Methacholine Challenge Test Reveals a High Prevalence of Subclinical Severe Airway Hyper-Reactivity in Children and Yound Adults with Sickle Cell Disease Which Correlates with Higher Sickle Acute Events and Acute Chest

Background: Although increase prevalence of airway hyper-reactivity (AHR) has been detected in children, and even adults with sickle cell disease (SCD) using methacholine provocative challenge test (MCT), AHR was not been found to be associated with clinical asthma.Hence, the clinical implications of AHR in SCD are unknown. Furthermore, AHR studies in SCD patients using MCT lack comparisons with a control population. We recently showed that increased placenta growth factor (PlGF) levels in sickle mice causes AHR by upregulating leukotriene synthesis and inflammation. We therefore designed a prospective study to determine the prevalence of AHR in children and young adults with SCD, and in unaffected siblings living in the same household as a control population, and also determine the biochemical and clinical implications of AHR in SCD.

Methods: A total of 67 patients with SCD and 14 unaffected siblings of the patients enrolled on to the study for determining baseline AHR (ClinicalTrials.gov Identifier: NCT00448370). Chief eligibility criteria included a diagnosis of SCD (HbSS, HbSC or HbS b-thalassemia) confirmed by hemoglobin electrophoresis or gene sequencing, ages 5.5 years to 30 years who were able to complete pulmonary function testing and were at baseline/steady state (no acute sickle events 3 weeks prior or acute chest syndrome (ACS) 4 weeks prior). Excluded were patients taking anti-leukotriene medications within 30 days prior, although patients with SCD who had a known history of asthma but not on leukotriene inhibitors were eligible. Patients with chronic transfusions, congenital lung or heart condition, or other medical condition that could affect the pulmonary function testing were also ineligible. Unaffected siblings of the 67 enrolled SCD patients living in the same household were offered enrollment for MCT and those consenting were enrolled.

Results: 64 SCD patients and 14 siblings underwent a MCT; 3 of 67 SCD patients had FEV1<70% of predicted at baseline, and therefore, for safety reasons, were not subjected to MCT. The degree of AHR was defined by the provocative concentration of methacholine that resulted in a 20% decline in FEV1 (PC20): no AHR=PC20 >16mg/mL; borderline AHR=PC20 4-16mg/mL; mild AHR=PC20 1-3mg/mL and moderate-severe AHR=PC20 <1mg/mL. All subjects were given a dose of albuterol after completion of MCT. MCT was safe in all subjects, and no AHR or acute sickle events occurred in any subject for three weeks after the MCT. Overall, 25 of 67 (37%) SCD patients had no AHR, 8 of 67 (12%) had borderline AHR (PC20 9±1.4mg/ml), 10 of 67 (15%) had mild AHR (PC20 1.72 ±0.16) and 24 of 67 (36%) had moderate-severe AHR (PC20 0.6 ±0.06). Of unaffected siblings, 10 of 14 (71%) had no AHR, 2 of 14 (14%) had borderline AHR (PC20 10±0.4) and 2 of 14 (14%) had mild AHR (PC20 1.7±0.4). Moderate to severe AHR was only seen in SCD patients and not sibling controls. Severe AHR prevalence was highest in the HbSS group. We analyzed the clinical characteristics of the SCD subjects, including acute sickle episodes, defined as admission for one of the following: pain crises, fever with or without a known source of infection, cough/wheezing/chest pain, impending acute chest syndrome, enlarged and splenic sequestration or priapism. SCD patients with AHR had significantly higher (P<0.05) admissions from acute sickle episodes (2.7±0.1) and acute chest syndrome (1.5±0.15). We next assessed PlGF and leukotriene levels in SCD subjects. PlGF levels were not quantifiable due to presence of hemolysis, which confounds PlGF ELISA. However, we found a trend towards higher (1.3-2 fold) expression of PlGF-induced target genes (VEGF, IL-1β, IL-8, CCL2) in the mononuclear cells of patients with severe AHR; Also, circulating cysteinyl leukotriene D4 levels in their plasma were significantly higher.

Conclusions: There was a higher prevalence of mild to severe AHR (51%) in patients with SCD, compared to a 14% prevalence of mild AHR in siblings living in the same household. The higher degree of AHR was associated with increased SCD morbidity - increased rates of admission from ACS and pain crisis, when compared to those with no AHR. AHR was associated with higher leukotrienes and a trend towards higher PlGF target gene induction. Together, our data suggests that AHR may be a biomarker for SCD morbidity, and may also be a therapeutic target, since blockade of PlGF or leukotriene synthesis in patients with severe AHR may reduce SCD morbidity.