The Effect Of Peripheral Leukocytapheresis On Endotoxemia-induced Lung Injury In Dog And The Mechanism Research

Acute lung injury (ALI) and its serious form, acute respiratory distress syndrome(ARDS) is a secondary inflammory lung injury characterized with obstinatehyppoxygenation induced by effect of cascade amplification involved multiple inflammorymedium and effector cells. ALI/ARDS is characterized by increased permeability of thealveolar-capillary barrier, resulting in an influx of protein-rich edematous fluid and aconsequent impairment in arterial oxygenation. The mortality of ALI/ARDS remains highin spite of sophisticated intensive care at high cost and improved strategies in preventionand treatments. Efforts for prevention and treatments have shown little improvement overthe past decades. Thus, there is an urgent need to explore potential novel preventative andtherapeutic strategies for patients with, or at high risk of developing, ALI/ARDS. It is stillone of the hottest and most difficult area of clinical critical care medicine research. It isgenerally accepted that most forms of ALI involve lung neutrophil entrapment andactivation, as well as neutrophil-mediated pulmonary injury. Various proteinaceousinflammatory mediators either directly or indirectly induce lung injury through activation ofneutrophils and induction of cytotoxic molecules. This is especially true of the serineprotease neutrophil elastase (NE) and oxidants produced by neutrophils in pulmonary andcirculating pools. Leukocytosis, a condition characterized by an elevated number ofleukocytes, especially neutrophils in the blood, is the prominent feature of bacteritic sepsis.In patients with sepsis-related ALI, significantly higher neutrophil counts in thebronchoalveolar lavage fluid (BALF) were consistently noted in non-survivors as comparedto survivors. It is well known that neutrophils in the BALF or lung tissue are recruited fromperipheral blood during the development of lung injury. Thus, inhibition and partialelimination of peripheral neutrophils may be a potential prophylactic or treatment strategyfor endotoxin-induced ALI/ARDS. Several in vivo studies have demonstrated thatinhibition of peripheral neutrophils by leukocytotoxic medicines or antibodies could attenuate neutrophil-mediated lung injury. One report showed similar result from removalof peripheral macrophages by physical method. While the interventions with chemicals orantibodies targeting at all neutrophils appeared to reduce inflammation, most of theseresults were from small rodents. Leukocytapheresis is a technology which could removeleukocytes by using specific devices in vivo or in vitro state to alleviate the harmful lesion.We hypothesize that partial removal of peripheral leucocytes, in particular neutrophils inblood in endotoxemia dogs, could alleviate the inflammatory damage of lung and acutelung injury caused by white blood cells in endotoxemia animal, and explore new preventiveand therapeutic strategy.Objective The present study utilized leukocytapheresis(LCAP) to investigate theeffect of decreased peripheral neutrophils in preventing the endotoxemia animals fromdeveloping into ALI/ARDS in our endotoxemia dog model, and explore the underlyingmechanisms.Methods A total of24healthy male mongrel dogs(weighing15-20kg) were enrolledand randomly divided into LPS, Sham LCAP and LCAP groups(8per group). Followinganesthesia, monitoring, ventilation and vessel preparation, animals in each group wereinjected with LPS (2mg/kg; E. coli O55:B5) to induce endotoxemia. LCAP was performedby an automated continuous-flow blood cell separator16h following the LPSadministration and stable hemodynamics was acquired. The mononuclear cell program wasselected to separate peripheral leucocytes. The separator automatically generated theparameters blood flow rate per minute, total separated cycles and blood volume accordingto the animal's gender, body weight, height, peripheral leucocyte count, hematocrit leveland the targeted value of the peripheral leucocyte count. The parameters would be adjustedif needed. The counts of leukocytes and neutrophils in the periphery and in the collectedstorage bag were sampled to estimate the efficiency of separation during the LCAP process.The sham LCAP group underwent extracorporeal circulation and other procedures asdescribed in the LCAP groups without removal of the leucocytes, but were carried out bycontinuous reinfusion of the collection in the storage bag containing separated leucocytes.The time point16h selected for LCAP was based on our preliminary data (not shown) thatthe peripheral leucocyte count recuperated to the basal value about16h following the LPSchallenge. The8.0×109/L served as a targeted peripheral WBC count was based on the lower limitation of normal WBC count (8.36to16.4×10~9/L)(10healthy dogs, data notshown). For hemodynamic stability, limited liquid infusion or vasoconstriction agents weregiven.The baseline levels of leucocytes and neutrophils in blood, myeloperoxidase(MPO)and neutrophil elastase (NE) in serum, arterial blood gas was detected, and repeated thedetections at0h,2h,6h,12h,14h,16h,24h and36h after administeration of LPS, theperipheral blood collected were centrifuged10min (3000r/min) at4℃immediately. Thesupernatant was stored at-80℃freezer for following detection of the level of NE andMPO by method of enzyme-linked immunosorbent assay (ELISA). At36h afteradministration of LPS, the right middle lobe lungs were harvested immediately foracquirement of BALF for detection of NE The right superior lobe lung was harvested andfixed in4%paraformaldehyde in PBS and2.5%glutaraldehyde for pathology observationfollowing hematoxylin and eosin stain under optical microscope and observation ofultrastructural pathology under electron microscope. The apoptosis of lung parenchyma wasdetected by method of TUNEL. The water content was expressed by the wet/dry ratio oflung tissue. And the nuclear factor-kappa B (NF-κB) subunit p65and tumor necrosis factoralpha (TNF-α) in lung tissues were measured by methods of Western Blot. Statisticalanalyses: All values are expressed as the mean±standard error (SE). The data wereanalyzed with Statistical Products and Service Solutions (SPSS)13.0for Windows software.Comparisons among the groups at the corresponding time points were performed usingStudent's t-test. The average levels of leukocytes, PMNs and NE were calculated and thecomparisons among groups were performed by one-way ANOVA. Incidences of ALI ingroups were analyzed with the chi-square test. Probability (P)-values less than0.05(P <0.05) were considered statistically significant.ResultsIncidence of ALIDiagnoses for ALI and ARDS followed the criteria set by the American-EuropeanConsensus Conference of1994. Accordingly,7animals in each of the LPS and shamgroups, and3in the LCAP group, developed ALI (P<0.05, LCAP vs LPS and shamgroups). No ARDS was found in any of the groups.The numbers of leucocytes and neutrophils in periphery and lung The counts of leucocytes and neutrophils in the periphery decreased rapidly followingthe injection of LPS, then increased slowly and returned to basal values about16hpost-injection, and afterward gradually increased further. The counts of peripheralleucocytes and neutrophils in the LCAP group were significantly reduced compared to theLPS or sham groups at20h(P <0.01) and24h(P <0.05). The average count of WBC andPMN in peripheral blood in the LCAP group was statistically lower than in the LPS or thesham-LCAP groups(P <0.05).There are no statistical differences among counts of neutrophils in lung tissues in threegroups, observated under the optical microscope.General pathological and apoptotic changes in lung parenchymaLung interstitial edema, hyperemia, and neutrophil detention in microvascularexudation in lung interstitial and alveolar spaces were found in all groups after the LPSchallenge. However, relatively minor abnormal lesions were observed in the LCAP group.The numbers of apoptotic lung parenchyma cells in the LCAP group were significantlylower compared to that in the LPS and the sham-LCAP groups (P <0.05).The ultrastructural injuries of lung tissues.Observation by Transmission Electron Microscope, the alveolar septa, capillaryendothelium and basilemma occurred in all groups. vacuolation, osmiophilic granulesdecrease in type II alveolar epithelial cell, the outline of the type II cell remains clear,injuries were relatively mild in group LCAP, Cell necrosis and osmiophilie granulesdisappear and the lamellar bodies appear discrimination, showed relatively serious lungparenchyma injuries in LPS and sham group.The wet/dry ratio of lung, protein content in BALFThe wet/dry ratio of lung and the protein content in BALF were significantly lowerthan in the LPS and sham groups(p<0.05), also the apoptotic cells in lung parenchyma(/HP)were significantly lower than in the LPS and sham groups.Additionally, the average levels of PMN and NE in blood in animals without ALI werealso lower than in animals with ALI (p<0.05).The level of NE in the serum following endtoxemia and LCAPThe NE levels in serum increased continuously since LPS administration, LCAP didn'tdecrease the absolute level of NE but clearly slowed down the ascending trend and achieved maximal efficacy about4h following LCAP. The NE levels in the serum at20hand24h in group LCAP were significantly lower than in LPS or sham groups at20h(p<0.01)and24h(p<0.05); Also the mean NE level of all time points in LCAP group wassignificantly lower than in the LPS and sham groups. While the NE levels in BALF weren'tsignificantly different among groups.The level of NF-κB p65and TNF-α in lung tissuesThe NF-κB p65expression in lung tissues in LCAP group were significantly lowerthan in the LPS and sham groups (p<0.05). While the TNF-α in the lung tissues amonggroups have no statistical differences (p>0.05).The correlation analysis among PMN, NE and PaO2/FiO2The mean counts of PMN in periphery are significantly correlated with mean NElevels in serum. Both the mean levels of PMN and NE in peripheral blood were inverselycorrelated with oxygenation indices at36h (p<0.01). However, the numbers of PMNs andthe levels of NE in BALF, and the PMNs, MDA and TNF-α in lung tissues, had nosignificant differences among groups (p>0.05)(data not shown).ConclusionsThe mongrel dog is relatively satisfactory animal in endotoxemia-associatedALI/ARDS model. The physique of dog is relatively big, and the physiological parametersof blood cell and the values of artorial blood gas are similar with those of human, thediagnose standard of ALI/ARDS could adopt the clinical one. The period is relatively longfor the appearance of ALI/ARD following the LPS administration, which is also similarwith the course of human. More importantly, different from small animals, the dog couldaccept supportive therapies after appearance of ALI/ARDS, thus live a relative long-termperiod, and replicate the complex features of this human disease more factually. Themonitoring of hemodynamic, respiratory support, management of airway and other nursingare essential during this process. The results suggested the counts of WBC and PMNdecreased rapidly following the administration of LPS, and increased slowly6-8hrsfollowing the LPS challenged, while returned to the baseline at16hrs or so after the LPSadministration. The counts would increase slowly from then on. We demonstrated that thetargeted WBC count in periphery and time point for LCAP in our research could decreasethe accidence of ALI. Our results also suggested that leukocytapheresis significantly improved neutrophilia and subsequently attenuate lung injuries partly through a mechanismof decreasing the level of invasive inflammatory mediators in serum and lung tissue,especially the neutrophil elastase in serum. The decrease of PMN in peripheral bloodfollowing LCAP may reduce the expression of NF-κB p65in lung parenchyma, which inturn lead to down-regulation of the inflammation in lung tissue and alleviate the lung injury.Our research demonstrated that LCAP seem a useful and potential preventive andtherapeutic strategy in endotoxemia-associated ALI/ARDS.