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Anafilaksija je resna preobčutljivostna reakcija, ki nastane v minutah po ponovnem kontaktu z alergenom in lahko povzroči tudi smrt. Glavne efektorske celice anafilaksije so mastociti in bazofilci. Oboji na površini izražajo visoko afinitetni receptor za IgE (FcεRI). Nedavne raziskave so pokazale, da med anafilaksijo bazofilci migrirajo iz krvi v tkivo, pri čemer naj bi imel ključno vlogo kemokin CCL2. Namen naše raziskave je bil določiti vpliv anafilaktičnih faktorjev in CCL2 v serumih bolnikov na migracijo bazofilcev in vitro. V raziskavo smo vključili zdrave darovalce, pri katerih smo z negativno imunomagnetno selekcijo iz krvi izolirali bazofilce. V poskusih migracije smo uporabili parne vzorce serumov bolnikov med anafilaktično reakcijo in po njej. Migracija bazofilcev je potekala v prilagojenih Boydenovih komorah pri 37 °C in 5 % CO2. Za blokado CCL2 smo uporabili nevtralizacijsko protitelo anti-CCL2. Analiza števila in aktivacije bazofilcev je potekala na pretočnem citometru. Rezultati raziskave so pokazali, da je za proučevanje migracije bazofilcev, kjer spremljamo tudi njihovo aktivacijo, ustreznejša uporaba bazofilcev, izoliranih iz heparinizirane krvi kot iz krvi, zbrane v EDTA. Serum bolnikov z anafilaksijo vsebuje faktorje, ki vplivajo na povečano migracijo bazofilcev in vitro. Z modelom migracije bazofilcev proti serumu nismo uspeli potrditi, da inaktivacija CCL2 v serumu vpliva na znižano migracijo. Rekombinantni CCL2 (rCCL2) v raztopini vpliva na povečano migracijo bazofilcev, blokada rCCL2 z anti-CCL2 pa jo zmanjša. Potrebne so dodatne raziskave, ki bodo potrdile natančno vlogo in vir CCL2 pri anafilaksiji. Anaphylaxis is a severe, potentially life-threatening allergic reaction, which can occur within seconds or minutes of exposure to an allergen. Mast cells and basophils are believed to be the main effector cells of anaphylaxis. Both cells express high affinity IgE receptor (FcεRI) on their surface. Recent data suggest a substantial migration of circulating basophils during anaphylaxis, which correlates with a significant increase in serum concentrations of the chemokine CCL2. The aim of our study was to determine the effect of serum anaphylactic factors and CCL2 on migration of basophils in vitro. Basophils were isolated with negative immunomagnetic selection from peripheral whole blood of healthy donors. Paired serum samples of patients taken during and after anaphylaxis were used. Migration of basophils took place in adapted Boyden chambers at 37 °C and 5 % CO2. Anti-CCL2 neutralizing antibody was used to block CCL2. Analysis of the number and activation of basophils was carried out on a flow cytometer. Our results showed that there was a significant difference in the basophil activation in heparinized blood compared to EDTA blood. We showed that serum anaphylactic factors have impact on basophil migration, but we could not confirm that the blockade of CCL2 in serum results in reduced migration. We also confirmed that human recombinant CCL2 (rCCL2) in buffer solution promotes basophil migration, whereas anti-CCL2 treatment reduces it. Further studies are needed to elucidate the exact role and source of CCL2 during anaphylaxis. |
