J. Lande, V. Gimino, T. Berryman, R.A. King, and M.I. Hertz

Department of Medicine and Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, United States


Supported by NIH PPG 5PO1-AI50162-02



Background:  Lung and heart-lung transplantation are effective treatments for many diseases unresponsive to other therapy.  However, long term survival of recipients is limited by the development of bronchiolitis obliterans syndrome (BOS).  Acute lung rejection is common and is the leading risk factor for the development of BOS.  Despite many investigations there are no reliable biomarkers of AR.   We used microarray analysis to investigate the gene expression of bronchoalveolar lavage (BAL) cells to identify biomarkers for acute rejection.


Methods: Gene expression microarrays were performed using RNA from BAL cells of lung transplant recipients with acute rejection (n=7) or with no rejection (n=27).  BAL cell and differential counts were done.  T-tests were used to compare signal values between groups. The relationships of significant transcripts with the groups were visualized with 2D hierarchical clustering (Cluster, Treeview), and relationships of significant genes across biologic pathways (GenMAPP) were investigated. To evaluate variability in microarray preparation, a single BAL sample from one patient was equally divided at the time of acquisition and the two aliquots were processed identically (divided aliquots). To evaluate how BAL sampling location might affect microarray results, we obtained separate BAL samples from the right middle lobe and lingula of healthy bilateral single lung transplant recipient (paired samples).  The samples were collected and processed separately and in parallel.


Results: The microarray signal value data from the divided aliquots showed very high correlation (r2 > 0.99). The microarray signal value data from the paired samples showed very high correlation (r2 > 0.98). The BAL cell counts were similar between both rejection groups.

135 genes were upregulated in the acute rejection samples as compared to the no rejection samples.  These included acute rejection response genes, immune-response genes with unknown roles in acute rejection, and genes of unknown function.  Clustering grouped all acute rejection samples, and the majority of the no rejection samples.  The acute rejection samples displayed 6 GenMAPP pathways (including TGF-β signaling, inflammatory response, and apoptosis) with significant changes in expression.


Conclusions:  There exists very little variation inherent in the techniques required for producing microarray results from BAL cells.  There is very high correlation between gene expression patterns in BAL cells obtained from both lungs of a lung transplant recipient at a given time.  These quality control studies support the fact that microarray analysis can be used to produce reliable and reproducible results from BAL cells.

Microarray analysis is a powerful tool to identify candidate genes involved in acute rejection of the lung allograft.   The individual genes, distinct patterns of gene expression, or biologic pathways identified may represent novel biomarkers for acute rejection or for the development of BOS.