A spontaneously arising porcine model of bullous pemphigoid.

Bullous pemphigoid (BP) is an IgG-mediated autoimmune blistering disease targeting the hemidesmosomal proteins bullous pemphigoid antigens 1 and 2. Currently, there is no active animal model in which to dissect the immunopathogenic mechanism. We noticed that cutaneous blistering arose spontaneously in 12 adult Yucatan minipigs. Skin lesions consisted of turgid, isolated or clustered vesicles that occasionally evolved from erythematous and pruritic patches. Histopathological examination revealed subepidermal vesicles rich in intact and degranulated eosinophils. Antigen mapping and transmission electron microscopy confirmed that dermoepidermal separation took place in the lamina lucida of the epidermal basement membrane zone. Direct immunofluorescence revealed the presence of IgG deposited linearly at the dermoepidermal junction in seven of nine skin specimens examined. Indirect immunofluorescence testing confirmed the presence, in the serum from eight of eight affected pigs, of circulating basement membrane-specific IgG autoantibodies (titers 1 : 50 to 1 : 250). Using uncleaved and salt-split lip substrates, the autoantibodies were shown to target antigens situated not only at the basal, but also at the lateral and apical aspects of stratum basale keratinocytes. Immunoelectron microscopy confirmed that circulating IgG autoantibodies recognized hemidesmosomal antigen(s). ELISA, immunoblotting and immunoadsorption demonstrated that five of eight serum samples exhibited high immunoreactivity against BPAG2-NC16A peptides. This novel porcine acquired blistering dermatosis could be proposed as a valuable model to conduct immunomechanistic studies on the natural progression of BP, correlation of autoreactive T cells or autoantibodies with disease activity, and the role of eosinophils in the blistering process, as these diseases cannot be modeled easily in human patients or in murine passive transfer models.

Toxicologic and carcinogenic effects of the type IV phosphodiesterase inhibitor RP 73401 on the nasal olfactory tissue in rats.

RP 73401, a type IV phosphodiesterase inhibitor, caused toxic effects in the nasal olfactory region of Sprague-Dawley rats when administered by either oral or inhalation exposure. A single oral administration of RP 73401 (at a dose of > or = 50 mg/kg) or 5-day inhalation exposure (1 hr/day) at a dose of approximately 1.0 mg/kg per day caused degeneration and sloughing of the olfactory surface epithelium. Degeneration and loss of Bowman's glands were noted in the underlying lamina propria and submucosa. Electron microscopy of these lesions demonstrated that sustentacular cells and the epithelial cells lining Bowman's glands were the primary target cells in the olfactory mucosa. The earliest ultrastructural changes detected in these cells were dilatation and vesiculation of the endoplasmic reticulum, suggesting that metabolic activation is important for the toxic effects. In repeated-dose studies, 13 wk of oral dosing at 2.0 or 6.0 mg/kg per day resulted in subtle disorganization of the olfactory epithelium, whereas basal cell hyperplasia in the olfactory epithelium was identified in a 6-month inhalation study at a dose of 1.0 mg/kg per day. A 2-yr inhalation carcinogenicity study resulted in tumors of the nasal olfactory region in rats treated at 0.5 and 1.0 mg/kg per day. Most tumors were classified as olfactory neuroblastomas, and immunohistochemistry on selected tumors was consistent with their being of neuroectodermal origin. Of the species studied (rat, mouse, and dog), the olfactory toxicity of RP 73401 was confined to the rat, and the toxicity was likely related to metabolic activation by olfactory epithelial cells rather than the phosphodiesterase activity of the compound.

Endotoxin infusion in anesthetized sheep is associated with intrapulmonary sequestration of leukocytes that immunohistochemically express tumor necrosis factor-alpha.

Plasma levels of tumor necrosis factor-alpha (TNF-alpha) peak between 2 and 4 h during a 12-h continuous infusion of endotoxin in awake sheep. We hypothesized that a source of this TNF-alpha is the pool of leukocytes that accumulate in the pulmonary circulation. To test this hypothesis, we physiologically monitored six anesthetized sheep during baseline and 4-h endotoxin infusion periods (10 ng/kg x min). We obtained open-lung biopsies at baseline and at 20 min and 2 and 4 h during the endotoxin infusion period for immunohistochemical localization of TNF-alpha. The plasma concentration of TNF-alpha increased from an average baseline concentration of 0.06 +/- 0.03 ng/ml (mean +/- SD) to a peak of 1.40 +/- 0.28 ng/ml at 2 h of the endotoxin infusion. We observed increased cytoplasmic TNF-alpha immunoreactivity in situ among neutrophils and intravascular mononuclear phagocytes during the endotoxin infusion compared with baseline. Also, the number of immunopositive leukocytes increased in the pulmonary circulation during the continuous infusion of endotoxin. We conclude that TNF-alpha-producing leukocytes accumulate in the pulmonary circulation during endotoxemia. These cells probably contribute to both the rise in the circulating levels of TNF-alpha and the development of acute lung injury.

Physical and cytochemical properties of neutrophils activated in situ in the lung during ZAP infusion in sheep.

Increased retention of activated neutrophils in the lungs contributes to endothelial cell injury. However, characterization of the morphological changes that occur in neutrophils during activation in the pulmonary microcirculation has not been fully determined in vivo. Therefore, the present study was designed to determine structural and cytochemical properties of neutrophils in situ in pulmonary arterioles and alveolar capillaries during the infusion of zymosan-activated plasma (ZAP) or plasma (control) in anesthetized sheep. Quantitative morphological methods showed that ZAP infusion caused significant retention of neutrophils in alveolar capillaries [2.19 +/- 0.40 (SD) x 10(8) neutrophils/ml of capillary blood volume] and pulmonary arterioles (1.02 +/- 0.46 x 10(8) neutrophils/ml of arterial blood volume) compared with plasma infusion (1.03 +/- 0.15 and 0.30 +/- 0.10 x 10(8) neutrophils/ml, respectively; P < 0.05). Harmonic mean diameter of ZAP-activated neutrophils in situ (7.19 +/- 0.44 microns) was significantly greater than the diameter of neutrophils in plasma-treated sheep (6.29 +/- 0.17 microns; P < 0.05). Neutrophil cross-sectional area (54 +/- 3 microns2) and volume (248 +/- 27 microns3) in situ in alveolar capillaries were also significantly greater in ZAP-treated sheep than in control sheep (41 +/- 4 microns2 and 184 +/- 9 microns3, respectively; P < 0.05). Similarly, microvascular neutrophils in ZAP-treated sheep were vacuolated and elongated, filamentous actin was redistributed peripherally, and the cells were degranulated. We conclude that during ZAP infusion, neutrophils become enlarged and degranulated in pulmonary microvessels, especially in alveolar capillaries. The structural and cytochemical changes that occur are consistent with the hypothesis that neutrophil activation is accompanied by alterations in neutrophil physical properties, alterations that may facilitate retention and contribute to endothelial cell injury.

Infusion of zymosan-activated plasma affects neutrophils in peripheral blood and bone marrow in sheep.

Intravenous infusion of zymosan-activated plasma (ZAP) in sheep results in acute lung injury mediated in part by free radical release from stimulated neutrophils that are retained in increased numbers in the pulmonary microcirculation. ZAP infusion is also associated with long-term reduction in elicited superoxide anion generation from segmented neutrophils in the circulating and bone marrow pools for at least 24 h after the infusion. The purpose of our study was to investigate the effect of ZAP infusion on leukocyte counts and neutrophil granule-associated enzyme activity in the circulating and bone marrow pools. Cytochemical methods were used to characterize enzyme activity in primary granules (acid phosphatase and myeloperoxidase) and secondary granules (alkaline phosphatase). During the infusion period, neutrophil differential counts decreased less in venous blood than in matched arterial blood. Release from bone marrow stores was evident as increased numbers of circulating band neutrophils during and after the infusion. Neutrophils in venous and arterial blood smears were degranulated acutely during and 1-3 h after the infusion was stopped. Band and segmented neutrophils in bone marrow also appeared slightly degranulated 1-2 h after the infusion. In vitro experiments showed that band and segmented neutrophils in bone marrow degranulated in response to ZAP incubation. Immature polymorphonuclear leukocytes did not degranulate. Five to 24 h after ZAP infusion, enzyme activity in circulating and bone marrow neutrophils was at baseline levels, suggesting that new cells were being released into the circulating pool because degranulated neutrophils do not synthesize new granules. Another indication of a long-term effect in bone marrow were slight decreases in the percentage of immature polymorphonuclear leukocytes and band neutrophils and a significant decrease in the percentage of eosinophils, all of which coincided with a significant increase in the percentage of segmented neutrophils. Our results demonstrate that circulating complement anaphylatoxins affect neutrophils in bone marrow as well as the vascular space.

Downregulation of blood and bone marrow neutrophils decreases expression of acute lung injury in sheep.

We have shown that infusion of zymosan-activated plasma (ZAP) in sheep causes acute lung injury and downregulates peripheral blood neutrophils in that elicited superoxide release is reduced for at least 24 h after the infusion. The present study was designed to test the following hypotheses: 1) peripheral blood neutrophils are representative of neutrophils marginated in the pulmonary circulation, 2) blood neutrophils are downregulated because neutrophils developing in bone marrow are similarly affected, and 3) downregulated neutrophils have a reduced capacity to produce tissue injury. In a series of experiments in 21 sheep, we showed that elicited superoxide release was similar in peripheral blood neutrophils and in marginated neutrophils washed out of the pulmonary vascular bed. Measurements of superoxide release from blood and bone marrow neutrophils collected 2-24 h after ZAP infusion revealed progressive downregulation with time and greater downregulation of superoxide release in bone marrow neutrophils compared with peripheral blood neutrophils. Finally, after downregulating peripheral blood neutrophils, subsequent infusion of ZAP in conscious sheep produced sequestration of neutrophils in the pulmonary circulation but failed to produce a sustained increase in lung lymph protein clearance. The results suggest that neutrophil downregulation, as measured in vitro, is expressed in vivo as reduced ability of neutrophils to produce tissue injury when challenged by an activating agent.

PGE1 inhibited PMN attachment to air emboli in vivo during infusion of ZAP without preventing lung injury.

Prostaglandin E1 (PGE1) treatment of neutrophils inhibits their adherence to substrates in vitro, including endothelial cell monolayers. Demonstration that PGE1 inhibits neutrophil adherence in vivo in the lung, however, is complicated by PGE1 effects on cells other than neutrophils, such as endothelial cells. To determine whether PGE1 inhibits neutrophil adherence properties in vivo, we used air emboli as intravascular targets for neutrophil attachment. Four experimental conditions were studied in anesthetized and awake sheep that were treated with 1) PGE1 and air emboli, 2) saline and air emboli, 3) PGE1 and zymosan-activated plasma (ZAP) + air emboli, and 4) saline and ZAP + air emboli. PGE1 (30 ng.kg-1.min-1) or saline was infused continuously 1 h before and 1 h during the infusion of air emboli (group 1; n = 13 sheep) or ZAP + air emboli (group 2; n = 13 sheep). The number of neutrophils (PMNs) attached to air emboli in four anesthetized sheep per condition was significantly less in sheep given PGE1 and ZAP + air emboli [8 +/- 3 (SD) PMNs/mm of embolus perimeter] than in the other three conditions (14-21 PMNs/mm; P less than 0.05). Repeated experiments in five awake sheep per group showed that PGE1 treatment did not prevent increased lung lymph protein clearance in either group compared with saline treatment. We conclude that PGE1 specifically inhibited attachment of ZAP-activated neutrophils to air emboli in vivo. The lack of pathophysiological protection suggests that PGE1-induced alterations in neutrophil attachment properties were independent of other cellular activation responses.

Calcium is required for PMA induced superoxide release from human neutrophils.

Experiments were designed to reexamine the relationship between extracellular calcium and superoxide generation in phorbol myristate acetate (PMA) stimulated neutrophils exploiting a newly adapted method to measure superoxide anion (O2-) generation from adherent cells stimulated at high and low cell density. Human neutrophils were plated in microtiter wells in cell densities of either 0.2 or 2.0 million cells/well. Superoxide release was measured sequentially over 60 min by reduction of ferricytochrome c. Cells were maintained in 1 mM Ca++ or 0 mM Ca++ Hanks' buffer for 60 min prior to activation as well as during measurement of O2-. In 1 mM Ca++, 2.0 million adherent neutrophils released 10.7 +/- 1.2 nmol O2- in 20 min (n = 4). O2- release was not significantly different for high density cells incubated and stimulated in 0 mM Ca++. In the presence of 1 mM Ca++, 0.2 million adherent neutrophils released 6.3 +/- 0.5 nmols O2- in 20 min. With cells stimulated at low density, PMA stimulated O2- release was significantly decreased (3.0 +/- 0.6 nmol O2- in 20 min) as was the initial rate of secretion of O2- in the absence of extracellular calcium. Basal release of superoxide was also greater in the presence of 1 mM Ca++ (0.96 nmol/20 min) compared to basal release in 0 mM Ca++ (0.22 nmol/20 min). Additional experiments with 0.2 million cells/well showed that extracellular Ca++ was required during stimulation with PMA and that prior incubation of cells for up to 60 min in 0 mM Ca++ had no effect on O2- release measured in the presence of calcium. Furthermore, PMA stimulated O2- was independent of verapamil (10(-5)-10(-7) M), suggesting that voltage-dependent calcium channels do not participate in this response. The planar areas for unstimulated neutrophils in 0 mM Ca++ increased after addition of PMA. Unstimulated cells in 1 mM Ca++ tended to be larger and planar areas did not increase after PMA. These studies demonstrate that PMA stimulated O2- secretion is dependent on extracellular calcium particularly when adherent neutrophils are stimulated at low cell density. Furthermore, extracellular calcium at a concentration of 1 mM primes neutrophils by increasing basal secretion of O2- and increasing superoxide release after a maximum stimulating dose of PMA.

Superoxide anion release from blood and bone marrow neutrophils is altered by endotoxemia.

In vivo endotoxin infusion produces neutrophil-mediated acute lung injury and increases superoxide anion release from phorbol myristate acetate (PMA)-stimulated blood neutrophils collected 18-24 hours after the infusion. Because the turnover time of circulating blood neutrophils is only 6-8 hours, it was hypothesized that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with increased superoxide anion release from bone marrow neutrophils. To test this hypothesis, two doses of Escherichia coli endotoxin (5.0 and 0.5 micrograms/kg) were infused into chronically instrumented awake sheep. Blood and bone marrow neutrophils were collected 24 hours after the infusion, and superoxide anion release from unstimulated and PMA-stimulated neutrophils was measured in vitro. Endotoxin infusion produced an increase in pulmonary microvascular permeability, in intravascular activation (degranulation) of blood neutrophils, and in circulating blood neutrophils 24 hours after the infusion. High-dose endotoxin (5.0 micrograms/kg; n = 4) increased superoxide anion release from unstimulated peripheral blood neutrophils (2.25 +/- 0.38 times baseline [p less than or equal to 0.05]) and from peripheral blood neutrophils stimulated with 10(-9) M PMA in vitro (1.46 +/- 0.55 times baseline). Low-dose endotoxin (0.5 micrograms/kg; n = 5), on the other hand, did not alter superoxide anion release from peripheral blood neutrophils. Bone marrow neutrophils could not be isolated reproducibly after high-dose endotoxin because of leukoaggregation. Bone marrow neutrophils were isolated after low-dose endotoxin infusion. Stimulation of these cells with 10(-9) M PMA in vitro resulted in a two- to fourfold increase above control release (p less than or equal to 0.05). Increased superoxide anion release from both peripheral blood and bone marrow neutrophils occurred in the absence of circulating endotoxin, as measured by a Limulus assay. These results show that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with an increase in the superoxide anion release from bone marrow neutrophils. Furthermore, the recruitment of affected bone marrow neutrophils into peripheral blood may explain the increased superoxide anion release from blood neutrophils 24 hours after endotoxin infusion.