The effect of leukocyte depletion on pulmonary M₂ muscarinic receptor function in parainfluenza virus‐infected guinea‐pigs

Abstract

Parainfluenza infections of the airways cause dysfunction of inhibitory M2 muscarinic receptors on the pulmonary parasympathetic nerves. To distinguish the direct effects of virus from the effects of virus‐induced airway inflammation on M2 muscarinic receptor function, guinea‐pigs were depleted of leukocytes by pretreating with cyclophosphamide (30 mg kg−1, i.p. daily for 7 days) after which they were infected with parainfluenza virus type 1 (Sendai virus). Guinea‐pigs were anaesthetized, tracheotomized, and ventilated. The vagus nerves were isolated and cut, and the distal ends were electrically stimulated causing bronchoconstriction. In control animals, pilocarpine (1–100 μg kg−1, i.v.) inhibited and gallamine (0.1–10 mg kg−1, i.v.) potentiated vagally‐induced bronchoconstriction by stimulating or blocking M2 muscarinic receptors on the vagus. These effects of pilocarpine and gallamine were almost completely lost in virus‐infected animals, demonstrating loss of M2 receptor function. Cyclophosphamide depleted peripheral blood leukocytes and inhibited the virus‐induced influx of inflammatory cells into the lung. Depletion of leukocytes protected M2 receptor function from viral infection in some, but not all, guinea‐pigs tested. Among infected animals that had been depleted of leukocytes, the viral content (expressed as the log of the number of tissue culture infectious doses per g lung tissue) of those that retained normal M2 receptor function was 4.29 ± 0.05 (mean ± s.e.mean), while the viral content of those that lost M2 receptor function despite leukocyte depletion was 5.45 ± 0.20 (P = 0.011). Thus the viral content of the lungs in which M2 receptor function was lost was 16 times greater than that of the lungs in which M2 receptor function was preserved. Viral content correlated with the inhibition of vagally‐mediated bronchoconstriction after the maximum dose of pilocarpine (100 μg kg−1; r2 = 0.81, P = 0.0004). In antigen‐challenged animals, inhibitory M2 muscarinic receptor function is restored when positively charged inflammatory cell proteins are bound and neutralized by heparin. However, heparin (2000 u kg−1, i.v.) did not reverse virus‐induced loss of M2 muscarinic receptor function, even in those guinea‐pigs with a lower viral titer. Because leukocyte depletion protected M2 muscarinic receptor function only in animals with mild viral infections, it appears that viruses have both an indirect, leukocyte‐dependent effect on M2 receptors and, in animals with more severe infections, a leukocyte‐independent effect on M2 receptors. The failure of heparin to restore M2 receptor function demonstrates that the leukocyte‐dependent loss of M2 receptor function is not mediated by positively charged inflammatory cell proteins.

Virus- and interferon-induced loss of inhibitory M2 muscarinic receptor function and gene expression in cultured airway parasympathetic neurons.

“…This appears to be an important mechanism of M 2 receptor dysfunction in antigen-challenged guinea pigs, as neutralizing the positively charged proteins using heparin reverses the loss of receptor function (21), while blocking the effect of eosinophil major basic protein using an antibody prevents antigeninduced loss of receptor function (22). However, the failure of heparin to reverse the effects of viral infections in virusinfected guinea pigs (13) suggests that the leukocyte-dependent mechanisms of M 2 receptor dysfunction after viral infection are not related to eosinophil proteins. Among the possible leukocyte-independent mechanisms, we showed that parainfluenza virus itself causes a 10-fold loss of agonist affinity for the M 2 receptor when incubated with the receptor in vitro (19).…”

“…We have shown previously that viral infections impair the function of M 2 receptors in vivo via mechanisms that are both leukocyte-dependent and leukocyte-independent (13,19). Among the effects of leukocytes on M 2 receptor function, we have shown that several positively charged proteins of the Figure 5.…”

“…Previously, we have suggested several mechanisms by which viral infections may impair the function of airway neuronal M 2 receptors. Studies in which guinea pigs were depleted of leukocytes before parainfluenza virus infection demonstrated that both leukocyte-dependent and leukocyte-independent mechanisms may be involved (13).…”

The rise of the phoenix: the expanding role of the eosinophil in health and disease

“…In normal, nonallergen-sensitized guinea-pigs, M2R dysfunction also develops after virus infection, but in contrast, virus-induced loss of M2R function in nonallergen-sensitized animals is not mediated by eosinophils (44,48). Such virus-induced M2R dysfunction is seen in nonatopic humans (38), which may be secondary to increased interferon (IFN)-c. The IFN-c is higher in nonallergen virus-infected animals (49) and humans (50), it positively correlates with AHR in humans (51), and it decreases M2R expression in nerve cell culture (52).…”

Viral infection induces dependence of neuronal M2 muscarinic receptors on cyclooxygenase in guinea pig lung.

“…Acute loss of M 2 muscarinic receptor function after infection with virus, or exposure to antigen or ozone, depends on lung inflammation (39,40). The similar chronic effects of both viral infection and ozone exposure on the cyclooxygenase de-pendence of M 2 receptor function suggest that inflammation is also important in causing these changes.…”