Red blood cells induce hypoxic lung inflammation

Abstract

Hypoxia, which commonly associates with respiratory and cardiovascular diseases, provokes an acute inflammatory response. However, underlying mechanisms are not well understood. Here we report that red blood cells (RBCs) induce hypoxic inflammation by producing reactive oxygen species (ROS) that diffuse to endothelial cells of adjoining blood vessels. Real-time fluorescence imaging of rat and mouse lungs revealed that in the presence of RBC-containing vascular perfusion, hypoxia increased microvascular ROS, and cytosolic Ca(2+), leading to P-selectin-dependent leukocyte recruitment. However, in the presence of RBC-free perfusion, all hypoxia-induced responses were completely inhibited. Because hemoglobin (Hb) autoxidation causes RBC superoxide formation that readily dismutates to H(2)O(2), hypoxia-induced responses were lost when we inhibited Hb autoxidation with CO or nitrite, or when the H(2)O(2) inhibitor, catalase was added to the infusion to neutralize the RBC-derived ROS. By contrast, perfusion with RBCs from BERK-trait mice that are more susceptible to Hb autoxidation and to hypoxia-induced superoxide production enhanced the hypoxia-induced responses. We conclude that in hypoxia, increased Hb autoxidation augments superoxide production in RBCs. Consequently, RBCs release H(2)O(2) that diffuses to the lung microvascular endothelium, thereby initiating Ca(2+)-dependent leukocyte recruitment. These findings are the first evidence that RBCs contribute to hypoxia-induced inflammation.

Figure 1

Endothelial ROS in lung microvessels in situ. (A,B) Color-coded endothelial DCF fluorescence in venules (V) and septal capillaries (S) under normoxic (left) and hypoxic (right) conditions. Vessel margins are depicted (white lines). The alveolar septum is indicated (arrows). Venules were given RBC-containing perfusion at indicated hematocrits. (C) Tracings of DCF fluorescence intensity from an identical endothelial cell at baseline and hypoxic conditions. Perfusate was RBC-free or RBC-containing (hematocrit, 20%) with or without catalase (50 U/mL) replicated 5 times. (D) Hematocrit (Hct) dependency of endothelial DCF fluorescence. Data obtained after 30 minutes of hypoxia are mean plus or minus SE (n = 4 each point; *P < .05 vs hematocrit of 20%). (E) Group data are for hypoxia-induced responses in venules given RBC-containing perfusions (hematocrit, 20%). The unfilled bar corresponds to pO₂ of 40 mmHg attained after 17 minutes of hypoxia. The bar immediately to the right corresponds to pO₂ of 21 mmHg at 30 minutes of hypoxia exposure. For the other bars, RBCs were perfused together with catalase (50 U/mL) or RBCs were pretreated with the indicated agents (L-NAME, 250 μM; azide, 1 mM; nitrite, 5 mM). Data are mean plus or minus SE; n = 5 for each bar (*P < .05 vs untreated RBC perfusion at pO₂ of 21 mmHg, hematocrit of 20%). (F) Data are for venules after 30 minutes of hypoxia. RBC indicates RBCs from wild-type (WT) and BERK-trait (BT) mice were perfused (hematocrit, 20%) in WT and BT lungs as indicated. Catalase (50 U/mL) was included in the perfusion as indicated. Data are mean plus or minus SE; n = 5 for each group (*P < .05 vs first bar; §P < .05 vs 3rd bar).

Figure 2

Heme degradation products in rat RBCs. Fluorescence of heme degradation products is shown for Hb incubated (12 hours, 37°C) at pO₂ of 100 (normoxic) or 21 (hypoxic) mmHg (n = 4; A), Hb of RBC perfused through lungs inflated under normoxic and hypoxic conditions (n = 4; B), and Hb of nontreated and azide- (C) or cyanate- (D) treated RBCs. The endothelial DCF response is also shown for cyanate-treated RBCs (E) and perfused through lungs inflated under normoxic and hypoxic conditions (n = 3). Data are mean plus or minus SE (#P < .05 vs baseline [panels A,B, 1st bar]; *P < .05 vs hypoxic RBC [panel C, 1st bar]; **P < .05 vs hypoxic RBC [panel E, 1st bar]).

Figure 3

Endothelial Ca²⁺cyt. Data are mean plus or minus SE; n = 5 for each bar (*P < .05 vs first bar). (A,B) Pseudocolor-coded 340:380 ratio for fura 2-loaded capillary endothelial cells under baseline (left) and hypoxic (right) conditions. Vessel margins are depicted (white lines). Alveolar septum is indicated (arrows). Venules are perfused RBC-containing or RBC-free solutions as indicated. (C) Tracings show time-dependent changes in the presence or absence of RBCs in vascular perfusion replicated 6 times. (D) Hematocrit (Hct) dependency of endothelial Ca²⁺ after 30 minutes of hypoxia exposure. *P < .05 vs HCT: 20%. (E) Bars represent venular group data for indicated perfusion conditions 30 minutes after hypoxic lung inflation. RBC indicates hematocrit = 20%; catalase, 50 U/mL; Ca²⁺-free, perfusion was Ca²⁺ depleted. (F) Bars represent RBC-containing perfusions under control and Ca²⁺-free conditions.

Figure 4

DAF fluorescence showing endothelial NO production in lung venular capillaries. (A) Capillaries loaded with DAF-FM. Images were obtained under normoxic conditions (left) and 0 (middle) and 30 (right) minutes after switching to hypoxia. (B) Tracings of DAF-FM fluorescence in a single endothelial cell in a venular capillary that was perfused at either 0% (gray) or 20% (black) hematocrit. (C) Group data show DAF-FM fluorescence as ratio of initial (0 minutes). Data are mean plus or minus SE; n = 4 each bar.

Figure 5

Leukocyte-endothelial cell interactions in lung capillaries. (A,B) Images show rhodamine-6G–labeled leukocytes (white dots) in venular capillaries margins of which are outlined (white lines). At hematocrit of 20%, whereas few leukocytes adhere to the venular endothelium under baseline conditions (top left), leukocyte attachment to the endothelium is greater after 30 minutes of hypoxia (top right). By contrast, at hematocrit of 0% hypoxia causes no leukocyte adhesion (bottom right). (C-K) Group data show leukocyte rolling (WBCrol/min) and sticking (WBCstick/min) rates and RBC velocity (k) for normoxia (baseline) and after 30 minutes of hypoxia (hypoxia) under the indicated conditions. Vessels were perfused at 20% hematocrit except as indicated. anti–P-sel indicates P-selectin mAb. #P < .05 vs baseline (panels C,D,I,J); *P < .05 versus WT-RBC in WT-lung (1st bar, panels G,H).