Bone marrow transplantation reveals an essential synergy between neuronal and hemopoietic cell neurokinin production in pulmonary inflammation (original) (raw)

PPT-A gene–deficient mice. PPT-A gene–deficient mice were generated by nonsibling heterozygous crossings of mice carrying a targeted deletion of the substance P and neurokinin A encoding regions of the PPT-A gene (these were a generous gift from A. Basbaum, University of California, San Francisco, San Francisco, California, USA) (17). Littermates lacking the mutant allele were used as matched WT controls for all the experiments. PPT-A gene–deficient mice cannot synthesize substance P, neurokinin A, and the neurokinin A extended peptides neuropeptide K and neuropeptide γ (2), leaving neurokinin B, which is encoded by the PPT-B gene, as the only potential ligand for the neurokinin receptors. In preliminary studies, we ascertained that these mice have a normal TNF-α serum surge after the administration of endotoxin (2.45 ± 0.03 ng/ml in PPT-A gene–deleted mice vs. 2.4 ± 0.02 ng/ml in WT mice 90 minutes after 10 mg of LPS/kg, n = 3, strain C57B/6J). We have also shown that macrophages isolated from the peritoneum of the same mice release TNF-α at concentrations commensurate with those of WT mice, independent of whether they were preincubated with substance P (data not shown).

The original line of PPT-A gene–deleted mice was both kept in a CD-1 background and backcrossed to inbred C57B/6J mice for eight or more generations. Experiments were performed separately in both strains to identify potential variability from gene epistasis. All animal care practices and experimental procedures were approved by the Washington University Institutional Animal Studies Committee.

Capsaicin-mediated sensory denervation. Selective denervation with capsaicin was used to define the contribution of neurokinins released by sensory fibers expressing the vanilloid TRPV1 receptor (18) to immune complex–mediated inflammation. Capsaicin causes release of substance P and other neurokinins from C-fiber terminals, producing in the process a durable and extensive functional ablation of these fibers (19, 20), which is most complete when the capsaicin is given during the newborn period (21). The mice, CD-1 or C57B/6J, were injected with capsaicin (50 mg/kg) subcutaneously at day 2 or 3 after birth and studied at 12 weeks of age. At the time of the study, capsaicin-treated mice exhibited normal behavior, and their circulating total and differential white cell counts (11.9 × 103 ± 1.4 × 103 vs. 11.8 × 103 ± 1.3 × 103 cells/μl, mean ± SE, n = 6), erythrocyte counts (9.2 × 106 ± 0.2 × 106 vs. 9.0 × 106 ± 0.2 × 106), and platelet counts (693 × 103 ± 63 × 103 vs. 498 × 103 ± 9 × 103) were similar to those of untreated mice of the same age and background.

Immune complex–mediated injury. The effects of immune complex–mediated injury were studied over a 4-hour period. Mice were anesthetized with 1.5% Avertin (1.5 ml/kg intraperitoneally), and their tracheae were cannulated through the mouth with a 22-gauge Angiocath, using a pediatric otoscope to visualize the glottis. Chicken ovalbumin (20 mg/kg in 0.5 ml PBS; Sigma-Aldrich, St. Louis, Missouri, USA) was injected into the tail vein, and a polyclonal rabbit-raised antibody against ovalbumin (10 mg/kg in 0.1 ml PBS; Chemicon International Inc., Temecula, California, USA) was instilled into the trachea immediately thereafter (7). The endotracheal tube was removed, and the mouse was returned to its cage, with free access to water and chow. Each mouse was again anesthetized 20 minutes before the 4-hour end-point by isoflurane inhalation in a Plexiglas induction box, and Evans blue (30 mg/kg in 0.1 ml PBS; Sigma-Aldrich) was injected intravenously (22) to assess the effect of the immune complex–mediated inflammation on alveolar-capillary permeability. After administration of an overdose of Na pentobarbital, the chest and neck were opened by midline incision, and a blood sample was drawn from the right ventricle to determine the serum concentration of Evans blue. The trachea was cannulated by tracheostomy, and the left main-stem bronchus was ligated. The right lung was then lavaged with PBS in three passes of 1 ml each. Finally, the left lung was removed and frozen for later analysis, and the right lung was fixated in situ with 1 ml of 4% paraformaldehyde instilled into the trachea and removed for sectioning.

Mechanical ventilation. After cannulation of the trachea with a 22-gauge Angiocath under anesthesia with Na pentobarbital (50 mg/kg intraperitoneally), the mouse was placed on the cradle of a small-rodent plethysmograph (Kent Scientific Corp., Litchfield, Connecticut, USA). The tracheal cannula was connected to the tubing of a Harvard rodent ventilator (Harvard Apparatus Inc., Holliston, Massachusetts, USA) using a Y-connector and tubing of minimal dead space. The ventilator was adjusted to provide an end-expiratory pressure of 2 cm H2O and a tidal volume of either 6 ml/kg (peak airway pressure 9–10 cm H2O) or 20 ml/kg (peak airway pressure 16–17 cm H2O), using air as the ventilation gas. The breathing rate was adjusted to either 150 or 45 breaths per minute, depending on tidal volume, to maintain minute ventilation constant at 900 ml/(kg×min). Preliminary experiments showed that these combinations of tidal volume and breathing rate maintained blood pH and PCO2 within physiological range. Next, the mouse was injected with 0.3 mg/kg of pancuronium bromide intraperitoneally for neuromuscular blockade. An additional dose of 25 mg/kg of Na pentobarbital was give 2 hours after the initial anesthetic dose or, at any point, if spontaneous movement was observed. Throughout the experiment, airway pressures and tidal volume were measured continuously using appropriate range differential-pressure transducers (Validyne Engineering Corp., Northridge, California, USA). Signals for pressure and volume (derived from the time integral of the signal from the plethysmograph’s calibrated-screen pneumotachometer) were displayed continuously and recorded on a computerized data-acquisition system (Biopac Systems Inc., Goleta, California, USA). At the end of a 4-hour period of mechanical ventilation, half of the mice were killed with an overdose of Na pentobarbital, and their lungs were lavaged with three passes of 1 ml PBS each, fixated, and excised. The other half of the mice had their tracheal cannulae removed and were returned to their cages until 24 hours later, when they were killed with an overdose of Na pentobarbital and their lungs processed following a similar protocol. Alveolar-capillary permeability was determined by injection of Evans blue 20 minutes before the end of the experiments, as described above.

Bone marrow reconstitution experiments. To define the contribution of PPT-A gene–expressing hemopoietic cells to the amplification of both immune complex–mediated and stretch-mediated injuries, we performed bone marrow reconstitution experiments based on three permutations of donor and recipient: WT to WT mice, which served as controls for the effects of conditioning radiation and the bone marrow transplantation process; WT to PPT-A gene–deleted mice, in which only bone marrow–derived cells gained the ability to synthesize PPT-A gene–encoded peptides; and PPT-A gene–deleted to WT mice, in which only bone marrow–derived cells lost the ability to synthesize PPT-A gene–encoded peptides. In the case of the CD-1 mice, littermates were used as donors; in the case of the C57BL/6 mice, pure-bred mice carrying a Ly-5 congenic marker were used as either WT donors or recipients to confirm the existence of complete chimerism in the circulating hemopoietic cells (23). In preparation, all recipient mice underwent conditioning with 1,000 cGy from a 137Cs source at a rate of 95 cGy/min the day before intravenous injection of the donor bone marrow. Femora from the donor mice were dissected and flushed with 1 ml Hebs (20 mM Hepes, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM dextrose). The resultant cell suspension was centrifuged at 400 g and resuspended in 10 ml PBS. The supernatant was centrifuged again at 350 g, and the pellet was resuspended in 1 ml PBS. Cells were counted and diluted to administer a total dose of 2 × 106 nucleated cells. A wait period of at least 145 days was allowed for bone marrow engraftment and for the turnover of any hemopoietic cells that survived the conditioning irradiation. Previous studies have shown that, while other bone marrow–derived cell compartments are repopulated quickly, lung macrophages may require up to 90 days to achieve complete chimerism after bone marrow transplantation (24).

Assessment of inflammatory injury and detection of substance P. Cells in the bronchoalveolar lavage fluid were counted in a hemocytometer after centrifugation (at 63 g for 5 minutes) in an automatic slide-plating system (Cytofunnel; Thermo Shandon Inc., Pittsburgh, Pennsylvania, USA), fixation, and staining with eosin and methylene blue. Evans blue concentrations in serum and bronchoalveolar lavage fluid were determined spectrophotometrically at 620 nm. Casein zymograms were performed as described previously to obtain a semiquantitative assessment of protease activity (9). Substance P, TNF-α, and macrophage inflammatory protein-1α (MIP-1α) concentrations in the bronchoalveolar lavage fluid were measured by duplicates with commercial ELISA kits (Cayman Chemical Co., Ann Arbor, Michigan, USA, for substance P; Quantikine M from R&D Systems Inc., Minneapolis, Minnesota, USA, for TNF-α and MIP-1α). Immunostaining for substance P was performed in paraffin-embedded sections using a guinea pig polyclonal primary antibody (Chemicon International Inc.) at 1:200 dilution and alkaline phosphatase immunohistochemistry (Vector Laboratories Inc., Burlingame, California, USA) for detection.

Differences between groups were tested by ANOVA or by the Kruskal-Wallis test, depending on whether the data followed a normal distribution. Tissues were cut in 10-μm sections and stained with H&E according to standard technique. For the ventilation studies, where the injury was less severe and differences in tissue inflammation more difficult to discern, we asked a colleague uninvolved in the work to rate blindly two equal random samples (n = 6 per group) of tissue sections from WT and PPT-A gene–deleted mice. A score of 0–2 was assigned to the inflammatory response observed in each slide (0, no inflammation; 1, mild infiltration with neutrophils or hemorrhage; and 2, severe infiltration with neutrophils or hemorrhage). The scores were compared by χ2 analysis.