Kendra J. Greenlee

Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation.

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2−/−/MMP9−/− mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2−/−/MMP9−/− allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.

Development of respiratory function in the American locust Schistocerca americana I. Across-instar effects

SUMMARY We tested the hypothesis that oxygen delivery from the atmosphere to the tissues becomes more difficult as grasshoppers increase in body size throughout development due to increases in tracheal length. If this is true, then older, larger grasshoppers should have smaller safety margins [higher critical oxygen partial pressures (PO2s)] for oxygen delivery than younger, smaller grasshoppers. We exposed grasshoppers of first, third and fifth instars and adults to decreasing levels of atmospheric O2 and measured their ventilatory responses. Contrary to our prediction, we found that larger grasshoppers had critical PO2s eight times lower than juveniles due in part to their threefold lower mass-specific metabolic rates and their ability to quadruple convective gas exchange. Adults more than doubled abdominal pumping frequency and increased tidal volume by 25% as PO2 decreased fourfold, whereas the youngest juveniles showed no such responses. This study indicates that juveniles may be more susceptible to hypoxia in natural situations, such as exposure to high altitude or restricted burrows. Also, larger size is not necessarily correlated with a smaller safety margin for oxygen delivery in insects.

Divergent functions for airway epithelial matrix metalloproteinase 7 and retinoic acid in experimental asthma

The innate immune response of airway epithelial cells to airborne allergens initiates the development of T cell responses that are central to allergic inflammation. Although proteinase allergens induce the expression of interleukin 25, we show here that epithelial matrix metalloproteinase 7 (MMP7) was expressed during asthma and was required for the maximum activity of interleukin 25 in promoting the differentiation of T helper type 2 cells. Allergen-challenged Mmp7−/− mice had less airway hyper-reactivity and production of allergic inflammatory cytokines and higher expression of retinal dehydrogenase 1. Inhibition of retinal dehydrogenase 1 restored the asthma phenotype of Mmp7−/− mice and inhibited the responses of lung regulatory T cells, whereas exogenous administration of retinoic acid attenuated the asthma phenotype. Thus, MMP7 coordinates allergic lung inflammation by activating interleukin 25 while simultaneously inhibiting retinoid-dependent development of regulatory T cells.

Respiratory changes throughout ontogeny in the tobacco hornworm caterpillar, Manduca sexta

SUMMARY The respiratory system of growing caterpillars is challenged in two distinct ways as they develop from hatchlings to fifth instars preparing for pupation. First, across instars, body sizes and tracheal lengths increase substantially. Second, within each instar, animal mass can more than double while major tracheal respiratory system structures, such as spiracles and large tracheae, are fixed in size until molting. To test whether these growth processes result in a decrease in O2 delivery capacity relative to tissue oxygen needs, we exposed feeding Manduca sexta larvae of various ages to decreasing levels of atmospheric O2 and measured their metabolic rate and ability to feed. We found that near the beginning of all instars, M. sexta were able to maintain gas exchange and feed down to approximately 5 kPa O2, indicating that these insects are able to match tracheal O2 delivery to increased metabolic rates across instars. However, gas exchange and feeding of caterpillars nearing the molt were limited at much higher O2 levels (up to 15 kPa O2), suggesting that caterpillars have limited capacities to increase tracheal O2 delivery as O2 consumption rates increase within instars. It seems possible that the safety margin for O2 delivery may disappear completely in the last hours before ecdysis, providing an ultimate if not proximate explanation for the necessity of molting.

Development of respiratory function in the American locustSchistocerca americana

SUMMARY We hypothesized that oxygen delivery becomes more difficult for insects and tracheate arthropods as they progress throughout an intermolt period. During this time, body mass can more than double, yet the major tracheae and spiracles cannot be increased in size until molting. Also, tissue growth could compress air sacs used for convective gas exchange. To test these possibilities, we investigated the effect of within-instar growth on respiratory parameters, including CO2 emission rate, ventilation frequency, tidal volume and critical oxygen partial pressure (PO) for first-, third- and fifth-instar juveniles and adults of the American locust Schistocerca americana. We found that late-stage grasshoppers tended to have 40% higher total CO2 emission rates but 15% lower mass-specific CO2 emission rates and 35% higher ventilation frequencies than early-stage animals. Maximal tracheal system conductance decreased by 20-33% at the end of an instar, possibly due to compression of air sacs. In addition, animals nearing the end of an instar had higher critical PO values for abdominal pumping, and late-stage adults had 50% lower tidal volumes, suggesting that increases in tissue mass throughout an instar may hinder the ability of animals to breathe deeply. Late-stage adults had lower critical PO values for CO2 emission, although this pattern was not found in any juvenile instars, indicating that late-stage juveniles compensate for decreased conductance by increasing ventilation frequency or the use of diffusive gas exchange. Our data suggest that late-stage arthropods are more vulnerable to hypoxia and may have reduced aerobic capacities and lower tissue PO s than early-stage arthropods.

Issues of convection in insect respiration: Insights from synchrotron X-ray imaging and beyond☆☆☆

While it has long been known that in small animals, such as insects, sufficient gas transport could be provided by diffusion, it is now recognized that animals generate and control convective flows to improve oxygen delivery across a range of body sizes and taxa. However, size-based methodological limitations have constrained our understanding of the mechanisms that underlie the production of these convective flows. Recently, new techniques have enabled the elucidation of the anatomical structures and physiological processes that contribute to creating and maintaining bulk flow in small animals. In particular, synchrotron X-ray imaging provides unprecedented spatial and temporal resolution of internal functional morphology and is changing the way we understand gas exchange in insects. This symposium highlights recent efforts towards understanding the relationship between form, function, and control in the insect respiratory system.

Synchrotron imaging of the grasshopper tracheal system: morphological and physiological components of tracheal hypermetry

As grasshoppers increase in size during ontogeny, they have mass specifically greater whole body tracheal and tidal volumes and ventilation than predicted by an isometric relationship with body mass and body volume. However, the morphological and physiological bases to this respiratory hypermetry are unknown. In this study, we use synchrotron imaging to demonstrate that tracheal hypermetry in developing grasshoppers (Schistocerca americana) is due to increases in air sacs and tracheae and occurs in all three body segments, providing evidence against the hypothesis that hypermetry is due to gaining flight ability. We also assessed the scaling of air sac structure and function by assessing volume changes of focal abdominal air sacs. Ventilatory frequencies increased in larger animals during hypoxia (5% O(2)) but did not scale in normoxia. For grasshoppers in normoxia, inflated and deflated air sac volumes and ventilation scaled hypermetrically. During hypoxia (5% O(2)), many grasshoppers compressed air sacs nearly completely regardless of body size, and air sac volumes scaled isometrically. Together, these results demonstrate that whole body tracheal hypermetry and enhanced ventilation in larger/older grasshoppers are primarily due to proportionally larger air sacs and higher ventilation frequencies in larger animals during hypoxia. Prior studies showed reduced whole body tracheal volumes and tidal volume in late-stage grasshoppers, suggesting that tissue growth compresses air sacs. In contrast, we found that inflated volumes, percent volume changes, and ventilation were identical in abdominal air sacs of late-stage fifth instar and early-stage animals, suggesting that decreasing volume of the tracheal system later in the instar occurs in other body regions that have harder exoskeleton.

Body size-independent safety margins for gas exchange across grasshopper species

Why is maximal insect body size relatively small compared to that of vertebrates? Possibly insect body size is limited by the capacity of the tracheal respiratory system to delivery oxygen down longer and longer tracheae to the tissues. If so, one possible outcome would be that larger insect species would have a smaller safety margin for oxygen delivery (higher critical PO2, Pc). We tested this idea by exposing inactive adult grasshoppers of a range of species and body sizes (0.07–6.4 g) to progressively lower oxygen atmospheres and measuring their ventilation frequency and their ability to maintain metabolic rate (indexed by CO2 emission rate). We analyzed effects of body size on these parameters by simple linear regressions, as well as methods to control for phylogenetic relatedness among species. We found interspecific variation in Pc, but Pc did not significantly correlate with body mass (average Pc across all species = 4 kPa). Maximal tracheal system conductance scaled approximately with mass0.7, and estimated ventilation in hypoxia (ventilatory frequency×tidal volume) scaled directly with mass, suggesting that convection is the major mechanism of gas exchange in all these species. These comparative data strengthen the growing body of evidence that body size does not affect the safety margin for oxygen delivery in insects.

Dual Protective Mechanisms of Matrix Metalloproteinases 2 and 9 in Immune Defense against Streptococcus pneumoniae

A localized and effective innate immune response to pathogenic bacterial invasion is central to host survival. Identification of the critical local innate mediators of lung defense against such pathogens is essential for a complete understanding of the mechanism(s) underlying effective host defense. In an acute model of Streptococcus pneumoniae lung infection, deficiency in matrix metalloproteinase (MMP)2 and MMP9 (Mmp2/9−/−) conferred a survival disadvantage relative to wild-type mice treated under the same conditions. S. pneumoniae-infected Mmp2/9−/− mice recruited more polymorphonuclear leukocytes to the lung but had higher bacterial burdens. Mmp2/9−/− mice showed significantly higher levels of IL-17A, IP-10, and RANTES in the lung. Although MMP2-dependent cleavage partially inactivated IL-17A, MMP9 was critical for effective bacterial phagocytosis and reactive oxygen species generation in polymorphonuclear neutrophils. These data demonstrate critical nonredundant and protective roles for MMP2 and MMP9 in the early host immune response against S. pneumoniae infection.

Hypoxia-induced compression in the tracheal system of the tobacco hornworm caterpillar, Manduca sexta

SUMMARY Abdominal pumping in caterpillars has only been documented during molting. Using synchrotron X-ray imaging in conjunction with high-speed flow-through respirometry, we show that Manduca sexta caterpillars cyclically contract their bodies in response to hypoxia, resulting in significant compressions of the tracheal system. Compression of tracheae induced by abdominal pumping drives external gas exchange, as evidenced by the high correlation between CO2 emission peaks and body movements. During abdominal pumping, both the compression frequency and fractional change in diameter of tracheae increased with body mass. However, abdominal pumping and tracheal compression were only observed in larger, older caterpillars (>0.2 g body mass), suggesting that this hypoxic response increases during ontogeny. The diameters of major tracheae in the thorax increased isometrically with body mass. However, tracheae in the head did not scale with mass, suggesting that there is a large safety margin for oxygen delivery in the head in the youngest animals. Together, these results highlight the need for more studies of tracheal system scaling and suggest that patterns of tracheal investment vary regionally in the body.