Involvement of CD40-CD40L signaling in postischemic lung injury

Timothy M. Moore, W. Bradley Shirah, Pavel L. Khimenko, Peyton Paisley, Robert N. Lausch, and Aubrey E. Taylor

Department of Physiology, University of Alabama College of Medicine, Mobile, Alabama 36688-0002


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 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).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta W/Delta t) during the last 2-min interval was analyzed to calculate the Kf,c. The Delta W/Delta 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 × 10-4 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 Delta Kf,c values calculated in lungs subjected to I/R alone, I/R with or without anti-IL-10, or I/R plus rIL-10 were also compared using ANOVA with a Student-Newman-Keuls post hoc test to determine significant differences (P < 0.05).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Relative percentages of CD4+ or CD8+ T lymphocytes and B cells in the whole blood and perfusate samples taken from in situ sites or ex vivo lungs, respectively

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 approx 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 (approx 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 (Delta 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): Delta Kf,c = 0.31 ± 0.02 ml · min-1 · cmH2O-1 · 100 g lung tissue-1; goat IgG + I/R (n = 4): Delta 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.


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 1.   Calculated changes in pulmonary microvascular permeability [change (Delta ) in filtration coefficient (Kf,c)] occurring over time (Time Con, n = 5 experiments) or in response to ischemia/reperfusion (I/R, n = 5), I/R + pretreatment with 0.8 µg/ml pAb L-17 (Anti-CD40, n = 5), and I/R + pretreatment with 0.2 µg/ml pAb K-19 (Anti-CD40L, n = 5). Delta Kf,c was calculated by subtracting the baseline Kf,c value for each lung from its corresponding Kf,c value measured after 45 min of ischemia and 30 min of reperfusion. * Significant difference from I/R (P < 0.05).

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.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2.   Baseline (filled bars) and 90-min experimental (open bars) Kf,c values for isolated lungs challenged with 2.57 × 10-4 M phorbol 12-myristate, 13-acetate (PMA) alone (PMA control, n = 5), approx 3.0 × 107 rat lymphocytes alone (Lymp control, n = 5), PMA-activated rat lymphocytes (Lymp activated, n = 5), or PMA-activated lymphocytes with pretreatment of 0.8 µg/ml anti-CD40 (Lymp activated + Anti-CD40, n = 5). P < 0.05, significant difference from the corresponding baseline value (*) and significantly different from Lymp activated Kf,c value at 90 min (#).

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.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   Perfusate concentrations of macrophage inflammatory protein (MIP)-2 as determined by ELISA vs. reperfusion time of postischemic lungs. Ischemic duration was 45 min for all I/R groups. Time control groups (n = 5) were perfused and ventilated without interruption for a time matched to the I/R control group (n = 5). Anti-CD40 and Anti-CD40L denote lung groups that were pretreated with pAb L-17 (0.8 µg/ml, n = 5) and pAb K-19 (0.2 µg/ml, n = 5), respectively. * P < 0.05, 30- and 90-min reperfusion MIP-2 levels were significantly elevated relative to time control values.

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 (Delta Kf,c 30 min = 0.25 ± 0.07 and Delta 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.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   Calculated changes in pulmonary microvascular permeability (Delta Kf,c) occurring in response to 45 min of ischemia followed by 30 and 90 min of reperfusion. Lungs were either untreated (ischemia/reperfusion control, n = 5) or were pretreated with 0.5 µg/ml of the anti-IL-10 antibody (n = 5). Delta Kf,c was calculated by subtracting the baseline Kf,c value for each lung from its corresponding Kf,c value measured after 30 (filled bars) and 90 (open bars) min of reperfusion. Significant difference from the Delta  Kf,c values at 30 (*) and 90 (**) min compared with I/R control group.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 5.   Calculated changes in pulmonary microvascular permeability (Delta Kf,c) occurring in response to I/R (I/R Con, n = 5) and I/R + pretreatment with incrementing doses of rat recombinant IL-10 (n = 5 for each group). Delta Kf,c was calculated by subtracting the baseline Kf,c value for each lung from its corresponding Kf,c value measured after 45 min of ischemia and 30 (filled bars) and 90 (open bars) min of reperfusion. P < 0.05, significant difference from the I/R control Delta Kf,c value at 30 min reperfusion (*) and significant difference from the I/R control Delta Kf,c value at 90 min reperfusion (**).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adkins, WK, and Taylor AE. Role of xanthine oxidase and neutrophils in ischemia-reperfusion injury in rabbit lung. J Appl Physiol 69: 2012-2018, 1990[Abstract/Free Full Text].

2.   Austrup, F, Vestweber D, Borges E, Lohning M, Brauer R, Herz U, Renz H, Hallmann R, Scheffold A, Radbruch A, and Hamann A. P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflammed tissues. Nature 385: 81-83, 1997[ISI][Medline].

3.   Berman, JS, Beer DJ, Theodore AC, Kornfeld H, Bernardo J, and Center DM. Lymphocyte recruitment to the lung. Am Rev Respir Dis 142: 238-257, 1990[ISI][Medline].

4.   Borges, E, Tietz W, Steegmaier M, Moll T, Hallmann R, Hamann A, and Vestweber D. P-selectin glycoprotein ligand-1 (PSGL-1) on T helper 1 but not on T helper 2 cells binds to P-selectin and supports migration into inflamed skin. J Exp Med 185: 573-578, 1997[Abstract/Free Full Text].

5.   Déchanet, J, Grosset C, Taupin JL, Merville P, Banchereau J, Ripoche J, and Moreau JF. CD40 ligand stimulates proinflammatory cytokine production by human endothelial cells. J Immunol 159: 5640-5647, 1997[Abstract].

6.   Durie, FH, Foy TM, Masters SR, Laman JD, and Noelle RJ. The role of CD40 in the regulation of humoral and cell-mediated immunity. Immunol Today 15: 406-410, 1994[ISI][Medline].

7.   Eppinger, MJ, Ward PA, Bolling SF, and Deeb GM. Regulatory effects of interleukin-10 on lung ischemia-reperfusion injury. J Thorac Cardiovasc Surg 112: 1301-1306, 1996[Abstract/Free Full Text].

8.   Fliegert, FG, Tschernig T, and Pabst R. Comparison of lymphocyte subsets, monocytes, and NK cells in three different lung compartments and peripheral blood in the rat. Exp Lung Res 22: 677-690, 1996[ISI][Medline].

9.   Granger, DN, McCord JM, Parks DA, and Hollwarth ME. Xanthine oxidase inhibitors attenuate ischemia-induced vascular permeability changes in the cat intestine. Gastroenterology 90: 80-84, 1986[ISI][Medline].

10.   Henn, V, Slupsky JR, Gräfes M, Anagnostopoulos I, Förster R, Müller-Berghaus G, and Kroczek RA. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature 391: 591-594, 1998[ISI][Medline].

11.   Hickey, MJ, Issekutz AC, Reinhardt PH, Fedorak RN, and Kubes P. Endogenous interleukin-10 regulates hemodynamic parameters, leukocyte-endothelial cell interactions, and microvascular permeability during endotoxemia. Circ Res 83: 1124-1131, 1998[Abstract/Free Full Text].

12.   Hirohata, S. Human Th1 responses driven by IL-12 are associated with enhanced expression of CD40 ligand. Clin Exp Immunol 115: 78-85, 1999[ISI][Medline].

13.   Hollenbaugh, D, Mischel-Petty N, Edwards CP, Simon JC, Denfeld RW, Kiener PA, and Aruffo A. Expression of functional CD40 by vascular endothelial cells. J Exp Med 182: 33-40, 1995[Abstract].

14.   Horie, Y, Wolf R, Chervenak RP, Jennings SR, and Granger DN. T-lymphocytes contribute to hepatic leukostasis and hypoxic stress induced by gut ischemia-reperfusion. Microcirculation 6: 267-280, 1999[ISI][Medline].

15.   Infante-Duarte, C, and Kamradt T. Th1/Th2 balance in infection. Springer Semin Immunopathol 21: 317-338, 1999[ISI][Medline].

16.   Jordan, SC, and Fredrich R. Cytokines and lymphocytes. In: Cytokines in Health and Disease (2nd ed.), edited by Remick DG, and Friedland JS.. New York: Dekker, 1997, p. 357-372.

17.   Karmann, K, Hughes CCW, Schechner J, Fanslow WC, and Pober JS. CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression. Proc Natl Acad Sci USA 92: 4342-4346, 1995[Abstract].

18.   Khimenko, PL, Moore TM, Wilson PS, and Taylor AE. Role of calmodulin and myosin light-chain kinase in lung ischemia-reperfusion injury. Am J Physiol Lung Cell Mol Physiol 271: L121-L125, 1996[Abstract/Free Full Text].

19.   Kokura, S, Wolf RE, Yoshikawa T, Ichikawa H, Granger DN, and Aw TY. Endothelial cells exposed to anoxia/reoxygenation are hyperadhesive to T-lymphocytes: kinetics and molecular mechanisms. Microcirculation 7: 13-23, 2000[ISI][Medline].

20.   Kunkel, EJ, Ramos CL, Steeber DA, Muller W, Wagner N, Tedder TF, and Ley K. The roles of L-selectin, beta 7 integrins, and P-selectin in leukocyte rolling and adhesion in high endothelial venules of Peyer's patches. J Immunol 161: 2449-2456, 1998[Abstract/Free Full Text].

21.   Lee, NA, and Lee JJ. The macroimportance of the pulmonary immune microenvironment. Am J Respir Cell Mol Biol 21: 298-302, 1999[Free Full Text].

22.   Lichtman, AH, and Abbas AK. T-cell subsets: recruiting the right kind of help. Curr Biol 7: R242-R244, 1997[ISI][Medline].

23.   Mach, F, Schönbeck RP, Fabunmi C, Murphy E, Atkinson JY, Bonnefoy P, Graber P, and Libby P. T lymphocytes induce endothelial cell matrix metalloproteinase expression by a CD40L-dependent mechanism. Am J Pathol 154: 229-238, 1999[Abstract/Free Full Text].

24.   Massey, KD, Strieter RM, Kunkel SL, Danforth JM, and Standiford TJ. Cardiac myocytes release leukocyte-stimulating factors. Am J Physiol Heart Circ Physiol 269: H980-H987, 1995[Abstract/Free Full Text].

25.   Miura, M, Fu X, Zhang QW, Remick DG, and Fairchild RL. Neutralization of groalpha and macrophage inflammatory protein-2 attenuates renal ischemia/reperfusion injury. Am J Pathol 159: 2137-2145, 2001[Abstract/Free Full Text].

26.   Moore, KW, O'Garra A, de Waal Malefyt R, Vieira P, and Mosmann TR. Interleukin-10. Annu Rev Immunol 11: 165-190, 1993[ISI][Medline].

27.   Moore, TM, Khimenko P, Adkins WK, Miyasaka M, and Taylor AE. Adhesion molecules contribute to ischemia and reperfusion-induced injury in the isolated rat lung. J Appl Physiol 78: 2245-2252, 1995[Abstract/Free Full Text].

28.   Pabst, R, Binns RM, Licence ST, and Peter M. Evidence of a selective major vascular marginal pool of lymphocytes in the lung. Am Rev Respir Dis 136: 1213-1218, 1987[ISI][Medline].

29.   Pabst, R, and Tschernig T. Lymphocytes in the lung: an often neglected cell. Anat Embryol (Berl) 192: 293-299, 1995[ISI][Medline].

30.   Perry, M, and Taylor AE. Phorbol myristate acetate-induced injury of isolated perfused rat lungs: neutrophil dependence. J Appl Physiol 65: 2164-2169, 1988[Abstract/Free Full Text].

31.   Punch, JD, and Bromberg JS. Cytokines and transplantation. In: Cytokines in Health and Disease (2nd ed.), edited by Remick DG, and Friedland JS.. New York: Dekker, 1997, p. 557-562.

32.   Reul, RM, Fang JC, Denton MD, Geehan C, Long C, Mitchell RN, Ganz P, and Briscoe DM. CD40 and CD40 ligand (CD154) are coexpressed on microvessels in vivo in human cardiac allograft rejection. Transplantation 64: 1765-1774, 1997[ISI][Medline].

33.   Romagnani, S. Th1/Th2 cells. Inflamm Bowel Dis 5: 285-294, 1999[ISI][Medline].

34.   Sawaya, DE, Jr, Zibari GB, Minardi A, Bilton B, Burney D, Granger DN, McDonald JC, and Brown M. P-selectin contributes to the initial recruitment of rolling and adherent leukocytes in hepatic venules after ischemia/reperfusion. Shock 12: 227-232, 1999[ISI][Medline].

35.   Seibert, AF, Haynes J, and Taylor A. Ischemia-reperfusion injury in the isolated rat lung. Role of flow and endogenous leukocytes. Am Rev Respir Dis 147: 270-275, 1993[ISI][Medline].

36.   Sekido, N, Mukaida N, Harada A, Nakaishi I, Watanabe Y, and Matsushima K. Prevention of lung reperfusion injury in rabbits by a monoclonal antibody against interleukin-8. Nature 365: 654-657, 1993[ISI][Medline].

37.   Smith, CA, Davis T, Anderson D, Solam L, Beckmann MP, Jerzy R, Dower SK, Cosman D, and Goodwin RG. A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins. Science 248: 1019-1024, 1990[ISI][Medline].

38.   Stein-Streilein, J. Immunobiology of lymphocytes in the lung. Reg Immunol 1: 128-136, 1988[Medline].

39.   Symon, FA, McNulty CA, and Wardlaw AJ. P- and L-selectin mediate binding of T cells to chronically inflamed human airway endothelium. Eur J Immunol 29: 1324-1333, 1999[ISI][Medline].

40.   Torres, RM, and Clark EA. Differential increase of an alternatively polyadenylated mRNA species of murine CD40 upon B lymphocyte activation. J Immunol 148: 620-626, 1992[Abstract/Free Full Text].

41.   Tumpey, TM, Cheng H, Yan XT, Oakes JE, and Lausch RN. Chemokine synthesis in the HSV-1-infected cornea and its suppression by interleukin-10. J Leukoc Biol 63: 486-492, 1998[Abstract].

42.   Wagers, AJ, Waters CM, Stoolman LM, and Kansas GS. Interleukin 12 and interleukin 4 control T cell adhesion to endothelial selectins through opposite effects on alpha-1, 3-fucosyltransferase VII gene expression. J Exp Med 188: 2225-2231, 1998[Abstract/Free Full Text].

43.   Wiley, JA, Geha R, and Harmsen AG. Exogenous CD40 ligand induces a pulmonary inflammation response. J Immunol 158: 2932-2938, 1997[Abstract].

44.   Wolber, FM, Curtis JL, Maly P, Kelly RJ, Smith P, Yednock TA, Lowe JB, and Stoolman LM. Endothelial selectins and alpha 4 integrins regulate independent pathways of T-lymphocyte recruitment in the pulmonary immune response. J Immunol 161: 4396-4403, 1998[Abstract/Free Full Text].

45.   Wolber, FM, Curtis JL, Milik AM, Fields T, Seitzman GD, Kim K, Kim S, Sonstein J, and Stoolman LM. Lymphocyte recruitment and the kinetics of adhesion receptor expression during the pulmonary immune response to particulate antigen. Am J Pathol 151: 1715-1727, 1997[Abstract].

46.   Yellin, MJ, Brett J, Baum D, Matsushima A, Szabolcs M, Stern D, and Chess L. Functional interactions of T cells with endothelial cells: the role of CD40L-CD40-mediated signals. J Exp Med 182: 1857-1864, 1995[Abstract].

47.   Zeevi, A, Fung JJ, Paradis IL, Dauber JH, Griffith BP, Hardesty RL, and Duquesnoy RJ. Lymphocytes of bronchoalveolar lavages from heart-lung transplant recipients. J Heart Transplant 4: 417-421, 1985[Medline].

48.   Zhang, XW, Liu Q, Wang Y, and Thorlacius H. CXC chemokines, MIP-2 and KC, induce P-selectin-dependent neutrophil rolling and extravascular migration in vivo. Br J Pharmacol 133: 413-21, 2001[Abstract/Free Full Text].

49.   Zhou, L, Stordeur P, de Lavareille A, Thielemans K, Capel P, Goldman M, and Pradier O. CD40 engagement on endothelial cells promotes tissue factor-dependent procaogulant activity. Thromb Haemost 79: 1025-1028, 1998[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 283(6):L1255-L1262
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society