Expression of hIL-1β in the Lungs of Infant Bitransgenic Mice
To verify that the absence of the β6 integrin subunit did not change the levels of hIL-1β in the lungs of bitransgenic pups, doxycycline was administered to pregnant and lactating dams from plug date until the pups were killed at PN7. The levels of mature hIL-1β mRNA and protein were similar in IL-1β/β6+/+ and IL-1β/β6−/− pups (hIL-1β/β-actin mRNA, IL-1β/β6+/+ 0.57 ± 0.34 [n = 7]; IL-1β/β6−/−, 0.61 ± 0.29 [n = 12], P = 0.8; hIL-1β protein/total protein, IL-1β/β6+/+ 67 ± 29 ng/mg [n = 9]; IL-1β/β6−/− 59 ± 11 ng/mg [n = 7], P = 0.4).
Absence of the β6 Integrin Subunit Improved the Survival of IL-1β–Expressing Mice
mice survived until PN7, whereas two control/β6+/+
pups died at or soon after birth (). Approximately 50% of the IL-1β/β6+/+
mice died by 7 days of age (, data from Bry and colleagues [18
]). In contrast, only 2% (one pup) of the IL-1β/β6−/−
mice died by 7 days of age ().
Figure 1. The absence of the β6 integrin subunit improved the survival and postnatal growth of IL-1β–expressing mice. Doxycycline was administered to the dams from the beginning of pregnancy until the pups were killed at Postnatal Day (PN) (more ...)
Absence of the β6 Integrin Subunit Improved Postnatal Growth in IL-1β–Expressing Mice
The birth weights of control/β6+/+, control/β6−/−, IL-1β/β6+/+, and IL-1β/β6−/− pups were similar (data not shown). IL-1β/β6+/+ and IL-1β/β6−/− mice weighed less than their littermate controls at PN7 (). Although control/β6−/− mice weighed less than control/β6+/+ mice, IL-1β/β6−/− mice weighed more than IL-1β/β6+/+ mice ().
Absence of the β6 Integrin Subunit Improved Alveolar Septation and Alveolar Wall Thinning in IL-1β–Expressing Mice
To examine whether the absence of the β6 integrin subunit affected alveolar development in infant mice expressing IL-1β, the alveolar chord length and alveolar wall thickness were studied in infant IL-1β–expressing and control mice with wild-type or null β6 loci. In control/β6+/+ and control/β6−/− pups, alveolar septation was underway at PN7, as expected, whereas alveolarization was disrupted in both IL-1β/β6+/+ and IL-1β/β6−/− mice (). On histological sections, the size of the distal airspaces appeared smaller in IL-1β/β6−/− than in IL-1β/β6+/+ mice (). This result was confirmed by alveolar chord length measurement that revealed shorter alveolar chord length in IL-1β/β6−/− mice than in IL-1β/β6+/+ mice ().
Figure 2. Alveolar septation and alveolar wall thinning in IL-1β–expressing mice was improved in mice deficient in β6 integrin subunit. Doxycycline was administered to the dams from the beginning of pregnancy until the pups were killed. (more ...)
In the absence of doxycycline administration, alveolar chord length was similar in control/β6+/+, control/β6−/−, IL-1β/β6+/+, and IL-1β/β6−/− mice (control/β6+/+, 35.0 ± 2.38 μm [n = 4]; control/β6−/−, 37.3 ± 1.42 μm [n = 5]; IL-1β/β6+/+, 37.5 ± 2.63 μm [n = 4]; IL-1β/β6−/−, 37.0 ± 0.97 μm [n = 5]).
Because alveolar wall thinning is an important part of normal lung development (33
), we measured alveolar wall thickness in the mice at PN7. The alveolar wall thickness was similar in control/β6+/+
mice (). IL-1β expression prevented alveolar septal thinning in both β6+/+
mice (). However, the alveolar walls were thinner in IL-1β/β6−/−
than in IL-1β/β6+/+
Absence of the β6 Integrin Subunit Suppressed IL-1β–Induced Neutrophil and Macrophage Infiltration into the Lung
Although the number of neutrophils within the alveolar septa was similar in control/β6−/− and control/β6+/+ pups, control/β6−/− pups had more neutrophils in the alveolar spaces than control/β6+/+ pups (). Although IL-1β caused infiltration of neutrophils into the lung in both β6+/+ and β6−/− mice, absence of the β6 integrin subunit suppressed the IL-1β–induced neutrophil migration into the alveolar spaces (). No difference was found in the number of alveolar septal neutrophils between IL-1β/β6−/− and IL-1β/β6+/+ mice (). Thus, the distribution of neutrophils between alveolar septa and lumen was different in β6−/− and β6+/+ mice. In IL-1β/β6+/+ mice, 69% of the neutrophils in the alveolar region of the lung were in the alveolar spaces, whereas, in IL-1β/β6−/− mice, only 39% of the neutrophils were intra-alveolar ().
Figure 3. Neutrophils and macrophages in the lungs of infant mice. Dams received doxycycline from the beginning of pregnancy until the pups were killed at PN7. Neutrophils and macrophages were detected by immunohistochemistry and counted in the alveolar airspaces (more ...)
Most of the macrophages in the distal lung were in the alveolar lumen in all animals (). The numbers of macrophages in the alveolar lumen and alveolar septa were similar in control/β6+/+ and control/β6−/− mice (). IL-1β increased the number of macrophages in both the alveolar lumen and alveolar septa in β6+/+ mice, but only in the alveolar lumen in β6−/− mice (). Absence of the β6 integrin subunit suppressed IL-1β–induced macrophage infiltration into both the alveolar lumen and the septa ().
Absence of the β6 Integrin Subunit Suppressed the IL-1β–Induced Expression of Chemoattractants, of the Acute-Phase Protein SAA3, and of Osteopontin in the Lungs of Infant Mice
The higher number of neutrophils in the alveolar spaces in control/β6−/− mice compared with control/β6+/+ mice was associated with increased mRNA and protein expression of the neutrophil-attractant CXC chemokines, KC and MIP-2, and mRNA expression of the CXC chemokine receptor, CXCR2, in the lungs of control/β6−/− mice compared with control/β6+/+ mice (). The mRNA expression of the monocyte chemokines, MCP-1 and MCP-3, and the protein levels of MCP-1, were also higher in control/β6−/− than in control/β6+/+ mice (). Similarly, the mRNA expression of the chemotactic and proinflammatory proteins, S100A8, S100A9, and SAA3, was up-regulated in control mice in the absence of the β6 integrin (). In contrast, absence of the β6 integrin subunit suppressed the mRNA expression and protein production of osteopontin in control mice ().
Figure 4. mRNA expression and protein levels of CC and CXC chemokines. Dams were given doxycycline throughout pregnancy and postnatally until the pups were killed at PN7. The IL-1β–induced mRNA expression and protein production of the CXC chemokines, (more ...)
Figure 5. mRNA expression and protein production of inflammatory mediators in the lungs of infant mice. Dams were given doxycycline from beginning of pregnancy until the pups were killed at PN7. The IL-1β–induced expression of calgranulin A (S100A8) (more ...)
Although IL-1β increased the expression and production of the neutrophil chemokines, KC and MIP-2, in both β6+/+ and β6−/− mice, the levels of these chemokines were lower in IL-1β/β6−/− mice than in IL-1β/β6+/+ mice (). Absence of the β6 integrin subunit also suppressed the IL-1β–induced mRNA expression of S100A8, S100A9, and SAA3, and the mRNA expression and protein production of osteopontin (). In contrast, absence of the β6 integrin potentiated the IL-1β–induced expression of the monocyte chemoattractants, MCP-1 and MCP-3, and the production of MCP-1 in the lungs of infant mice (). IL-1β increased CXCR2 mRNA expression similarly in β6−/− and β6+/+ mice ().
Absence of the β6 Integrin Subunit Prevented Airway Remodeling and Suppressed the Expression of the Chitinase-Like Lectins, Ym1 and Ym2, in IL-1β–Expressing Mice
The airways of control/β6+/+ and control/β6−/− mice were structurally normal (). Expression of IL-1β caused thickening and goblet cell hyperplasia of the airway epithelium and induced accumulation of inflammatory cells in the airways in mice with wild-type β6 loci (). Large numbers of lymphocytes were seen around the airways in IL-1β/β6+/+ mice (). Absence of the β6 integrin subunit suppressed these pathological changes in the airways of IL-1β–expressing mice (). A majority of the airways in IL-1β/β6+/+ had more than 80% goblet cells, whereas a majority of the airways in IL-1β/β6−/− had less than 20% goblet cells.
Figure 6. Airway structure and expression of the chitinases, Ym1 and Ym2. Dams were given doxycycline from the beginning of pregnancy until the pups were killed at PN7. (A–D) Airway histology, periodic acid Schiff (PAS) staining. The airways of control/β6 (more ...)
Because the chitinase-like lectins, Ym1 and Ym2, are up-regulated in airway inflammation (34
), we studied their expression in the lungs of infant mice. The mRNA expression of Ym1 and Ym2 was lower in control/β6−/−
than in control/β6+/+
mice (). The expression of both Ym1 and Ym2 was increased roughly 50-fold in IL-1β/β6+/+
mice compared with control/β6+/+
mice (). Although the expression of these chitinases was increased by IL-1β in mice lacking the β6 integrin subunit, the expression of Ym1 and Ym2 was much lower in IL-1β/β6−/−
mice than in IL-1β/β6+/+
Apoptosis and Proliferation in the Lungs of Control and IL-1β–Expressing Mice
Dysregulated apoptosis and/or proliferation may contribute to the structural alterations in BPD (36
). In addition, the numbers of inflammatory cells in the lungs could be affected by apoptosis. For these reasons, we studied apoptosis and proliferation in the lungs of newborn mice at PN0 and PN7. As previously described, a peak of apoptosis was seen in the lungs of control/β6+/+
mice directly after birth (39
) (). Interestingly, apoptosis in the lungs at PN0 was suppressed by IL-1β in β6+/+
, but not in β6−/−
Figure 7. Apoptosis and proliferation in the distal lung. Dams were given doxycycline from the beginning of pregnancy until the pups were killed at PN0 or PN7. (A) Total number of distal terminal transferase dUTP nick-end labeling–positive cells (apoptotic (more ...)
As expected (39
), apoptosis in control lungs decreased from PN0 to PN7 (). IL-1β enhanced apoptosis in the lungs of both β6+/+
mice at PN7 (). In addition, the lungs of control/β6−/−
mice had more apoptosis than control/β6+/+
mice at PN7 (). At PN7, IL-1β increased the numbers of apoptotic cells in the airspace in both β6+/+
mice (). However, there was no difference in apoptosis between IL-1β/β6+/+
mice in the airspaces (). Thus, differences in apoptosis did not explain the differences in the numbers of inflammatory cells in the alveolar spaces between IL-1β/β6+/+
mice. Absence of the β6 integrin increased the number of apoptotic cells within the alveolar septa in both control and IL-1β–expressing mice at PN7 (). This suggests that increased apoptosis in the septa of IL-1β/β6−/−
mice may have contributed to the septal thinning in these mice.
The number of proliferating cells, as studied by Ki-67 labeling, rose sharply from PN0 to PN7 in control/β6+/+
lungs (). This is consistent with previous results showing a large increase in cell proliferation in the lung after birth (39
). The total number of proliferating cells within the distal lung of control/β6+/+
mice was approximately 30%, similar to results previously obtained in wild-type murine lungs at PN7 (40
). No significant differences were seen in the numbers of proliferating cells between the four different genotypes at either PN0 or PN7 ().
Lack of Spontaneous Inflammation in Fetal β6 Integrin–Deficient Mice
β6 integrin deficiency leads to spontaneous inflammation in adult mice (22
). Similarly, more neutrophils and higher expression of inflammatory proteins were present in the lungs of the infant control/β6−/−
mice than in control/β6+/+
mice at PN7 (, , and ). Because spontaneous inflammation in β6 integrin–deficient mice might protect their lungs against subsequent inflammation, we studied whether lack of β6 integrin caused inflammation in fetal mice at E14, the time at which hIL-1β production is first initiated by doxycycline administration (18
). In this experiment, no doxycycline was given to pregnant mice, and the lungs of fetuses were harvested at E14. Importantly, there were no differences at this time point between β6+/+
in the numbers of macrophages or neutrophils (). Furthermore, we found no difference in the mRNA expression of the following inflammatory genes in the lungs of β6+/+
mice: KC, MIP-2, MCP-3, osteopontin, S100A8, S100A9, SAA3, Ym1, and Ym2 (). The expression of MCP-1 was higher in β6+/+
than in β6−/−
mice (1.44 ± 0.05 versus 1.19 ± 0.07; P
< 0.01) (). Thus, we found no evidence of enhanced inflammation in β6−/−
fetuses compared with β6+/+
fetuses at E14. The suppression of IL-1β–induced inflammation in β6 integrin–deficient infant mice is therefore unlikely due to a previous inflammation in the mice.
LACK OF SPONTANEOUS INFLAMMATION IN β6-DEFICIENT MICE AT EMBRYONIC DAY 14
Active TGF-β1 in the Lungs of IL-1β–Expressing and Control Infant Mice
Because the αvβ6 integrin is an in vivo activator of TGF-β1, we measured the levels of active and total TGF-β1 in IL-1β/β6+/+ and IL-1β/β6−/− mice and their controls at PN7. IL-1β increased the levels of active TGF-β1 in both β6+/+ and β6−/− mice (). IL-1β–expressing mice deficient in β6 integrin tended to have lower levels of active TGF-β1, but the difference was not statistically significant (). The levels of total TGF-β1 did not significantly differ between the different genotypes ().
Figure 8. Active and total transforming growth factor (TGF)–β1 in the lungs of infant mice. Dams were given doxycycline from the beginning of pregnancy until the pups were killed at PN7. (A) The level of active TGF-β1 was increased in the (more ...)