Bronchial lesions of mouse model of asthma are preceded by immune complex vasculitis and induced bronchial associated lymphoid tissue (iBALT)

Abstract

We systematically examined by immune histology the lungs of some widely used mouse models of asthma. These models include sensitization by multiple intraperitoneal injections of soluble ovalbumin (OVA) or of OVA with alum, followed by three intranasal or aerosol challenges 3 days apart. Within 24 h after a single challenge there is fibrinoid necrosis of arterial walls with deposition of immunoglobulin (Ig) and OVA and infiltration of eosinophilic polymorphonuclear cells that lasts for about 3 days followed by peribronchial B-cell infiltration and slight reversible goblet cell hypertrophy (GCHT). After two challenges, severe eosinophilic vasculitis is present at 6 h, increases by 72 h, and then declines; B-cell proliferation and significant GCHT and hyperplasia (GCHTH) and bronchial smooth muscle hypertrophy recur more prominently. After three challenges, there is significantly increased induced bronchus-associated lymphoid tissue (iBALT) formation, GCHTH, and smooth muscle hypertrophy. Elevated levels of Th2 cytokines, IL-4, IL-5, and IL-13, are present in bronchial lavage fluids. Sensitized mice have precipitating antibody and positive Arthus skin reactions but also develop significant levels of IgE antibody to OVA but only 1 week after challenge. We conclude that the asthma like lung lesions induced in these models is preceded by immune complex-mediated eosinophilic vasculitis and iBALT formation. There are elevations of Th2 cytokines that most likely produce bronchial lesions that resemble human asthma. However, it is unlikely that mast cell-activated atopic mechanisms are responsible as we found only a few presumed mast cells by toluidine blue and metachromatic staining limited to the most proximal part of the main stem bronchus, and none in the remaining main stem bronchus or in the lung periphery.

Figure 1. Vascular and bronchial inflammation in ovalbumin sensitized mice

100X and 200X pictures of H&E stained microscopic slides after single (first row), double (second row), and triple (third row) intranasal ovalbumin (OVA) challenges of OVA sensitized mice. Normal and day 0 (before 3rd challenge) are shown in the first column for comparison. At 6 hours after a single challenge there is essentially no change in pulmonary arteries from normal. However after a second challenge there is severe fibrinoid necrosis and polymorphonuclear infiltrate. After 3 challenges there is little fibrinoid necrosis but a marked perivascular infiltrate of mononuclear cells. After 1 and 2 challenges the vasculitis resolves with slight residual round cell infiltrate. However, after three challenges there is extensive iBALT formation at 24–72 hours that then resolves over the next two weeks.

Figure 2. Bronchial mucosal lesions

A. 40X; B–H. 400X. A and B: normal lung. C, D: 1 challenge; E, F: 2 challenges; G, H: 3 challenges. A. Shows selection of tissue section including left main stem bronchus were changes can be measured accurately. B and F includes ocular micrometer scale used to count nuclei and measure thickness of mucosa and submucosal muscle. Table 5 shows the measurements and statistical analysis. Clearly by the second challenge there are lesions associated with asthma including mucosal hyperplasia and hypertrophy and hypertrophy of submucosal smooth muscle. There is a significant increase in mucosal cell nuclei at 48 hours after 2 and 3 challenges (p<01); in mucosal thickness and submucosal smooth muscle thickness after each challenge (p<01) as well as significant increases from one challenge to the next.

Figure 3. Eosinophilic inflammation and mast cells

A–D. Demonstration of eosinophilic vasculitis by Congo Red stain after double challenge and E, F: bronchial eosinophilia 24 hours after triple challenge. G–J. Mast cells in normal and challenged mice. A–D. Double challenge A. 24 hrs.; B. 48 hrs.; C. 72 hrs.; D. 1 week. E, F. Bronchi 24 hrs. after triple challenge. B shows high percentage of Congo Red positive eosinophils at day 2 and C shows lesser numbers of eosinophils and more mononuclear cells. These are replaced by round cells by 1 week (D). Only a few eosinophils remain by day 14 (not shown). E and F show eosinophils in bronchi after 3 challenges. There are few perivascular eosinophils after 3 challenges (see Fig 3). G. a mast cell in normal lung. H 3 mast cells 48 hours after triple challenge. I Low magnification showing 5 mast cells in proximal left main stem bronchus. J. High magnification of mast cell. Mast cells are only found in the most proximal main stem bronchus and not in lung periphery in both normal, sensitized, as well as sensitized and challenged mice.

Figure 4. Immunoperoxidase labeling for OVA (A–D) and Ig (E–L) after pulmonary challenge

200X or 400X. A and E show faint perivascular labeling around small vessels (arrows) at 6 hours after a single challenge. This is seen more clearly in enlargements in supplemental Fig. 2. At 24 hours most of the OVA labeling is in macrophages both in zones of perivascular inflammation (B, arrow) and scattered in alveoli (C, arrows). This is also seen 6 hrs after 3 challenges (D). At 24 hours Ig is seen as deposits in walls of vessels with inflammation. (F, G), but not in macrophages. 6 hrs after 3 challenges Ig is seen in dense deposits around vessels. D and H are pictures of serial sections showing perivascular localization of Ig and OVA in alveolar macrophages. We did not see OVA labeling at 72 hrs or later. After 72 hours labeling of Ig is mostly cellular. I, J: 1 challenge, 72 hrs and 1 week; K, L: 3 challenges, 72 hrs and 1 week.

Figure 5. iBALT formation after single (A–D), double (E–L) and triple challenge (M–Q)

A–D. Single challenge, 72 hrs. E–H. Double challenge, 6 hrs. I–L Double challenge, 72 hrs. M–Q, Triple challenge, 24 hrs. The first evidence of iBALT is a peribronchial cuff of B-cells 72 hours after a single challenge (A–D). This is also present 6 hours after double challenge (E–H) and expands into larger zones of B-cells (Pax5+) at 72 hrs. (I–L). Peribronchial iBALT contains few, if any, eosinophils (N, L), although eosinophils are seen in adjacent Pax5- zones (compare J with L). From 24 to 72 hours after triple challenge, well-formed B-cell follicles appear around vessels next to bronchi (M–Q). These consist of zones of Pax5+ B-cells (white outline in M, Pax 5 stain in N) and Ig+, Pax 5- cells (black outline in M, Ig stain in 0), with less frequent T-cells (CD3, P), and macrophages (F4/80, Q). The two top arrows in M point from Pax5- zones to Ig+ zones in O. The three lower arrows in M point from a Pax5+ zone to corresponding Ig- zone in O; CD3+ zone in P and F4/80 zone in Q. Note mucous hyperplasia in M.

Figure 6. Cells in bronchial lavage fluid (BAL) and IgE antibody to OVA in sera

A–D Cells in BAL at 0, 24, 72 hours and 2 weeks. E. Graph of percentage of cells: eosinophils-light blue; monocytes-dark blue; lymphocytes-red; neutrophils-yellow. The points in E represent the average of 3 mice. F. shows development of IgE antibody to OVA at 48 hours and 1 week after 1, 2 or 3 challenges. Significant increases first occur 1 week after the first challenge or 48 hours after the second challenge which is about the same time as 48 hours after the second challenge (5 days after the first challenge).

Figure 7. Precipitating antibody (gel diffusion) and cutaneous Arthus reaction

A. Double-diffusion-in-agar showing precipitating antibody to OVA in sensitized mice. B. Gross picture of skin showing area of inflammation after injection of OVA into skin of sensitized mouse (Arthus reaction). C. H&E and D, Congo Red staining of skin section of Arthus reaction showing predominance of neutrophils and few eosinophils (arrows in I) in dermis of skin 6 hours after injection of OVA (Arthus reaction). Insert in C shows polychromatic staining of mast cells in the skin. E. OVA and F. Ig immunoperoxidase staining.