Adaptation to chronic hypoxia involves immune cell invasion and increased expression of inflammatory cytokines in rat carotid body

Abstract

Exposure to chronic hypoxia (CH; 3-28 days at 380 Torr) induces adaptation in mammalian carotid body such that following CH an acute hypoxic challenge elicits an abnormally large increase in carotid sinus nerve impulse activity. The current study examines the hypothesis that CH initiates an immune response in the carotid body and that chemoreceptor hyperexcitability is dependent on the expression and action of inflammatory cytokines. CH resulted in a robust invasion of ED1(+) macrophages, which peaked on day 3 of exposure. Gene expression of proinflammatory cytokines, IL-1beta, TNFalpha, and the chemokine, monocyte chemoattractant protein-1, was increased >2-fold after 1 day of hypoxia followed by a >2-fold increase in IL-6 on day 3. After 28 days of CH, IL-6 remained elevated >5-fold, whereas expression of other cytokines recovered to normal levels. Cytokine expression was not restricted to immune cells. Studies of cultured type I cells harvested following 1 day of in vivo hypoxia showed elevated transcript levels of inflammatory cytokines. In situ hybridization studies confirmed expression of IL-6 in type I cells and also showed that CH induces IL-6 expression in supporting type II cells. Concurrent treatment of CH rats with anti-inflammatory drugs (ibuprofen or dexamethasone) blocked immune cell invasion and severely reduced CH-induced cytokine expression in carotid body. Drug treatment also blocked the development of chemoreceptor hypersensitivity in CH animals. Our findings indicate that chemoreceptor adaptation involves novel neuroimmune mechanisms, which may alter the functional phenotypes of type I cells and chemoafferent neurons.

Fig. 1.

Immunofluorescence in rat carotid body of type I cell marker tyrosine hydroxylase (TH; red) and immune cell antigen ED1 (green). A: normal. B, C, and D: 1, 3, and 7 days of chronic hypoxia (CH), respectively. Scale bar = 50 μm.

Fig. 2.

Time course of CH-induced inflammatory cytokine and TH gene expression in rat carotid body. Quantitative PCR (qPCR) data are normalized to 18S RNA and expressed relative to mRNA levels in normal tissue. MCP-1, monocyte chemoattractant protein-1. *, **, And *** indicate P < 0.05, 0.01, and 0.001 vs. normal, respectively. CH: exposure at 380 Torr for time indicated.

Fig. 3.

Effect of exposure to hypoxia at 565, 515, or 380 Torr for 7 days on expression of TH and inflammatory cytokines in rat carotid body. Data are expressed relative to normal nonhypoxic rats. *, **, And *** indicate P < 0.05, 0.01, and 0.001, respectively, vs. normal; +++P < 0.001 vs. 515-Torr group.

Fig. 4.

Effect of 24-h in vivo hypoxia on expression of TH, the chemokine, MCP-1, and inflammatory cytokines IL-1β, IL-6, and TNFα in rat type I cells. Data are obtained from 15 normal and 15 hypoxic cells using amplified RNA (aRNA)/qPCR technology. See text for details. *, **, And *** indicate P < 0.05, 0.01, and 0.001, respectively, vs. normal.

Fig. 5.

In situ hybridization histochemistry for cytokine IL-6 in normal (A) and 3-day CH (380 Torr; B) carotid body. CH induces increased IL-6 expression in slender cell processes, consistent with the morphology of type II cells (arrows in A vs. B). Upregulation of IL-6 expression is also indicated in type I cells (enveloped by type II cell processes). Incubation in “sense probe” (C) shows background staining. Scale bar = 20 μm.

Fig. 6.

Left: carotid sinus nerve (CSN) activity evoked by a standard hypoxic stimulus (indicated by separate trace of bath Po₂) in normal (N) vs. 8- to 10-day CH preparations. CH elicits a robust increase in hypoxic sensitivity and basal resting activity; however, note that hypersensitivity to hypoxic challenge is absent in CH animals concurrently treated with dexamethasone (Dex; 0.1 mg·kg⁻¹·day⁻¹) or ibuprofen (Ib; 4 mg·kg⁻¹·day⁻¹). Right: summary data (averaged evoked impulses per second) from 4 or 6 preparations in each group: drug-free (top) or concurrently treated with either dexamethasone or ibuprofen (middle and bottom). ***P < 0.001 vs. normal.

Fig. 7.

Effect of anti-inflammatory drugs on CH-induced immune cell activity in rat carotid body. Green cells are immunostained for CD45, a universal leukocyte marker; red indicates TH. A: normal carotid body contains a few immune cells that weakly express CD45. Following 3 days of CH (D), the tissue contains numerous cells that express higher levels of CD45. In normoxic carotid bodies from animals treated with dexamethasone (0.1 mg·kg⁻¹·day⁻¹; B) or ibuprofen (4 mg·kg⁻¹·day⁻¹; C) few or no detectable CD45⁺ immune cells are visible. Likewise, in CH animals treated with dexamethasone (E) or ibuprofen (F), CD45⁺ cells are virtually absent. Notice that TH fluorescence is increased in animals treated with dexamethasone.

Fig. 8.

Effect of ibuprofen (ib; 4 mg·kg⁻¹·day⁻¹) on 7-day CH-induced gene expression of TH and cytokines in rat carotid body. *, **, And *** indicate P < 0.05, 0.01, and 0.001, respectively, vs. normal; +, ++, and +++, P < 0.05, 0.01, and 0.001, respectively, vs. 7-day CH (CH7).

Fig. 9.

Effect of dexamethasone (Dx; 0.1 mg·kg⁻¹·day⁻¹) on CH-induced gene expression of TH and cytokines in rat carotid body. A: 3-day CH (CH3). B: CH7. *, **, And *** indicate P < 0.05, 0.01, and 0.001, respectively, vs. normal; +, and +++, P < 0.05 and 0.001, respectively, vs. CH.