Sickle erythrocytes and platelets augment lung leukotriene synthesis with downregulation of anti-inflammatory proteins: relevance in the pathology of the acute chest syndrome

Abstract

Initiation, progression, and resolution of vaso-occlusive pain episodes in sickle cell disease (SCD) have been recognized as reperfusion injury, which provokes an inflammatory response in the pulmonary circulation. Some 5-lipoxygenase (5-lox) metabolites are potent vasoconstrictors in the pulmonary circulation. We studied stimulation of production of the inflammatory eicosanoids leukotrienes (LTs) and prostaglandin E2 (PGE2) by isolated rat lungs perfused with sickle (HbSS) erythrocytes. Our hypothesis is that HbSS erythrocytes produce more LTs than normal (HbAA) erythrocytes, which can induce vaso-occlusive episodes in SCD patients. Lung perfusates were collected at specific time points and purified by high-pressure liquid chromatography, and LTC4 and PGE2 contents were measured by enzyme-linked immunosorbent assay (ELISA). Rat lung explants were also cultured with purified HbAA and HbSS peptides, and 5-lox, cyclooxygenase 1/2, and platelet-activating factor receptor (PAFR) proteins were measured by Western blotting, while prostacyclin and LTs produced by cultured lung explants were measured by ELISA. Lung weight gain and blood gas data were not different among the groups. HbSS-perfused lungs produced more LTC4 and PGE2 than HbAA-perfused lungs: 10.40 ± 0.62 versus 0.92 ± 0.2 ng/g dry lung weight (mean ± SEM; P = 0.0001) for LTC4. Inclusion of autologous platelets (platelet-rich plasma) elevated LTC4 production to 12.6 ± 0.96 and 7 ± 0.60 ng/g dry lung weight in HbSS and HbAA perfusates, respectively. HbSS lungs also expressed more 5-lox and PAFR. The data suggest that HbSS erythrocytes and activated platelets in patient's pulmonary microcirculation will enhance the synthesis and release of the proinflammatory mediators LTC4 and PGE2, both of which may contribute to onset of the acute chest syndrome in SCD.

Keywords: 5-lipoxygenase; PAF receptor; cyclooxygenase; leukotriene receptors; prostaglandins; pulmonary circulation.

Figure 1

Scheme of leukotriene biosynthesis and physiological effects in the lung. Arachidonic acid released by action of phospholipase A₂ (PLA₂) is acted upon by 5-lipoxygenase (5-Lox) producing 5-HPETE (5-hydroperoxyeicosatetraenoic acid), which is metabolized to various leukotrienes (LT), including LTC₄, which binds to its CystLT₁ receptors to evoke some physiological responses, including inflammation and vasoconstriction. FLAP: 5-Lox activating protein; 5-HETE: 5-hydroxyeicosatetraenoic acid; CystLT: cysteinyl leukotriene.

Figure 2

Profile of leukotriene C₄ (LTC₄) produced by isolated rat lungs perfused with buffer alone or with erythrocytes, with metabolites measured at 15 minutes of perfusion. Three separate groups of lungs were perfused with buffer (GPBS; n = 6), normal erythrocytes (HbAA-RBC; n = 6), or sickle cell erythrocytes (HbSS-RBC; n = 12). Perfusates were collected as described in “Methods,” and the LTC₄ was extracted, purified by high-pressure liquid chromatography, and measured by enzyme-linked immunosorbent assay. Data are means + SEM. A single asterisk indicates significant (P < 0.05) difference from LTC₄ production in GPBS control; double asterisks indicate significant (P < 0.05) difference from both GPBS controls and the HbAA-RBC group. GPBS: phosphate-buffered saline containing 131 mM NaCl, 5.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 1% bovine serum albumin, and 8.3 mM glucose.

Figure 3

Profile of leukotriene C₄ (LTC₄) produced by isolated rat lungs perfused with GPBS or with HbAA (AA) or HbSS (SS) erythrocytes plus autologous platelets (Plts), measured at 0 and 15 minutes of perfusion. See Figure 2 for experimental details and other abbreviations. Data are means + SEM. An asterisk indicates significant (P < 0.05) difference from LTC₄ production in the same group at 0 minutes. A pound sign (#) indicates significant (P < 0.05) difference from LTC₄ production in all other groups.

Figure 4

Profile of leukotriene C₄ (LTC₄) produced by isolated rat lungs perfused with GPBS or with HbAA or HbSS erythrocytes plus autologous platelets (Plts or PRP [platelet-rich plasma]) for 15 minutes at the initial perfusion rate (open bars) and for 15 more minutes at twice that rate (filled bars). See Figure 2 for experimental details and other abbreviations. Data are means + SEM. A single asterisk indicates significant (P < 0.05) difference from LTC₄ production at the initial perfusion rate; double asterisks indicate significant (P < 0.05) difference from both GPBS controls and the HbAA group.

Figure 5

Profile of prostaglandin E₂ (PGE₂) produced by isolated rat lungs perfused with buffer alone or with erythrocytes, with metabolites measured at 15 minutes of perfusion. See Figure 2 for experimental details and abbreviations. Data are means + SEM. ND: none detected. An asterisk indicates significant (P < 0.05) difference from PGE₂ production in GPBS control.

Figure 6

Profile of prostaglandin E₂ (PGE₂) produced by isolated rat lungs perfused with GPBS or with HbAA (AA) or HbSS (SS) erythrocytes plus autologous platelets (Plts), measured at 0 and 15 minutes of perfusion. See Figure 2 for experimental details and other abbreviations. Data are means + SEM. A single asterisk indicates significant (P < 0.05) difference from PGE₂ production in the same group at 0 minutes; double asterisks indicate significant (P < 0.05) difference from PGE₂ production in all other groups.

Figure 7

Profile of prostaglandin E₂ (PGE₂) produced by the isolated rat lungs perfused with GPBS or with HbAA or HbSS erythrocytes for 15 minutes at the initial perfusion rate (open bars) and at twice that rate (filled bars). See Figure 2 for experimental details and other abbreviations. Data are means + SEM. A single asterisk indicates significant (P < 0.05) difference from PGE₂ production at the initial perfusion rate; double asterisks indicate significant (P < 0.05) difference from PGE₂ production in both GPBS controls and the HbAA group.

Figure 8

Expression of inflammatory protein 5-lipoxygenase (5-LOX) by rat lung explants stimulated in vitro with normal (HbAA) or sickle cell (HbSS) peptides. Adult rat lungs were prepared for studies as described in “Methods.” Proteins were prepared, and 5-LOX was measured by Western blotting. HbSS peptide increased expression of 5-LOX. Data are means + SEM. An asterisk indicates significant (P < 0.05) difference from Krebs buffer control.

Figure 9

Expression of inflammatory protein platelet-activating factor receptor (PAFR) by rat lung explants stimulated in vitro with normal (HbAA) or sickle cell (HbSS) peptides. Adult rat lungs were prepared for studies as described in “Methods.” Proteins were prepared, and PAFR was measured by Western blotting. HbSS peptide increased expression of PAFR. Data are means + SEM. An asterisk indicates significant (P < 0.05) difference from Krebs buffer control; a pound sign (#) indicates significant (P < 0.05) difference from effects of both Krebs buffer control and HbAA peptide.

Figure 10

Expression of cyclooxygenase 1 (COX1) protein by adult rat lungs stimulated in vitro with normal (HbAA) or sickle cell (HbSS) peptides. Adult rat lungs were prepared for studies as described in “Methods.” Proteins were prepared, and COX1 expression was measured by Western blotting. There was no significant difference in COX1 expression among the 3 groups of lung explants. “Krebs” denotes the Krebs buffer control. Data are means + SEM.

Figure 11

Expression of cyclooxygenase 2 (COX2) protein by adult rat lungs stimulated in vitro with normal (HbAA) or sickle cell (HbSS) peptides. Adult rat lungs were prepared for studies as described in “Methods.” Proteins were prepared, and COX2 expression was measured by Western blotting. HbSS peptide increased expression of COX2 protein. Data are means + SEM. An asterisk indicates significant (P < 0.05) difference from effects of both Krebs buffer control and HbAA peptide.

Figure 12

Amount of 6-keto-PGF_1α measured from lung explants cultured with normal (HbAA) or sickle cell (HbSS) peptides. HbSS peptide stimulated release of 6-keto-PGF_1α at a level comparable to effect of A23187 and higher than the effect of HbAA peptide or Krebs buffer control. Data are means + SEM. ND: none detected. An asterisk indicates release significantly (P < 0.05) greater than that for Krebs control; a pound sign (#) indicates release significantly (P < 0.05) greater than that for both HbAA peptide and Krebs control.

Figure 13

Amount of leukotrienes measured from lung explants cultured with normal (HbAA) or sickle cell (HbSS) peptides. HbSS peptide stimulated release of leukotrienes at a level comparable to effect of A23187 and higher than the effect of HbAA peptide or Krebs buffer control. Data are means + SEM. A single asterisk indicates release significantly (P < 0.05) greater than that for Krebs control; double asterisks indicate release significantly (P < 0.05) different from that under all other treatments; a pound sign (#) indicates release significantly (P < 0.05) greater than that for both HbAA peptide and Krebs control.