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. Author manuscript; available in PMC: 2010 Mar 1.
Published in final edited form as: J Thorac Cardiovasc Surg. 2009 Mar;137(3):695–702. doi: 10.1016/j.jtcvs.2008.10.044

CD4+ T Lymphocytes Mediate Acute Pulmonary Ischemia-Reperfusion Injury

Zequan Yang 1, Ashish K Sharma 1, Joel Linden 2, Irving L Kron 1, Victor E Laubach 1
PMCID: PMC2670927  NIHMSID: NIHMS102742  PMID: 19258091

Abstract

Objective

Post-ischemic reperfusion of the lung triggers pro-inflammatory responses that stimulate injurious neutrophil chemotaxis. We hypothesized that T lymphocytes are recruited and activated during reperfusion and mediate subsequent neutrophil-induced lung ischemia-reperfusion injury (IRI).

Methods

An in vivo mouse model of lung IRI was employed. C57BL/6 mice were assigned to either sham group (left thoracotomy) or 7 study groups which underwent one hour left hilar occlusion followed by 1-24 hours reperfusion. Following in vivo reperfusion, the lungs were perfused ex vivo with buffer whereby pulmonary function was assessed. Lung vascular permeability, edema, neutrophil accumulation and cytokine/chemokine production (TNF-α, IL-17, CCL3, and CXCL1) were assessed by Evans blue dye leak, wet/dry weight ratio, myeloperoxidase, and ELISA, respectively.

Results

A preliminary study showed that 2-hr reperfusion resulted in greater pulmonary dysfunction than 1-hr or 24-hr reperfusion. The 2-hr reperfusion period was thus used for the remaining experiments. Comparable and significant protection from IR-induced lung dysfunction and injury occurred after antibody-depletion of neutrophils or CD4+ T cells, but not CD8+ T cells (p<0.05 vs. IgG control). Lung IRI was proportional to the infiltration of neutrophils but not T cells. Moreover, pulmonary neutrophil infiltration and the production of CXCL1 (KC) were significantly diminished by CD4+ T cell depletion, but not vice versa.

Conclusions

Both CD4+ T lymphocytes and neutrophils accumulate during reperfusion and contribute sequentially to lung IRI. The data suggest that neutrophils mediate IRI; however, CD4+ T cells play a critical role in stimulating chemokine production and neutrophil chemotaxis during IRI.

Introduction

Respiratory failure remains the most common complication in the perioperative period after lung transplantation. One of the major causes of respiratory failure and complications acutely observed after transplantation is ischemia-reperfusion injury (IRI)1, which has been reported to be responsible for up to 30% of patient mortality within 30 days2. An increasing body of evidence has shown that IRI is associated with enhanced inflammatory responses during reperfusion. Our previous animal experiments have shown that alveolar macrophages and circulating leukocytes contribute importantly to lung IRI, with macrophages serving as triggers and leukocytes, mainly neutrophils, as end effectors3-6. Furthermore, we recently reported that alveolar epithelial cells, especially type II cells, interact with alveolar macrophages to initiate the inflammatory responses during IRI7. However, the signaling pathways between alveolar macrophages and neutrophils remain to be defined.

There is growing evidence that T cells may also participate in the pathogenesis of lung IRI8-10. T cells are found to infiltrate the lung and are activated during reperfusion earlier than neutrophils10. Lymphocyte-deficient rats or mice have decreased IRI9, 10. Cytokines and chemokines that stimulate T cell chemotaxis and activation, such as IL-8, IL-12, IL-18, CCL5, and CCL2, are elevated during lung IRI7, 9, 11-13. T cells are known to amplify inflammatory responses through the secretion of lymphokines including IFN-γ, IL-2, IL-4, IL-17 and GM-CSF9, 14. These stimulate the chemotaxis of neutrophils and monocytes to site(s) of injury.

Whether T cells participate importantly in the inflammatory cascade that results in lung IRI is unclear. In the current study, we used an in vivo mouse model of lung IRI to examine the role of T cells in lung IRI. Since neutrophils are end-effectors of lung IRI, we also examined the effect of lymphocyte depletion of neutrophil trafficking into the lung. Monoclonal antibodies were used in order to render mice deficient in neutrophils, CD4+ T cells or CD8+ T cells.

Materials and Methods

Animals

This study employed a total of 74 (8-12 week old) male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine) which were assigned to seven IRI study groups and one sham group that underwent surgery but not hilar clamping. This study conformed to the “Guide for the Care and Use of Laboratory Animals” published by the National Institute of Health (NIH publication No. 85-23, revised 1985) and was conducted under protocols approved by the University of Virginia’s Institutional Animal Care and Use Committee.

In Vivo depletion of neutrophils

Rat anti-mouse Gr-1 mAb was used to deplete circulating neutrophils in mice as reported by others15. Briefly, 10μg anti-Gr-1 mAb (eBioscience, San Diego, CA) was injected via tail vein 24 hours prior to lung ischemia. Perioperatively, blood (30-40 μl) was obtained by puncturing the left external jugular vein, and leukocyte counts were performed using a HemaVet Hematology System (CDC Technologies, Oxford, CT).

In Vivo depletion of CD4+ or CD8+ T lymphocytes

Depletion of CD4+ or CD8+ T cells was achieved by using selective antibodies as reported previously16. Anti–CD4 mAb (GK1.5) or anti–CD8a mAb (53-6.7) (eBioscience, San Diego, CA) was injected intraperitoneally on two consecutive days at a dose of 0.2 mg/mouse/day. Two days after the second injection these animals underwent lung ischemia.

In vivo model of lung ischemia-reperfusion

Mice were anesthetized with inhalation isoflurane, intubated with PE-60 tubing and connected to a pressure-controlled ventilator (Harvard Apparatus Co, South Natick, MA). Mechanical ventilation was performed with room air as adjusted to a rate of 150 strokes/min, a stroke volume of 1.0 cc, and peak inspiratory pressure less than 20 cm H2O. Heparin (20 U/kg) was administered via external jugular injection. Left thoracotomy was performed by cutting the left 4th rib, and the left hilum was exposed. A 6-0 prolene suture was placed around the hilum facilitated by a tip-curved (22G) gavage needle. Both ends of the suture were then threaded through a 5-mm long PE-50 tubing. Occlusion was achieved by pulling up on the suture and thus pushing the tube against the hilum to initiate ischemia. A small surgical clip was applied to the suture on top of the tube to maintain tension of the tube against the hilum. The thoracotomy was then suture-closed, and the mouse was extubated, place in a cage, and allowed to awaken during the 1-hr hilar occlusion period. Five minutes before reperfusion, the mouse was re-anesthetized and re-intubated. Reperfusion was achieved by removing the clip and the tube/suture. Again, the chest was suture-closed. The mouse was extubated and returned to a cage until pulmonary function testing. Temperature was monitored during surgery by an anal probe and maintained between 36.5 to 37.5°C. Sham animals received only thoracotomy without hilar occlusion. To minimize pain and discomfort, an analgesic (Buprenorphine, 0.2mg/kg) was administered to all animals at the beginning of surgery.

Measurement of pulmonary function

At the end of scheduled reperfusion, pulmonary function was evaluated using an isolated, buffer-perfused mouse lung system (Hugo Sachs Elektronik, March-Huggstetten, Germany) as previously described by our laboratory6. Briefly, mice were anesthetized with ketamine and xylazine. A tracheostomy was performed, and animals were ventilated with room air at 100 strokes/min, a tidal volume of 7 μl/g body weight with a positive end-expiratory pressure of 2 cmH2O. The animals were exsanguinated by inferior caval transection. The pulmonary artery was cannulated via the right ventricle and the left ventricle was immediately tube-vented through a small incision at the apex of the heart. The lungs were then perfused at a constant flow of 60 μl·g body wt−1·min−1 with Krebs-Henseleit buffer containing 2% albumin, 0.1% glucose, and 0.3% HEPES (335–340 mosmol/kgH2O). The perfusate buffer and isolated lungs were maintained at 37°C throughout the experiment by use of a circulating water bath. Once properly perfused and ventilated, the lungs were maintained on the system for a 5-min equilibration period before data was recorded for an additional 10 min. Hemodynamic and pulmonary parameters were recorded during this period by the PULMODYN data acquisition system (Hugo Sachs Elektronik).

Bronchoalveolar lavage (BAL)

After pulmonary function measurements, the left lungs were lavaged with 0.4 ml normal saline. A micro clamp was used to occlude the right hilum prior to lavage. The BAL fluid was immediately centrifuged at 4°C (500 g, 5 min), and the supernatant was stored at −80°C until further analysis.

Lung wet/dry weight ratio

In separate groups, the left lung was harvested, weighed, and then placed in a vacuum oven (at 58°C) until a stable dry weight was achieved. The ratio of lung wet weight to dry weight was then calculated.

Pulmonary microvascular permeability

IRI-induced microvascular permeability in the lung was determined using the Evans blue dye extravasation technique17. Evans blue (20 mg/kg; Sigma-Aldrich) was injected i.v. 30 min before euthanasia. The pulmonary vasculature was then perfused for 15 min using the isolated, buffer-perfused lung system to remove intravascular dye. Lungs were then homogenized in PBS to extract the Evans blue and centrifuged. The absorption of Evans blue was measured in the supernatant at 620 nm and corrected for the presence of heme pigments: A620 (corrected) = A620 - (1.426 × A740 + 0.030).

Immunohistochemistry

A standard immunohistochemistry protocol for paraformaldehyde-fixed tissue was employed as detailed previously16. Briefly, the left lung was harvested, cut into 4 short-axis slices, and immediately fixed in 1% paraformaldehyde in PBS (pH 7.4) for paraffin embedding. Immunostaining was performed with rat anti-mouse neutrophil antibody (Serotec Inc), anti-CD3 antibody (Santa Cruz Biotechnology) or anti-Mac2 antibody (Accurate Chemical & Scientific Corp.). Three lung tissue slides (1 slide per mouse) from each group were used for semi-quantitative cell counts in peripheral lung tissue. These cell counts did not distinguish among cells in various components of the lung (e.g. airspace, interstitial or marginated) but included all cells in peripheral (alveolar) lung tissue. On each slide the lung tissue was divided into 4 to 6 parallel zones, and one photo was taken at each zone where the target cells were found in highest numbers under 100X magnification.

Measurement of myeloperoxidase (MPO)

MPO was measured in BAL fluid using a mouse MPO ELISA kit (Cell Sciences, Canton, MA).

Measurement of cytokines/chemokines

Cytokines/chemokines in BAL fluid were quantified using the Bioplex Bead Array technique with a multiplex cytokine panel assay (Bio-Rad Laboratories, Hercules, CA) as previously done by our lab7. The samples were analyzed as instructed using the Bioplex array reader, which is a fluorescent-based flow cytometer employing a bead-based multiplex technology, each of which is conjugated with a reactant specific for a different target molecule.

Statistical analysis

All data are presented as the mean ± SEM (standard error of the mean). Data were compared using one-way ANOVA followed by the Student’s t-test for unpaired data with Bonferroni correction. Square roots of tissue cell counts were compared using one-way ANOVA.

Results

Time course of reperfusion injury after one-hour hilar ligation

To define the time point during reperfusion when lung injury was most significant, we evaluated pulmonary function after 1, 2, and 24 hrs reperfusion following 1 hr ischemia. At each time point, significant and roughly comparable elevations in pulmonary artery pressure (PAP) and airway resistance (AR) occurred (data not shown). Lung compliance (LC) was significantly worse after 2 and 24 hrs reperfusion versus sham (2.34±0.19 and 4.02±0.19 vs. 5.61±0.36 μl/cmH2O, respectively, p<0.05). LC was not significantly reduced after 1 hr reperfusion (4.52±0.65 μl/cmH2O), but was significantly worse at 2 hrs reperfusion compared to 1 and 24 hrs (p<0.05). Based on these data, 1 hr ischemia and 2 hrs reperfusion were used for the remainder of the study.

Changes in circulating leukocyte numbers

Whole blood samples were collected from antibody-treated mice before ischemia and after 2 hrs reperfusion, and blood cells were counted with a HemaVet Hematology System (Fig. 1). In neutrophil-depleted mice there was >80% reduction in the number of neutrophils (p<0.05); however, there was also a significant reduction in monocytes (80%) compared to IgG isotype controls (Fig. 1A). After reperfusion, the total number of circulating white blood cells and lymphocytes were significantly reduced in both IgG control and neutrophil-depleted mice compared to before ischemia (Fig 1B). Monocytes were further significantly reduced in IgG control mice after reperfusion, but not in neutrophil-depleted mice. On the contrary, neutrophils were significantly increased by 2-fold after reperfusion in IgG control mice (compared to pre-ischemia), and not significantly increased in neutrophil-depleted mice (Fig. 1B).

Figure 1.

Figure 1

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Complete blood cell counts in antibody-treated mice. (A) Prior to hilar ligation there was an 80% reduction in neutrophils in neutrophil-depleted mice compared to IgG control mice, a 19% reduction in lymphocytes in CD8-depleted mice, and a 26% reduction of lymphocytes in CD4-depleted mice. *p<0.05 vs. IgG control. (B) Reperfusion caused a significant drop in total circulating leukocytes and lymphocytes in all antibody-treated mice. However, circulating neutrophils also increased after reperfusion, but this was significant only in IgG control mice. #p<0.05 vs. corresponding group in (A). WBC = total white blood cells.

In CD8- or CD4-depleted mice prior to ischemia, there were no significant changes in the number of total white blood cells, lymphocytes, neutrophils or monocytes (Fig. 1A). Two hrs after reperfusion (compared to pre-ischemia) there were significant reductions in total white blood cells (42%) and lymphocytes (70%) in CD8-depleted mice and 56% and 75% in CD4-depleted mice, respectively (p<0.05, Fig. 1B). Similar to what was observed in IgG isotype control mice, neutrophils were elevated in CD8- and CD4-depleted mice after reperfusion, but these levels were not significant (Fig. 1B). There were no differences in the level of hemoglobin and platelets among all antibody-treated mice before or after reperfusion (data not shown).

Changes in leukocyte numbers and BAL myeloperoxidase (MPO)

In sham and antibody-treated mice, leukocytes in peripheral (alveolar) lung tissue were semi-quantitatively evaluated in the left lung via immunohistochemistry. There were no significant differences in macrophage numbers between antibody-treated (after IRI) and sham mice (Table 1 and Fig. 2B bottom row). Both CD4+ and CD8+ T cells express CD3 antigen and thus immunohistochemistry using anti-CD3 antibody was used to assess combined CD4+ and CD8+ T cells. CD3+ T cells were significantly increased after IR in IgG control and neutrophil-depleted mice compared to sham (Table 1). CD3+ T cell counts were not altered in CD8-depleted mice but were significantly reduced in CD4-depleted mice (Table 1 and Fig. 2B middle row). Neutrophil numbers were significantly increased in IgG control, CD8- and CD4-depleted mice, but not in neutrophil-depleted mice compared to sham. However, neutrophil numbers were significantly lower in CD4-depleted mice compared to IgG control and CD8-depleted mice (Table 1 and Fig. 2B top row). MPO levels in BAL fluid, an indicator of neutrophil infiltration into alveolar airspace, showed no difference between sham, neutrophil-depleted and CD4-depleted mice, but was significantly increased in IgG control and CD8-depleted mice (Fig 2A).

Table 1.

Counts of leukocyte cell infiltration in peripheral lung tissue.

Groups
(n=3-5)		 Sham IgG control Neutrophil depleted CD8+ T cell depleted CD4+ T cell depleted
Neutrophils 4.2±0.7 30.8±2.7* 6.4±0.9φ 24.5±1.2* 17.5±1.7*#
CD3+ T cells 6.4±0.5 10.3±0.9* 9.4±0.7* 6.8±0.5 2.7±0.4φ
Macrophages 5.2±0.6 3.7±0.3 3.6±0.5 5.2±0.3 3.5±0.4
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*

p<0.05 vs. sham;

#

p<0.05 vs. IgG control and CD8 depletion;

φ

p<0.05 vs. all groups.

Figure 2.

Figure 2

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MPO levels and leukocyte subtype infiltration into peripheral lung tissue after reperfusion. (A) MPO levels in BAL fluid after reperfusion. *p<0.05 vs. sham. (B) Examples of immunohistochemical staining for neutrophils, CD3+ T cells, and macrophages in the study groups.

Pulmonary function during reperfusion in leukocyte-depleted mice

As expected, pulmonary function was significantly impaired in IgG control lungs after IR compared to sham (Fig. 3). AR was significantly increased, LC was significantly decreased, and PAP was significantly increased in IgG control lungs. All parameters of pulmonary function were partially but significantly improved in neutrophil-depleted and CD4-depleted mice when compared to IgG control (Fig. 3). No protection was observed in CD8-depleted mice.

Figure 3.

Figure 3

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Effect of leukocyte subtype depletion on pulmonary function after reperfusion. Airway resistance, lung compliance, and pulmonary artery pressure were significantly improved in neutrophil-depleted and CD4+ T cell-depleted mice. No protection was observed in CD8+ T cell-depleted mice. *p<0.05 vs. Sham, #p<0.05 vs. IgG control and CD8-depleted.

Pulmonary microvascular permeability

Evans blue content in left lung tissue was measured to assess pulmonary microvascular leak. As expected, there was significantly higher Evans blue content after IRI (IgG control) versus sham (Fig. 4A). Evans blue content was partially but significantly reduced in both neutrophil-depleted and CD4-depleted mice compared to IgG control or CD8-depleted mice (Fig 4A).

Figure 4.

Figure 4

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Microvascular permeability and pulmonary edema. (A) Evans blue content in lung tissue as an indicator of microvasular leak. In antibody-treated mice, Evans blue content was more significantly increased in IgG control and CD8-depleted mice than in neutrophil-depleted or CD4-depleted mice. (B) Lung wet/dry weight ratio, as an indicator of edema, was significantly increased in IgG control and CD8-depleted mice, but not in neutrophil-depleted or CD4-depleted mice (Fig 5B). *p<0.05 vs. sham, #p<0.05 vs. IgG control and CD8-depleted mice.

Lung wet/dry weight ratio

Lung wet/dry weight ratio, used as an indicator of edema, was significantly increased in IgG control and CD8-depleted mice versus sham. Importantly, wet/dry weight ratio was significantly reduced in neutrophil-and CD4-depleted mice (Fig 4B).

Changes in BAL cytokine/chemokine expression

CCL3 (MIP-1), TNF-α, IL-17 and CXCL1 (KC) levels in BAL fluid were significantly increased after IRI in IgG controls, neutrophil-depleted and CD8-depleted mice (Fig. 5). No significant induction of IFN-γ or IL-12 was observed after IRI (data not shown). Neutrophil depletion significantly reduced expression of IL-17. Depletion of CD4+ T cells resulted in significantly reduced CCL3, TNF-α, IL-17, and CXCL1 versus IgG control. Depletion of CD8+ T cells did not significantly alter cytokine/chemokine expression versus IgG control.

Figure 5.

Figure 5

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Expression of cytokines/chemokines in BAL fluid. CCL3 (MIP-1), TNF-α, IL-17 and CXCL1 (KC) were all significantly increased in IgG control, neutrophil-depleted and CD8-depleted mice compared to Sham. In CD4-depleted mice there was no significant difference in CCL3, IL-17, and CXCL1 compared to Sham. CXCL1 in CD4-depleted mice was significantly lower than all other antibody-treated mice. *p<0.05 vs. IgG control, #p<0.05 vs. CD8 depletion, †p<0.05 vs. neutrophil-depletion.

Discussion

The current study employed an in vivo mouse model to show that significant lung IRI occurs as early as 1 hr after left hilar ligation. Lung IRI was characterized by pulmonary dysfunction, edema, microvascular leak, and leukocyte infiltration. Lung IRI was significantly reduced in neutrophil- and CD4+ T cell-depleted mice, but not in CD8+ T cell-depleted mice. Circulating T cells were found to be decreased after reperfusion, possibly due to vascular margination or re-distribution to the lung or other tissues. Compared to sham mice, significant infiltration of CD3+ T cells occurred in control, neutrophil-depleted and CD8-depleted mice, but not in CD4-depleted mice. These results suggest that lung IRI is directly related to the level of infiltrating neutrophils, but not T cells. The leukocyte counts performed in this study did not distinguish among cells in various components of the lung (e.g. airspace, interstitial or marginated) and thus encompassed all peripheral (alveolar) lung tissue. The results demonstrate that pulmonary neutrophil infiltration is dependent on CD4+ T cells, but not vice versa. Cytokine/chemokine measurements revealed significantly increased CCL3 (MIP-1), TNF-α, IL-17 and CXCL1 (KC) production after reperfusion which were all significantly reduced by depletion of CD4+ T cells. Taken together, these results indicate that both neutrophils and CD4+ T cells contribute importantly to acute lung IRI.

Mouse model of lung IRI

Inflammatory responses during lung IRI play a critical role in early graft failure after lung transplantation1, 2, 4, 9, 11, 14, 18. Most in vivo lung IRI models entail ligation of the left hilum for 60-90 min followed by release of the ligature. Here, the mice are typically maintained on ventilation until some point during reperfusion, and thus ventilator-induced lung injury could have significantly contributed to these models19. The current study employed a model with similar surgical intervention, however the mechanical ventilation time was shortened to less than 20 min to minimize the potential of ventilator-induced lung injury. The absence of ventilator-induced injury is reflected in the current study by the sham animals which display minimal lung injury and well preserved lung function compared to the IR group. In addition, lung function is relatively stable throughout 2 hrs of continuous ventilation and perfusion in sham animals (data not shown) which would be expected to increase significantly over time if ventilator-induced injury was a significant component of this model.

Neutrophils are end-effectors during lung IRI

Studies have suggested that neutrophil accumulation appears to be the prime cellular mediator of pulmonary tissue destruction during IRI1-4. Lung IRI induces all the characteristics of an acute inflammatory response such as oxidative stress, activation of complement, macrophages and mast cells, elaboration of cytokines and chemokines, release of chemotactic factors, expression of cell adhesion molecules, neutrophil infiltration and pulmonary necrosis1, 3, 4, 6, 10, 20, 21. Compelling evidence from a variety of animal models and clinical studies indicates that neutrophils are the principle end-effectors of IRI3, 20, 21. Significant numbers of activated neutrophils accumulate in the lung after reperfusion as determined by tissue immunostaining10 or elevated activity of MPO20 which is found almost exclusively within neutrophils22. Neutrophils are generally believed to exacerbate tissue injury through the release of a variety of cytotoxic mediators such as reactive oxygen species and proteases23, 24. The current study is entirely consistent with a critical role of neutrophils in causing lung IRI. Over 80% of neutrophils were successfully depleted with anti-Gr-1 antibody. However, in contrast to other reports15, a significant number of circulating monocytes (80%) was also lost (Fig. 1A). The reduction in monocytes may have been due to use of intravenous injection rather than intraperitoneal injection used previously15. There was no significant reduction in lymphocytes. In the neutrophil-depleted mice, lung IRI was significantly reduced despite significant infiltration of CD3+ T cells. Furthermore, we found that protection from lung IRI correlated with a significant reduction in neutrophil infiltration but not other subtypes of leukocytes. These results demonstrate that activated neutrophils are end-effectors which directly cause lung IRI. Although T lymphocytes may have some direct toxic effects on pulmonary tissues, they predominantly appear to amplify an inflammatory response that is chemotactic to neutrophils.

CD4+ T lymphocytes mediate inflammatory responses during reperfusion

An increasing body of evidence has shown that T cells contribute importantly to lung IRI. T cells are activated and infiltrate into the lung during reperfusion earlier than neutrophils10. Inhibition of T cells before reperfusion has been shown to attenuate inflammation and decrease lung IRI20, 25. These data are consistent with the current results which suggest that CD4+ T cells, or a subset of these cells, are activated during lung IRI.

To identify the role of CD4+ T cells in lung IRI, we employed mice with antibody-induced depletion of either CD4+ or CD8+ T cells. After reperfusion, significantly less infiltration of CD3+ T cells were found in CD4- and CD8-depleted mice than in IgG control and neutrophil-depleted mice (Table 1). In sham mice there were higher numbers of peripheral lung T cells and macrophages than neutrophils. There was no significant change in the number of alveolar macrophages after 2 hrs reperfusion in all IRI groups; however, in the IgG control group, there was a further increase in the number of infiltrating neutrophils and T cells, with the change of neutrophils being greater (Table 1). This increase in T cell numbers may represent chemotaxis of circulating lymphocytes since circulating lymphocytes were significantly reduced during reperfusion. Although significant numbers of neutrophils are trapped (marginated or infiltrated) in the lungs, circulating neutrophil numbers were increased after reperfusion (statistical significance reached only in IgG control mice). The reason for the increase in circulating neutrophil numbers is not clear but possibly reflects mobilization from bone marrow in response to GM-CSF released by activated CD4+ T cells. Compared to CD8+ T cell-depleted mice, CD4+ T cell-depleted mice had significantly less infiltration of CD3+ T cells and neutrophils with correspondingly less lung IRI. However, neutrophil-depleted mice still exhibited increased numbers of CD3+ T cells as seen in IgG control mice. Thus neutrophils have no effect on T cell activation during lung IRI. Taken together, these results demonstrate that CD4+ T cells, not CD8+ T cells, are activated during lung IRI and play a critical role in amplifying an inflammatory response which culminates in the activation of neutrophils.

In lung IRI, CD4+ T cells could become activated through either antigen-independent or -dependent pathways. Antigen-independent mechanisms for T-cell activation have been described which involve IL-12, TNF-α, CCL2, CCL3, CCL5 and IFN-γ inducible protein-10 (IP-10)12, 13, 26. We found that CCL3, TNF-α, IL-17, and CXCL1 were significantly increased after IR in all antibody-treated mice except CD4-depleted mice. In CD4-depleted mice, production of IL-17 and CXCL1 were significantly lower than other antibody-treated mice after IR (except for IL-17 in neutrophil-depleted mice), indicating that CD4+ T cell activation precedes CXCL1 induction. These results are consistent with an antigen-independent pathway leading to CD4+ T cell activation during lung IRI. IL-17 activates alveolar macrophages and epithelial type II cells to induce CXCL1 which is a strong chemotactic mediator for neutrophil infiltrartion7, 27. Another possibility is that CD4+ T cells are activated directly by factors produced during reperfusion to release cytokines/chemokines that transactivate other leukocytes and epithelial cells.

Clinical impact

Identification of the initiatory signaling cascade via CD4+ T cells and macrophages will likely lead to specific pharmacological interventional targets for the amelioration of lung IR injury. For example, recent studies by our group have identified one such potential therapeutic agent to be the adenosine A2A receptor (A2AAR) which resides on leukocytes including CD4+ T cells and neutrophils. Here, we have shown that agonists which specifically activate A2AARs significantly reduce neutrophil infiltration and attenuate lung IR injury after transplantation4, 28. The possible protective role of A2AARs on CD4+ T cells in the setting of lung IR injury and the underlying molecular mechanisms are yet to be elucidated and are currently under investigation.

Conclusion

The current study suggests that sequential activation of CD4+ T lymphocytes and neutrophils occurs during lung IRI. CD4+ T cells accumulate in the lung during reperfusion but have little direct toxic effect. Instead, CD4+ T cells orchestrate the chemotaxis of circulating neutrophils to the lung. Activated neutrophils are end-effectors which carry out damage-producing tasks, while CD4+ T cells play a central role in mediating this injury process. The results underscore the importance of CD4+ T lymphocytes as mediators of lung IRI.

Acknowledgments

Funding sources: This study was funded by NIH/NHLBI grants RO1 HL077301 (VEL) and P01 HL073361 (JL).

Footnotes

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