Intravenous Immunoglobulins Reverse Acute Vaso-Occlusive Crises in Sickle Cell Mice through Rapid Inhibition of Neutrophil Adhesion.

Previous studies using intravital microscopy in a sickle cell disease (SCD) mouse model suggest that adherent leukocytes (WBCs) play a key role in vaso-occlusion by capturing circulating erythrocytes (RBCs) in venules. Commercial intravenous human gamma globulin (IVIG) given prior to the inflammatory stimuli increased microcirculatory blood flow and improved survival (Blood 2004 103(6):2397–400). To mimic the clinical situation where SCD patients seek medical attention after the onset of symptoms, we have developed an in vivo model in which the therapeutic intervention was administered after in the inflammatory challenge. Berkeley SCD bone marrow was transplanted into irradiated C57BL/6 to generate age- and gender-matched genetically identical cohorts of SCD mice. Fully engrafted male SCD mice were treated with TNF-α and prepared for intravital microscopy examination of the cremaster muscle. Seventy minutes following administration of TNF-α (0.5 μg i.p.), IVIG (800 mg/kg) or an equivalent volume of control PBS or albumin (800 mg/kg) was administered by a programmable syringe pump. Twenty minutes after IVIG, PBS, or albumin exposure, 8–10 venules were recorded over a period of 60 min with each venule recorded continuously for at least 3 min. IVIG significantly increased the number of rolling leukocytes (2–3 fold, p<0.05) compared to control groups treated with albumin or PBS. Moreover, further analyses of leukocyte behavior revealed that IVIG significantly increased rolling velocities, indicating that it alters adhesion pathways involved in slow rolling. In contrast, the number of WBCs adherent to the endothelium was reduced by ∼50% in the IVIG group (p<0.05). The effect of IVIG on leukocyte adhesion was rapid, being statistically significant in the first ∼10 min after completion of IVIG infusion. The number of adherent WBCs increased progressively over time in the PBS group but remained lower throughout the recording period in the IVIG group. To assess the effect of IVIG on sickle RBC adhesion, we quantified the interactions between circulating RBCs and adherent WBCs, and found that IVIG dramatically reduced the number of RBC-WBC interactions by 10–19 fold (p<0.05). These effects resulted in improved microcirculatory blood flow and Kaplan-Meier curves revealed a dramatic improvement in survival of sickle cell mice in the IVIG-treated sickle cell mice compared to control groups. Longer survival correlated positively with blood flow (p=0.003) and negatively with the number of adherent WBCs (p=0.01) and RBC-WBC interactions (p=0.05). In order to exclude the possibility that irradiation and graft-versus-host disease were confounding factors, we treated parental (non-transplanted) sickle mice with IVIG, and found similar results, although statistical significance was only reached for RBC-WBC interactions (p<0.001) due to the small number of mice. In addition, IVIG treatment reduced the number of adherent WBCs recruited in wild-type C57BL/6 mice (p<0.002). Using multichannel digital fluorescence videomicroscopy that we recently developed (Nat Methods. 2007 4:219–22), we found that IVIG affected specifically the recruitment of adherent neutrophils, but did not change the number of adherent monocytes or lymphocytes. Thus, these results suggest that IVIG may improve VOC by inhibiting neutrophil activation, and underscore the encouraging possibility that therapeutic interventions initiated when patients have an established crisis may change the outcome of acute episodes, preventing their progression into life-threatening complications.