifugation. Heparinized blood (15-20 ml) was diluted to 25 ml of total volume with sterile saline (0.9 g/dl). With a solution of Ficoll and sodium
diatrizoate adjusted to a specific density of 1.023 (Histopaque-1023; Sigma Diagnostics, St. Louis, MO), 15 ml were placed in the bottom of a
50-ml plastic centrifuge tube, and 25 ml of diluted whole blood were
layered on top. Centrifugation in a horizontal rotor (400 g
at room temperature for 40 min) separated the cellular components into
two distinct groups. The mononuclear cells (lymphocytes and monocytes)
and platelets are less dense than Histopaque and were isolated between
the top layer of plasma and the layer of Histopaque. The denser layer
of polymorphonuclear cells and erythrocytes was pulled to the bottom of
the tube. After the plasma was removed, the mononuclear cell layer was
aspirated, washed two times with normal saline, and then resuspended in
normal saline. Cell counts were performed on every sample using a
hemocytometer to determine total numbers of isolated cells, and a final
concentration of 3 × 107 cells was obtained by
appropriate dilution with saline. These cell suspensions, which
consisted of >95% lymphocytes and the balance being monocytes, were
then introduced in the venous reservoirs of isolated lungs according to
the specific protocols as designed.
Statistical Analysis
Comparisons between right and left ventricular blood lymphocyte cell counts, percentages, and CD4-to-CD8 ratios were performed using an unpaired t-test. Similar analyses were used to compare the results obtained within the zone II isolated lungs (i.e., control vs. ischemic) and within the zone III isolated lungs. Significance was defined as P < 0.05. Comparisons were made among isolated lungs subjected to I/R with or without anti-CD40, anti-CD40L, or control IgG using ANOVA with Student-Newman-Keuls as a post hoc test when differences were found. Significance was defined as P < 0.05. For studies in which lungs were subjected to activated lymphocyte challenge with or without pretreatment with anti-CD40, baseline vs. experimental Kf,c values for each experimental condition were compared using a paired t-test with significance being defined as P < 0.05. Kf,c values were tested for differences between groups using ANOVA. When differences were found, a Student-Newman-Keuls post hoc test was applied, and significance was defined as P < 0.05. The ![]() |
RESULTS |
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Table 1 shows the
results using whole blood and isolated lung perfusates to determine the
relative proportions of CD4+ lymphocytes, CD8+
lymphocytes, and B cells circulating through the in vivo and isolated
lung circulations. In the in situ control lung, total lymphocyte
numbers for the right (pulmonary inflow) and left ventricular (pulmonary outflow) blood samples averaged 8.8 ± 0.7 × 106 and 8.2 ± 0.3 × 106 cells/ml,
respectively. The percentages of CD4+,
CD8+, or B cells obtained from the left ventricular samples
were not different compared with the right ventricular samples.
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In the isolated lung preparations, two different experimental
conditions were studied to examine the effects of available vascular
surface area for resident lymphocyte sequestration. Isolated lungs
perfused in a manner such that a relatively low vascular recruitment
state was achieved, i.e., overall zone II conditions, exhibited an 36% drop in the total numbers of lymphocytes harvested from the venous effluent after 45 min of ischemia (control = 4.5 ± 0.2 × 104 and postischemic = 2.9 ± 0.4 × 104 cells/ml). However, no
differences were observed between the percentages of CD4+,
CD8+, or B cells in the venous effluent of control lungs
vs. lungs subjected to ischemia. Isolated lungs perfused in a
manner to increase the degree of vascular recruitment, i.e., overall
zone III conditions, likewise exhibited a significant drop
in the total numbers of harvested lymphocytes after ischemia
(control = 3.9 ± 0.6 × 104 and
postischemic = 1.7 ± 0.7 × 104
cells/ml). However, this drop in the overall number of circulating lymphocytes (
55%) was slightly higher than that observed for the
zone II perfused lungs, and a significant drop in the
percentage of CD4+ lymphocytes was seen.
We next conducted experiments to determine whether retained lymphocytes
can influence I/R-induced microvascular injury. Because T cells utilize
CD40-CD40L signaling processes to initiate and regulate different types
of inflammatory responses and because CD40 expression occurs within the
ischemic coronary microcirculation, we hypothesized that
CD40-CD40L interactions may be involved in modulating pulmonary
postischemic microvascular permeability. Figure
1 shows the calculated changes in
microvascular permeability (Kf,c) occurring
in response to 45 min of ischemia followed by 30 min of
reperfusion. All lungs were studied under zone III
conditions. Anti-CD40 or anti-CD40L antibodies were used to pretreat
lungs before subjecting them to ischemia. As shown, I/R
produced an increased microvascular permeability indicated by the
significant change in Kf,c compared with the
non-I/R value. However, significantly less microvascular injury
occurred in lungs that were treated with either the anti-CD40 or CD40L
antibodies. The protective effects of anti-CD40 and anti-CD40L were not
reproduced when equal doses of preimmune rabbit and goat IgG,
respectively, were studied in place of the specific antibodies [rabbit
IgG + I/R (n = 4):
Kf,c = 0.31 ± 0.02 ml · min
1 · cmH2O
1 · 100 g lung tissue
1; goat IgG + I/R (n = 4):
Kf,c = 0.43 ± 0.06 ml · min
1 · cmH2O
1 · 100 g lung tissue
1]. These data suggest that CD40- and
CD40L-dependent mechanisms contribute to the I/R-induced microvascular
injury in the lung.
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To explore further whether CD40 ligation can lead to alterations in
pulmonary microvascular permeability, we conducted additional experiments in which activated lymphocytes were introduced in the
isolated lung with and without pretreatment with anti-CD40. Figure
2 shows data from lungs that were
isolated and perfused continuously under zone III
conditions. Lungs were challenged with a lymphocyte-enriched perfusate,
PMA only, or a PMA-activated lymphocyte-enriched perfusate. Lungs
perfused with the lymphocyte-enriched perfusate alone displayed no
change in microvascular permeability over time. Lungs perfused
with our normal nonblood perfusate, i.e., not lymphocyte enriched,
displayed a significant increase in microvascular permeability after
PMA challenge. The degree of microvascular injury was exacerbated
significantly when PMA-activated lymphocytes were introduced in the
lung perfusates. However, when lungs were pretreated with anti-CD40
antibody before the introduction of PMA-activated lymphocytes, no
changes in microvascular permeability were observed. These data are
consistent with our initial findings of lymphocyte involvement in acute
I/R-induced lung microvascular injury and support our hypothesis that
I/R-induced sequestered lymphocytes contribute to microvascular
dysfunction through CD40-CD40L mechanisms.
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The events occurring in response to CD40-CD40L signaling to produce
postischemic alterations in microvascular permeability are
unknown. However, we tested whether CD40-CD40L signaling might result
in chemokine production in the isolated lung, as has been reported
previously for cultured endothelial cells (5, 10, 13).
Figure 3 shows that perfusate MIP-2
levels gradually increased over time in the isolated rat lung, and I/R
produced a significant increase in the production of MIP-2 over the
basal level. Pretreatment of lungs with either anti-CD40 or anti-CD40L
antibodies resulted in postischemic venous effluent MIP-2
levels not different from basal levels obtained from noninjured control
lungs, which were significantly less (P < 0.05) than
those measured from the I/R lung perfusates. These data suggest that a
consequence of CD40-CD40L signaling in the intact I/R lung is to
enhance chemokine production, consistent with similar data obtained
from cultured endothelial cells. Coupled with previous data implicating
acute neutrophil involvement in altering lung microvascular
permeability after I/R injury (1, 27, 35), these data also
suggest that the mechanism downstream of CD40-CD40L signaling leading
to altered microvascular integrity may involve chemokine production to
recruit and activate granulocytes.
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Previous studies have shown that IL-10, a putative anti-inflammatory
cytokine, attenuates I/R-induced lung injury (7). Although
the mechanism of protection afforded by IL-10 is unknown, it is
possible that IL-10 could modulate lymphocytic processes involved with
producing acute microvascular injury. Although basal IL-10 production
was not determined quantitatively in these studies, isolated lungs that
were perfused with anti-IL-10 displayed no changes in microvascular
permeability over time (data not shown), thereby suggesting the absence
of some basal control of IL-10 on microvascular permeability. Lungs
that were subjected to I/R exhibited the typical progressive increase
in Kf,c over time representative of
microvascular injury. This level of injury was not affected by
pretreatment with preimmune IgG, since Kf,c
measurements were not different from those calculated in untreated
lungs subjected to I/R (Kf,c 30 min = 0.25 ± 0.07 and
Kf,c 90 min = 0.62 ± 0.10 ml · min
1 · cmH2O
1 · 100 g
1, n = 3). However, Fig.
4 shows that pretreatment of lungs with the specific anti-IL-10 antibody resulted in a significant exacerbation in microvascular damage occurring at both 30 and 90 min reperfusion, thereby suggesting endogenous IL-10 production occurred with I/R to
limit the severity of I/R-induced injury. Furthermore, as shown in Fig.
5, microvascular injury occurring in
response to I/R could be prevented when recombinant rIL-10 was
supplemented to isolated lungs before subjecting them to
ischemia. These protective effects were dose dependent, with
IL-10 at concentrations of 10 ng/ml providing near-maximal protection
against microvascular injury at both 30 and 90 min reperfusion time.
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DISCUSSION |
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Reactive oxygen species, granulocyte activation, and endothelial cell contractile proteins have all been shown to contribute to post-I/R microvascular integrity alterations (1, 9, 18, 27). However, a precise mechanism by which all these factors interact to produce microvascular damage is unclear, and the initial events leading to activation of the postischemic inflammatory cascade are unknown.
Our present studies examine possible mechanism(s) by which lymphocytes and other cell types may be involved with actively producing acute lung I/R microvascular injury. General support for the idea of a lymphocytic contribution to regulation of microvascular dynamics comes from studies of lung lymphocyte trafficking and identification of pulmonary lymphocyte subsets. It is well established that the lung harbors a significant reservoir of lymphocytes of all lineages, and several studies have examined subset distribution profiles and circulation kinetics (3, 8, 21, 27, 29, 38). Lymphocytes are divided between three intrapulmonary compartments, including bronchus-associated lymphoid tissue, lymphocytes located in the lumen of the lower respiratory tract, and vascular and interstitial marginated pools. These lymphocyte pools are retained in the lung even after excision of the lungs for use in ex vivo studies.
Lymphocyte retention in the ex vivo lung preparation has also been studied with respect to washout kinetics. These studies have shown that isolated lungs release sequestered lymphocytes for as much as 6 h after instituting a nonblood vascular washout procedure. Data obtained from isolated rat lung studies in particular (8) have shown that T lymphocytes predominate in the nonwhole blood perfusate of isolated lungs. In addition, the isolated lung perfusate contains a higher proportion of circulating CD4+ cells relative to CD8+ cells and the CD4+-to-CD8+ ratio obtained in brochoalveolar lavage fluid is greater than that observed in the lung perfusate. These observations suggest that CD4+ cells may exhibit selective trafficking properties within the lung's pulmonary and systemic circulations to respond acutely to inflammatory stimuli generated from within the intrapulmonary mileu or the airways, respectively. If so, then microvascular endothelial cell mechanisms should be in place to facilitate and coordinate CD4+ lymphocyte movement as necessary.
Our present data suggest that the resident CD4+ lymphocyte population within the lung may respond to I/R. The consequences of their responsiveness may be to directly or indirectly regulate postischemic changes in microvascular permeability and in turn regulate lung edema. Significant retention of CD4+ cells was shown to occur after 45 min of global ischemia in our isolated rat lung model. Unexpectedly, this sequestration occurred only when Ppv was >0 cmH2O. Because the total numbers of lymphocytes retained with I/R increased significantly when Ppv were elevated to 5 cmH2O, it would be reasonable to conclude that the CD4+ cell population was more readily retained under these conditions secondarily to more endothelial surface area being available for tethering. The precise mechanisms leading to CD4+ lymphocyte retention within the lung will require further studies.
We tested whether the observed lymphocyte retention influenced I/R-induced changes in microvascular permeability. Our hypothesis was that lymphocytes, particularly CD4+ cells, mediate postischemic microvascular injury in part through CD40-CD40L signaling and chemokine generation. Our data indicated that interfering with CD40-CD40L signaling during I/R significantly attenuated postischemic increases in microvascular permeability and prevented I/R-induced chemokine production associated with I/R. Lymphocyte-mediated lung injury manifested as approximately fivefold increases in permeability and lung injury and were prevented with anti-CD40. Thus our data strongly support the hypothesis that pulmonary lymphocyte retention observed with I/R is consequential and CD40- and CD40L-dependent mechanisms participate in producing alterations in postischemic microvascular permeability.
Activated "Th1-like" CD4+ cells express a cell surface ligand designated CD40L, which was first identified as a costimulatory molecule to stimulate B cells to undergo Ig class switching (6, 16, 37, 40). The receptor for CD40L is CD40. Endothelial cells express CD40 constitutively in many microvascular beds, including the lung (43, 46), and CD40 expression can be upregulated in the coronary microcirculation after brief durations of global cardiac ischemia (32). CD40 expression is elicited in cultured endothelial cells after proinflammatory cytokine challenge. Ligation of cultured endothelial cell-expressed CD40 results in cell activation cascades that include the generation of local proinflammatory cytokines, matrix metalloproteinase secretion, and expression of endothelial cell adhesion molecules (5, 10, 13, 17, 23, 49). These events, when occurring in situ, can lead directly or indirectly to alterations in normal microvascular fluid and protein permeability. Furthermore, pulmonary endothelium-expressed CD40 could be stimulated by activated Th1-like CD4+ cells. Elucidation of these and other mechanisms with respect to in situ CD40-CD40L involvement in I/R-induced lung injury requires additional studies.
Downstream cascades that propagate the postischemic inflammatory response are also likely to occur. The highly neutrophil-selective chemokine IL-8 is produced in response to CD40 stimulation (10). IL-8 production has been shown to promote neutrophil retention and plasma protein extravasation in association with lung I/R injury (36). MIP-2 is the murine equivalent to human IL-8 (24, 41). MIP-2 elicits P-selectin-dependent neutrophil rolling in vivo (48) and is associated with the development of renal injury after I/R (25). Thus CD40-dependent liberation of neutrophil attractants by the pulmonary microvascular endothelium may also be necessary for postischemic microvascular lung injury, since neutrophil dependence in post-I/R changes in the pulmonary Kf,c have been described previously (1, 27, 35).
Exogenously administered IL-10 has been shown to halt the progression of various pulmonary inflammatory conditions, including that associated with I/R (7, 11, 41). It is also known that IL-10 is released by a number of cell types found in the lung, including CD4+ lymphocytes, alveolar macrophages, and fibroblasts (26). Thus we tested the possibility that lymphocyte-derived anti-inflammatory cytokines may be coincidentally produced with proinflammatory lymphocytic mechanisms that in turn result in a balanced postischemic inflammatory response.
We observed that anti-IL-10 administered to isolated lungs subjected to I/R significantly potentiated the microvascular injury. Furthermore, when I/R isolated lungs were supplemented with additional, exogenous IL-10, I/R-induced increases in microvascular permeability were prevented in a dose-dependent manner. These data are consistent with previous findings evaluating IL-10 in lung I/R injury, except that our studies used an isolated lung preparation, whereas beneficial effects of IL-10 were previously demonstrated using a whole animal model and over a much longer experimental time frame for I/R (7). When taken together, these studies indicate the importance of IL-10 in preventing lung injury associated with certain types of inflammatory conditions such as I/R. The mechanism by which IL-10 affords protection is unknown, although IL-10 could act to suppress MIP-2 production and subsequent activation of granulocytes (41).
In summary, our data suggest that lymphocytes are involved in the sequence of events associated with lung I/R-induced microvascular injury. Specifically, lymphocyte sequestration and CD40-CD40L signaling appear to be linked to these alterations in microvascular permeability. However, the mechanisms involved with how the permeability increase occurs in response to CD40-CD40L signaling are unclear, but chemokine production and neutrophil activation are likely involved. Concurrently, the anti-inflammatory cytokine IL-10 may be produced by selected cell types in the lung and serve to limit the severity of I/R-induced microvascular injury. Future strategies directed at interfering with early lymphocyte retention and activation or increasing tissue IL-10 levels are necessary in understanding the mechanisms associated with I/R-induced microvascular injury.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. E. Taylor, Dept. of Physiology, MSB 3024, Univ. of South Alabama College of Medicine, Mobile, AL 36688-0002 (E-mail: ataylor{at}jaguar1.usouthal.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 9, 2002;10.1152/ajplung.00016.2002
Received 14 June 2002; accepted in final form 1 August 2002.
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