| Objective Not only plays the role in clinical treatment, allogeneic blood transfusion(ABT) also has many immune-related regulatory functions. Transfusion-relatedimmunomodulation (TRIM) is the syndrome of immune system changed by bloodtransfusion, which characterized by immunosuppression. However,the exact mechanismunderlying TRIM is not clear. The most severe consequences of transfusion relatedimmune suppression in clinical setting are post-operation infection and recurrence,metastasis of tumor.On the one side, the biological active factors that were transfused may be thereasons of the adverse TRIM, like TRALI(Transfusion-related acute lung injury) andSIRS(systemic inflammatory response syndrome).At present, due to the complexityof clinical blood transfusion, the reports about the occurrence of TRIM remaincontroversial. More fundamental studies are needed to exploit the inflammatoryresponse of recipients caused by the RBCs transfusion.The focus of our study is on the mechanism underlying the inflammation causedby red blood cell transfusions, which has not been elucidated. Present study showsthat all stimuli signals must bind their receptors on the target cells membrane and playbiological effects through the specific signal transduction pathways. The nuclearfactor KB (nuclear factor kappa B, NF-KB), as the hub of multiple signal transductionpathways, is the center and common downstream pathways of inflammatorystimulations. In quiescent state, NF-KB complex binds with inhibitory factor IKB andkeeps the non-active state. Upon different stimuli, upstream signals can causephosphorylation and ubiquitination of IKB, and resulted in its degradation by theproteasome. Afterward, NF-KB is released and transferred into nuclear, and triggerstarget genes’ transcription, in which shows that the degradation of IKB andtransference into nuclear of NF-KB are the key step of NF-KB activation. SN50, theinhibitor of NF-KB, can specifically block NF-KB transfering from cytoplasm intonuclear, and inhibit the ability of NF-KB binding DNA. In our study, NF-KB signalpathway involved in the inflammatory response in recipients caused by RBCtransfusion is determined by detecting the changes at IKB-a protein level and NF-KB specific blocking with SN50.On the other hand, NK cells can directly kill tumor cells and pathogens infectedcells, and play an important role in immune responses and inflammatory.Currentstudies from treatment of solid tumor showed that alloreactive natural killer (NK)cells display higher killing capacity than self or compatible NK cells. Thus, it isplausible that activation of alloreactive NK cell response during blood transfusion is akey step to prevent and reverse transfusion-associatedimmunomodulation.Usually,Killer cell immunoglobulin-like receptors, KIR, whichlocated on NK cells surface, can specifically recognize and bind HLA-I molecule onthe surface of target cells, transfer inhibitory or activate signal into cells and decidealloreactive NK cells. There are two types of KIR, inhibitory and activated. Both ofthem can bind with specific ligands and mediate inhibitory and activated response,respectively. When the inhibitory receptor from donor NK cells can not be bindedwith HLA-I ligand or be incompatible with HLA-I molecule from recipient, as well assimultaneously HLA-I antigen from donor and recipient can not be matched, therewill give rise to an anti-leukemia alloreactive NK cells. Just as convenient as bloodtype cross matching test, to detect the match degree of donor KIR and recipientHLA-I, utilization of NK cells allogeneic activity can provide new insight forpreventing and reversing transfusion-related immunosuppression.MethodsPart 1:1 .The prepared RBC suspension (CPDA is the RBC maintenance solution) wasstored at 4 0C .Then 30m1 blood was taken out under sterile conditions at the indicatedtime points。、1 St day, 21 St day and 3 5 th day (D 1，D21 and D35) respectively, andcentrifuged at 1500 rpm for 20min. The supernatant was centrifuged at 12500 rpmagain for Smin and cells were removed. At last, the supernatant was aliquoted andstored at -20 0C.2. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolatedfreshly, cultured at the density of 2X106 cells per well in 24-well plate in complete1640 medium with a final volume of 1.5m1. PBMCs were divided into two groups asfollows. First, the non-LPS stimulation group, in brie仁supernatant of RBCs from Dl，D21 and D35 was added to the cultured PBMCs respectively with the volume equal to20% (V/V) of the total volume and the cell culture supernatants were collected after48hrs incubation and frozen for detection. Second, LPS stimulation group, briefly, supernatant of RBCs from Dl，D21 and D35 was added to the cultured PBMCsrespectively with the volume equal to 20% (V/V) of the total volume and a finalconcentration of 100ng/ml of LPS was added after 24hrs incubation, then cultured foranother 24hrs to collect cell culture supernatants and frozen for detection. Blankcontrols were set up in parallel with both groups (treating in the same way as testgroups but without RBCs supernatant).3 .Concentration of free hemoglobin,K+,Ca2+,Na+and Cl in 10 RBC supernatantsof D 1，D21 and D35 were detected respectively, including 7 samples for cell culture.4. The concentration of IL-1p，IL-6, IL-10, IL-17, TNF-a, TGF一p and GM-CSFd insupernatants were detected by ELISA.Part 2:1 .Cell culture was set up according to the method described in Part 1，and the cellswere collected after 48hrs incubation. Thereafter, IKB一a protein levels were detectedby Western blot.2. TNF-a concentration in cell culture supernatant was detected after treated withSN50, a specific inhibitor of NF-KB. Cells were divided into 4 groups as follows: firstLPS group; second, SN50 group;third, SN50 pretreatment group. Blank controlswere set up in parallel with each groups ((treating in the same way as test groups butwithout RBCs supernatant)Part 3:KIR was typed by PCR-SSP, patient cases were divided into groups by matchingdegrees of KIR from donor with HLA-I molecule from recipient, and retrospectivecase-control study was carried out. 53 cases were collected in leukemia group and 27in solid tumor group. Peripheral blood was collected prior to transfusion and within 5days after transfusion, respectively, the percentage of CD3+, CD3+CD4+, CD3+CD8+,CD19+ and CD3-CD56+ subgroup cells were detected by flow cytometry and theconcentration of TNF一a, IFN-y were detected by ELISA.Results1 The content of FHb, K+, Ca2+ in RBC suspension supernatants increased graduallyover time of the storage (P<0.05), whereas Na+ decreased(P<0.05). All detectedmarkers were within the normal range of the national blood quality standards.2. The RBC suspension supernatant of D35 can promote the secretion of TNF一a andIL-6 from PBMCs induced by LPS(P<0.05), whereas TGF一pwas inhibited (P<0.05)3 .Along with the storage time extension of RBC suspension supernatants added， he IKB一a degradation of PBMC increased under LPS induction.4. Compared to LPS, the introduction of SN50 can decrease the concentration ofTNF一a secreted by PBMCs护<0.05).5. The concentration of IFN-y in patient’s serum increased with the mismatcheddegree of KIR and HLA-I molecule护<0.05). After receiving blood transfusion, thechanges of IFN-y concentration in patients’ serum were significantly correlated to thechanges of NK cells.Conclusions1 .With the time extension of RBC storage, blood transfusion would increase theinflammatory response in recipients. The result layed a theoretical basis for furtherelucidating the mechanism underlying TRIM in patients with severe trauma afterblood transfusion.2. The gradually increase of IKB-a degradation indicated that RBCs supernatant canpromote the effect of NF-KB activation induced by LPS, whereas SN50 inhibit thateffect. The result suggested that NF-KB signaling pathway was involved in enhancingthe inflammatory response in blood transfusion recipients induced by LPS.3 .The changes of cancer patients’ immune response after blood transfusion wascorrelated with the mismatch degree of KIR from donor with HLA-I molecule fromrecipient, which provided a theoretical basis for reversing transfusion-associatedimmunosuppression using allogeneic NK cells... |
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