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The Journal of Immunology, 2003, 171: 3655-3667.
Copyright © 2003 by The American Association of Immunologists

Global Gene Expression Profiles During Acute Pathogen-Induced Pulmonary Inflammation Reveal Divergent Roles for Th1 and Th2 Responses in Tissue Repair1

Netanya G. Sandler*, Margaret M. Mentink-Kane*, Allen W. Cheever{dagger} and Thomas A. Wynn2,*

* Immunopathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and {dagger} Biomedical Research Institute, Rockville, MD 20852


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T helper 1 responses are typically proinflammatory, while Th2 responses have been considered regulatory. Interestingly, Th2 responses characterize a number of pulmonary diseases, many of which terminate in tissue remodeling and fibrosis. We developed a mouse model using Schistosoma mansoni eggs and cytokine-deficient mice to induce highly polarized Th1- or Th2-type inflammation in the lung. In this study, we examined the pathology and cytokine profiles in Th1- and Th2-polarized environments and used oligonucleotide microarray analysis to decipher the genes responsible for these effects. We further elaborated on the results using IL-10- and IL-13-deficient mice because these cytokines are believed to be the central regulators of Th2-associated pathology. We found that the Th1-polarized mice developed small granulomas with less fibrosis while expressing genes characteristic of tissue damage. Th2-polarized mice, in contrast, formed large granulomas with massive collagen deposition and up-regulated genes associated with wound healing, specifically, arginase, collagens, matrix metalloproteinases (MMPs), and tissue inhibitors of MMP. In addition, several members of the chitinase-like family were up-regulated in the lung following egg challenge. We also developed a method of defining the net collagen deposition using the expression profiles of several collagen, MMP, and tissue inhibitors of MMP genes. We found that Th1-polarized mice did not elaborate collagens or MMPs and therefore did not have a significant capacity for repair in this model. Thus, Th1-mediated inflammation is characterized by tissue damage, while Th2 directs wound healing and fibrosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Fibrosis develops in a number of inflammatory diseases of the lung. Idiopathic pulmonary fibrosis, for example, results from Th2-mediated inflammation, leading to progressive deposition of extracellular matrix, pulmonary fibrosis, and destruction of pulmonary parenchyma (1, 2, 3). In radiotherapy of thoracic malignancies, the Th2 response can cause pneumonitis and subsequent pulmonary fibrosis, limiting the dose of radiation (4). Furthermore, eosinophilia, subepithelial fibrosis, mucus overproduction, and airway hyperresponsiveness characterize asthma (5). Th2 lymphocytes are crucial in affecting all these sequelae (6, 7, 8).

Our lab has focused on the Th2 response elicited by the parasite Schistosoma mansoni. Intravenous injection of S. mansoni eggs, which are trapped in the lung, is a well-documented model of Th2 granuloma formation used to study basic mechanisms of asthma, allergy, and other Th2-mediated inflammatory diseases. Neutralization of IL-4, which drives the Th2 response, results in smaller granulomas and diminished Th2 responses (9, 10, 11). In contrast, neutralization of IL-12, which drives the Th1 response, up-regulates granuloma size and Th2 cytokine production. Interestingly, while IL-10 predisposes the mouse to the development of Th2 responses, it also regulates both Th1 and Th2 responses (12, 13, 14). In the absence of IL-10, a mixed Th1/Th2 environment results, characterized by increased IL-4 and IL-12 (15, 16). Consequently, we have developed IL-10/IL-4 and IL-10/IL-12 double knockout (KO)3 mice, which form extremely polarized Th1 and Th2 responses, respectively, to study these different responses. The simultaneous elimination of IL-4 and IL-10 results in very small granulomas with little eosinophilia, while IL-12- and IL-10-deficient mice develop large eosinophil-rich lesions.

Although we have documented the effects of immune polarization on pulmonary granuloma formation, the detailed mechanism by which this occurs remains unknown. Only a small percentage of the genes potentially responsible for extracellular matrix remodeling, collagen deposition, inflammatory cell infiltration, and resolution of the inflammatory response have been identified in the lung. Indeed, little is known about what accounts for the differential pathology of Th1- and Th2-polarized mice, and which Th1- and Th2-type genes account for the intermediate pathology of wild-type (WT) animals. We endeavored to define the genes responsible for the acute pathology in Th2-polarized mice and lack thereof in Th1-polarized mice to understand more about the processes responsible for the pathology seen in Th2-type diseases.

WT, IL-10/IL-4 KO, and IL-10/IL-12 KO mice were sensitized i.p. and challenged i.v. with S. mansoni eggs, then pulmonary mRNA expression profiles were examined via microarray analysis. We analyzed mRNA from these mice at days 0, 4, 8, and 14 postchallenge with oligonucleotide microarrays printed with ~7000 genes and confirmed the observations with real-time PCR. We discovered various clusters of genes that indicate the similarities in these three strains, and more importantly, the differences between Th1- and Th2-polarized mice, which most likely account for their unique pathological responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Induction of pulmonary granulomas

Six- to eight-week-old C57BL/10, C57BL/10Ai KO IL-10, C57BL/6Ai-(KO) IL-10/IL-4 mice, and C57BL/6Ai-(KO) IL-10/IL-12 were obtained from Taconic Farms (Germantown, NY). Breeding pairs of WT F2 and IL-13 KO (129Ola x C57BL/6 (F2)) were provided by A. McKenzie (Medical Research Council, Cambridge, U.K.). All mice were housed under specific pathogen-free conditions at the National Institutes of Health in an American Association for the Accreditation of Laboratory Animal Care-approved animal facility. S. mansoni eggs were extracted from the livers of infected mice at the Biomedical Research Institute and enriched for mature eggs. For the sensitization, mice were immunized with 5000 eggs i.p., and then challenged with 5000 eggs i.v. 14 days later.

Histopathology

For measurement of granulomas, the right lung was inflated with Bouin-Hollande fixative, and histologic sections were processed and stained with Giemsa and picrosirius red (collagen stain) (Histo-Path of America, Clinton, MD). The diameter of at least 30 granulomas was measured in each animal, and the average volume was calculated assuming a spherical shape for the lesions. Eosinophils were enumerated in the same slides.

Lymphocyte culture and cytokine detection

Periaortic lymph nodes were removed aseptically, and single-cell suspensions were prepared. Cells were plated in 24-well tissue culture plates at a final concentration of 3 x 106 cells/ml in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 50 µM 2-ME, and antibiotic-antimycotic solution (all from Life Technologies, Gaithersburg, MD). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2. Cells were stimulated with soluble S. mansoni egg Ag (20 µg/ml) or medium alone. Supernatant fluids were harvested at 72 h and assayed for IL-4, IL-5, IL-10, and IFN-{gamma} using paired Abs (BD PharMingen, San Diego, CA). IL-13 was determined using a detection kit from R&D Systems (Minneapolis, MN). Cytokine levels were calculated with standard curves constructed using recombinant murine cytokines.

RNA isolation and cDNA synthesis

At each time point (days 0, 4, 8, and 14 postchallenge for WT, 10/4 KO, 10/12 KO), 0.2 g of lung from five mice in each genotype was homogenized in TRIzol (Life Technologies) with a tissue polytron (Omni International, Warrenton, VA). Total RNA isolation was performed, as recommended (Life Technologies). A total of 50 µg of RNA from each experimental sample was used to generate a reference pool, allowing gene expression profiles to be compared with the average of all samples at all time points. Fluorescent cDNA targets were prepared from 50 µg of the experimental RNA sample (dUTP-Cy5; Amersham, Piscataway, NJ) and 50 µg of reference RNA (dUTP-Cy3; Amersham) in a reverse-transcription reaction using SuperScript II (Life Technologies).

Oligonucleotide microarray

Glass microarrays spotted with ~7000 genes printed in duplicate were obtained from the National Institute of Allergy and Infectious Disease Microarray Facility. The GEO accession assigned to this platform was GPL476. The microarrays were hybridized with fluorescently labeled cDNA targets, as prepared above, and scanned using a GenePix 4000A microarray scanner (Axon Instruments, Union City, CA). Image analysis was performed using GeneSpring (Silicon Genetics, Redwood City, CA), BRB Array Tools (Biometric Research Branch, National Cancer Institute), and DAVID (Glynn Dennis, Laboratory of Immunopathogenesis and Bioinformatics, National Institute of Allergy and Infectious Diseases (NIAID)).

Real-time PCR

For real-time PCR detection of RNA transcripts from WT, 10/12 KO, 10/4 KO, IL-10 KO, and IL-13 KO, total RNA was isolated from ~0.2 g lung tissue placed individually in 1 ml RNAStat60 (Tel-Test, Friendswood, TX). The sample was homogenized (Omni International), and total RNA isolation was performed using RNeasy Mini Kit (Qiagen Sciences, Germantown, MD). Total RNA was used to generate cDNA in a reverse-transcription reaction with Superscript II (Life Technologies). The cDNA was analyzed in an ABI PRISM 7900 sequence detection system using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-12, MMP-13, and tissue inhibitor of MMP-1 (TIMP-1) primers have previously been described. The real-time PCR primers (Invitrogen Life Technologies, Carlsbad, CA) were designed as set forth in Table I.


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  		Table I. Primers used in this studya

 
Statistical analyses

Student’s t test was used to compare RT-PCR results. A p value <0.05 was considered significant. For microarray analysis, hybridization signals (Cy5/Cy3 ratios) were normalized to the median signal on each array. In GeneSpring, microarrays were also normalized to negative controls. In BRB Array Tools, genes were excluded if they had a p value >0.0005 in all arrays or if they were missing values in three or more arrays. Red or green signals <10, spot sizes <10, and flagged genes were also excluded, yielding ~1200 genes. In both programs, calibrated ratio values were log (base 2) transformed. Hierarchical clustering based on one minus correlation with average linkage was performed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cytokine responses are exaggerated in immune-polarized mice

Lymph node cultures reveal the different cytokines induced upon egg challenge (Fig. 1). WT mice develop a dominant Th2 response, which peaks on day 7 and declines by day 14, with significant egg Ag-specific IL-4 and IL-13 and relatively low IFN-{gamma} and IL-10 (data not shown) protein levels present. IL-10/IL-12 KO mice display even higher IL-4 and IL-13 levels, approaching 6-fold higher on day 14, with almost no IFN-{gamma}. In contrast, IL-10/IL-4 KO mice have virtually no IL-4 or IL-13, with significantly more IFN-{gamma} than WT controls at both time points.



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  		FIGURE 1. Enhanced cytokine production in immune-polarized mice following challenge with S. mansoni eggs. Groups of five WT, IL-10/IL-4 KO, and IL-10/IL-12 KO mice were sensitized with 5000 freshly isolated eggs of S. mansoni and challenged i.v. 14 days later with 5000 eggs. On days 7 and 14 postchallenge, mice were sacrificed and single-cell suspensions of periaortic lymph nodes were collected after stimulation with soluble egg Ag for 72 h. Stimulated samples were analyzed for the presence of IFN-{gamma}, IL-4, and IL-13 by ELISA, as detailed in Materials and Methods.

 
Mice that generate a highly polarized Th1 (IL-10/IL-4 KO) or Th2 (IL-10/IL-12 KO) response react quite differently to pulmonary S. mansoni egg challenge. Th1-polarized mice form small granulomas with little eosinophil infiltration, while Th2-polarized mice elaborate significantly larger granulomas with marked eosinophilia (Fig. 2). Tissue sections stained with picrosirius red show little collagen deposition in the lungs of IL-10/IL-4 KO mice, while IL-10/IL-12 KO mice develop significant fibrosis with rapid onset (Fig. 2). WT mice, interestingly, develop a pathology intermediate of the Th1- and Th2-polarized mice, although more closely resembling the Th2 mice in granuloma formation and eosinophilia. The granuloma size in all three strains peaked at day 8 postchallenge, with involution occurring by day 14. Both the IL-10 KO and IL-13 KO mice parallel this pattern, however; the IL-13 KO mice generated smaller granulomas, and IL-10 KO mice on average formed slightly larger granulomas than WT mice (data not shown). Consequently, both IL-10 KO and IL-13 KO mice form granulomas intermediate of the Th2-polarized and Th1-polarized mice. Likewise, lung eosinophils continue to increase in WT and Th2-polarized mice between days 8 and 14, but decrease markedly in Th1-polarized mice during this period, and the eosinophilia of both IL-10 KO and IL-13 KO mice closely resembles that of WT mice (data not shown). Interestingly, mice deficient only in IL-10 develop more IL-5, IL-13, and IFN-{gamma}, particularly at day 14, while IL-13-deficient mice have IL-5, IL-10, and IFN-{gamma} levels comparable to WT at day 7, but with less IL-5 at day 14 (data not shown).



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  		FIGURE 2. Granuloma volume and tissue eosinophils. Lung tissue from WT, IL-10/IL-4 KO, and IL-10/IL-12 KO mice (five mice per group/time point) was fixed in Bouin-Hollande solution and stained with Giemsa. Average granuloma volumes (A) and percentage of eosinophils ± SE (B) are shown. Additional slides were stained with picrosirius red to illustrate the marked difference in collagen deposition in the immune-polarized mice. Representative day 8 granulomas from IL-10/IL-12 KO (C) and IL-10/IL-4 KO (D) mice are shown at x10 magnification.

 
Oligonucleotide microarrays reveal distinct patterns of gene expression between Th1- and Th2-polarized mice following S. mansoni egg challenge

To investigate possible mechanisms for this differential pathology, we used oligonucleotide microarrays to evaluate gene expression profiles for WT, Th1-polarized, and Th2-polarized mice at days 0, 4, 8, and 14 postchallenge. We performed hierarchical clustering analysis, cutting the dendogram at ~0.8 correlation yielding 171 clusters. A subset of Th1 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)- and Th2 (30, 31, 32, 33)-associated gene clusters is shown in Fig. 3. Grouping together adjacent clusters with similar expression profiles, we isolated five unique patterns reflecting genes that may account for both the similarities and the differences in pathology in these mice.



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  		FIGURE 3. Several unique gene clusters characterize Th1- and Th2-polarized pulmonary inflammation. Total RNA isolated from whole lung tissue at days 0 (unchallenged mice), 4, 8, and 14 postchallenge was used to generate labeled cDNA for hybridization to NIAID oligonucleotide arrays. Hierarchical clustering was performed using BRB Array Tools. Genes that showed reduced (green) or increased (red) expression relative to the reference RNA are included. Individual gene clusters are numbered on the left with representative examples of Th1-associated (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 ) and Th2-associated (30 31 32 33 ) gene clusters shown in A and B, respectively.

 
Groups 1, 2, and 3 display genes that are regulated similarly in all three strains

First, we investigated which genes were characteristic of inflammation in general. We were curious which genes were up-regulated independent of whether a Th1- or Th2-polarized inflammatory response was generated (Table II). Analysis revealed that chemokines were up-regulated initially in all three strains of mice (full data set available on line, upon request). In particular, monocyte chemotactic protein-1 (MCP-1), macrophage-inflammatory protein 1{beta} (MIP-1{beta}), and thymus- and activation-regulated chemokine (TARC) were expressed at day 4 (group 1). Interestingly, their expression in WT and Th1-polarized mice declines by day 8, but continues to be strong in the Th2-polarized mice through day 8. Together, these chemokines recruit T cells, monocytes, basophils, NK cells, and dendritic cells. In addition, genes reflective of cytokine induction are present, including TGF-{beta}-induced gene, IL-4-induced gene, cytokine-inducible Src homology 2-containing protein 1, and IL-6, as well as other genes characteristic of the response to a biotic stimulus, such as complement component 1q and an IgG FcR. The expression of these genes in Th2-polarized mice at day 8 suggests the persistence of activation of the immune response.


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  		Table II.
 
By days 8 and 14, a different set of genes is up-regulated in all three strains of mice (group 2). In particular, the expression of genes involved in organogenesis was increased, including homeobox C4 and B9. Genes for proteins that require calcium or that regulate calcium flux were also up-regulated, e.g., CCR9- and Ca2+-dependent activator protein for secretion, respectively. Also within this latter group are desmocollins 1 and 3 and protocadherin {alpha}7, all involved in desmosome formation. Importantly, transcription of four proteins implicated in airway smooth muscle hyperreactivity is increased, including three types of potassium channels, mitogen-activated protein kinase 1, and calcitonin. Furthermore, phospholipase A2 and PGD2 are increased, reflective of eicosanoid activation. Strikingly, androgen, progesterone, and estrogen receptors are all up-regulated. The expression pattern of all these genes appears to peak at day 8 in Th1-polarized mice and day 14 in WT and Th2-polarized mice.

In contrast, all three mouse strains down-regulate some genes upon egg challenge (group 3). Interestingly, genes involved in skeletal and cardiac muscle contraction, e.g., actin, myosin, and troponin, are down-regulated. Similarly, expression of genes responsible for arachidonic acid epoxygenation and PG inactivation is decreased, including several members of the cytochrome P450 family and hydroxyprostaglandin dehydrogenase 15. In addition, expression of oxygen radical scavengers, such as GST, oxygen radical generators such as aldehyde oxidase, and genes involved in oxidative metabolism including flavin-containing mono-oxygenases, is diminished. Finally, hemoglobin {beta}, X, and Z are all down-regulated.

Group 4 exhibits genes associated with Th1-polarized phenotype

WT mice 4 days postchallenge and Th1-polarized mice throughout the time following challenge express proteins already known to associate with a Th1 response, such as IFN-{gamma}-induced proteins and TNF-{alpha}-induced protein 2 (Fig. 3A and Table II). A variety of chemokines was expressed as well, such as IFN-inducible protein-10 and RANTES, recruiting monocytes, T cells, dendritic cells, and NK cells. Furthermore, NK cell ligands are up-regulated, and macrophage activation is reflected by the up-regulation of MIP-3{alpha}, macrophage-expressed gene 1, macrosialin, and macrophage C-type lectin.

Th1-polarized mice, and WT mice early on, also display a predisposition to the acute-phase response. Both IL-1{beta} and its activator caspase 1 are increased, as well as serum amyloids A2 and A3. A number of genes involved in cell cycle regulation, particularly in mitosis, were up-regulated solely in the Th1 mice. Cyclin A, which facilitates the transition from S phase into G2 phase, and cyclin B, which facilitates progression from G2 to M phase, are both up-regulated. Also, cell division cycle 2 homologue A, which binds cyclin A, is increased. Most fascinating, however, was the increase in cytotoxic genes, including granzymes A, B, and K; calgranulins A and B; caspases 1 and 3; and programmed cell death 1 ligand (Table II). Furthermore, genes responsible for intracellular protein degradation, e.g., ubiquitin D, ubiquitin-conjugating enzyme 8, and cathepsin D, are up-regulated.

Group 5 portrays genes up-regulated in Th2-polarized mice

Group 5 contains genes reflecting the Th2 immune response in both the WT and Th2-polarized mice (Fig. 3B). Chemokines that recruit Th2 effector cells, such as MCP-2, are up-regulated, as are proteins induced by Th2 cytokines, such as TGF-{beta}-induced and IL-4-induced gene 1. Importantly, IL-13 expression, a key mediator of fibrosis, increases. Eosinophilia is suggested not only by the expression of chemokines for eosinophils, including several eotaxins, but also by the increased transcription of eosinophil-synthesized proteins. Indeed, eosinophil-associated ribonucleases 1, 2, and 5 are all up-regulated. In addition, alternatively activated macrophages also appear to be present, as their marker arginase is up-regulated. Furthermore, synthesis of leukotrienes and thromboxanes is suggested by the induction of arachidonate 15-lipoxygenase and platelet thromboxane A synthase 1 (Table II).

Of note, a variety of proteins involved in wound healing appears in a sixth group. Not only are MMP-12 and MMP-13 increased, but also TIMP-1, the protein that degrades the majority of MMPs. Examination of the profiles on GeneSpring further revealed maximum induction of MMP-9, MMP-13, and TIMP-1 at day 4, followed by TIMP-2 at day 8, with MMP-12 continuing to rise at day 14 in WT and Th2-polarized mice (Fig. 4A). Interestingly, precursors of elements of the extracellular matrix, namely, procollagens, follow this pattern as well. Indeed, procollagen, types I, V, and XVIII, is expressed early, at day 4, with procollagen, type XVIII, peaking at day 8 (Fig. 4B). Moreover, coagulation factors III (thromboplastin), VII, and X are all up-regulated, suggesting activation of the extrinsic pathway of the coagulation cascade, which is triggered by tissue injury. Furthermore, expression of four members of the small proline-rich protein family, involved in the formation of cornified epithelium, was increased.



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  		FIGURE 4. MMPs, procollagens, and several members of the chitinase-like protein family are up-regulated in WT and Th2-polarized mice. Total RNA isolated from whole lung tissue at days 0 (unchallenged mice), 4, 8, and 14 postchallenge was used to generate labeled cDNA for hybridization to NIAID oligonucleotide arrays. GeneSpring was used to graph the expression profiles and the data for MMP (A), procollagen (B), and chitinase-like (C) genes illustrated as fold changes over time, using the pooled RNA from all time points as the reference. Greater than 2.5-fold change in expression was considered significant.

 
Strikingly, several chitinase-like proteins were induced in Th2-polarized and WT mice. Chitinase 3-like 3 (Ym1), resistin-like {alpha} (FIZZ1), and acidic chitinase all showed dramatically increased induction over time (Fig. 4C). Two other chitinase-like proteins, oviductal glycoprotein and chitinase 3-like 1, did not follow this trend.

Confirmation of extracellular matrix-related gene expression

To confirm and expand upon the collagen, MMP, and TIMP results, we performed real-time PCR on lung tissue. We analyzed the expression of several procollagens over the 2-wk time course. We found that WT mice displayed a 5-fold increase in procollagen type III message at day 4, a greater than 50-fold increase in procollagen type IV through day 14, and a 4-fold elevation in procollagen type V at days 4 and 8 postchallenge relative to WT naive mice (Fig. 5A). In contrast, the procollagens V, XIV, and XVIII were the most highly induced procollagens in Th2-polarized mice. Interestingly, procollagen IV expression actually declines upon egg challenge, although Th2-polarized mice induce significantly more of this procollagen at day 14 than the other strains. Strikingly, Th1-polarized mice do not up-regulate procollagen expression at all following egg challenge. In contrast, IL-10 KO mice significantly increase their synthesis of procollagen I, III, and XIV mRNA upon egg challenge, expressing significantly more procollagen III and IV than WT mice. Furthermore, IL-13 KO mice synthesized markedly less mRNA of procollagens I, III, and V than WT mice at day 7 (Fig. 5B), but procollagens V and XVIII were significantly induced upon egg challenge in the IL-13 KO mice. As in the immune-polarized mice, procollagen IV appears to be down-regulated following challenge in both IL-10 KO and IL-13 KO mice.



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  		FIGURE 5. Real-time PCR confirms the procollagen mRNA expression profiles delineated by microarray analysis. The values were normalized to hypoxanthine phosphoribosyltransferase (HPRT), and fold changes were generated by comparing with WT unchallenged mice. Greater than 2.5-fold change in expression was considered significant. A, Fold change in mRNA expression for procollagen genes in WT, IL-10/4 KO, IL-10/12 KO, IL-10 KO mice. B, Fold change in mRNA expression for procollagen genes in WT and IL-13 KO mice.

 
As with the procollagens, we performed real-time PCR to examine the expression of several MMPs at days 4, 8, and 14 following egg challenge (Fig. 6A). The WT mice displayed a minimal increase in MMP-3 expression following egg challenge, but a greater than 25-fold induction of MMP-9 and a >200-fold increasein MMP-13 at day 8 postchallenge. Even more striking was the exponential up-regulation of MMP-12 to 3000-fold the levels of the naive WT mice. Up to a 20-fold increase was seen in TIMP-1 over the time course. Similar to egg-challenged WT mice, Th2-polarized mice displayed increased MMP-9 expression relative to WT naive mice, although it peaked earlier and at a lower level. Like the WT mice, the Th2-polarized mice displayed a sharp exponential increase in MMP-12 continuing through day 14, yet MMP-13 mRNA peaked at a dramatically higher level than WT. In contrast to WT mice, Th2-polarized mice down-modulate MMP-3. Furthermore, the baseline TIMP-1 expression in naive IL-10/IL-12 KO mice is much lower than in WT naive mice, but quickly increases to 3000-fold its basal level upon challenge. In contrast, very little change was observed in the MMP message levels in Th1-polarized mice. We observed increased expression of MMP-9 at day 4, MMP-3 at day 8, and TIMP-1 throughout the time course, but these changes were all less than 10-fold. No alteration was observed in expression of MMP-12 or MMP-13. Likewise, TIMP-1 as well as most MMPs were relatively unchanged in IL-10 KO mice over the time course. Interestingly, MMP-2 expression showed an increase, while MMP-9 expression declined. In contrast, the absence of IL-13 resulted in a dramatic decrease in MMP-3, MMP-13, and TIMP-1 at both days 7 and 14 postchallenge, with a significant decrease in MMP-12 at day 7 (Fig. 6B). In contrast to the IL-10-deficient mice, MMP-9 expression increased in the IL-13 KO mice at day 14.



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  		FIGURE 6. Real-time PCR confirms the MMP and TIMP mRNA expression profiles delineated by microarray analysis. The values were normalized to HPRT, and fold changes were generated by comparing with WT unchallenged mice. Greater than 2.5-fold change in expression was considered significant. A, Fold change in mRNA expression for MMPs and TIMP-1 in WT, IL-10/4 KO, IL-10/12 KO, IL-10 KO mice. B, Fold change in mRNA expression for procollagen genes in WT and IL-13 KO mice.

 
Because collagens are degraded by MMPs, and TIMPs inhibit MMP activity, net collagen deposition depends on the dynamic interactions of all three of these proteins. We expressed this relationship by first determining the relative change in MMP activity, expressed as a ratio of the fold change in MMP to the fold change in TIMP. Next, to determine the ratio of collagen production to degradation relative to WT naive mice, we divided the fold change in procollagen mRNA by this relative MMP activity: fold change in procollagen mRNA/(fold change in MMP)/(fold change in TIMP).

Because all of these values are relative to WT day 0, the procollagen/MMP:TIMP ratio is 1 for the naive control, assumed to have no net collagen deposition or degradation. Hence, values greater than 1 reflect a tendency toward collagen deposition relative to steady state, and numbers less than 1 reflect matrix degradation. Inspecting ratios of different collagens to the specific MMPs that degrade them, we discovered a remarkable pattern. For all five collagens examined, the net collagen/MMP:TIMP ratio was markedly highest in 10/12 KO mice, irrespective of the type of collagen or MMP (Fig. 7A). Furthermore, in most cases, IL-10/IL-4 KO mice had the lowest ratio of the three strains, with WT mice in the middle. In addition, the highest ratios were observed with collagens I, IV, V, and XIV in the Th2-polarized mice (Fig. 7A), while collagens I, IV, and V were intermediate in WT mice. Just as MMPs were relatively unchanged in IL-10 KO mice, the ratios were relatively unchanged over the time course in these mice. Impressively, for most profiles examined, the IL-13 KO mice had significantly lower collagen/MMP:TIMP ratios than WT mice, particularly at day 7 (Fig. 7B).



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  		FIGURE 7. Collagen/MMP:TIMP ratios parallel the pathology seen upon egg challenge. Relative MMP expression was calculated by determining the fold change of the MMP mRNA expression relative to that of its inhibitor, TIMP-1, based on real-time PCR results. The fold change value for procollagen mRNA was divided by the fold change value for the relevant MMP to yield the fold change in the ratio of collagen deposition to collagen degradation compared with steady state. A, Collagen/MMP:TIMP ratios in WT, IL-10/12 KO, IL-10/4 KO, IL-10 KO mice (legend same as shown in Fig. 5A). B, Collagen/MMP:TIMP ratios in WT and IL-13 KO mice.

 
Confirmation of chitinase-like gene expression

Finally, the observed up-regulation of acidic chitinase, chitinase 3-like 3 (Ym1), and resistin-like {alpha} (FIZZ1) was confirmed with real-time PCR (Fig. 8A). Indeed, the mRNA of all three chitinase-like proteins was increased in WT and Th2-polarized mice. Ym1 had a 15-fold increase in expression in both the Th2-type and WT mice at days 4 and 8, but while the expression of Ym1 in WT mice leveled off by day 14, it continued to increase in Th2-polarized mice. FIZZ1 displayed an even more dramatic 125-fold boost in mRNA levels in WT mice at day 4, with a greater than 50-fold induction in Th2-polarized mice at this time point. Like Ym1, FIZZ1 expression declined in WT mice by day 14 while continuing to rise in the Th2 mice. Similarly, acidic chitinase displayed a ~10-fold increase in WT mice, with Th2-polarized mice expressing a ~20-fold increase over naive WT mice by day 14. Of note, the levels of mRNA of all three chitinases increased significantly in the Th2-polarized mice between days 8 and 14. In marked contrast, chitinase-like gene expression did not change much in the Th1-polarized mice. Interestingly, IL-10 KO mice had a significantly lower basal expression of Ym1, FIZZ1, acidic chitinase, and chitinase 3-like 1. Although acidic chitinase did increase dramatically in IL-10 KO upon challenge, Ym1 and FIZZ1 displayed only slight inductions. In the absence of IL-13, Ym1 and FIZZ1 expression was significantly inhibited and acidic chitinase was ablated (Fig. 8B). In agreement with the microarray results, chitinase 3-like 1 was not increased following egg challenge.



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  		FIGURE 8. Real-time PCR confirms the chitinase-like protein mRNA expression profiles delineated by microarray analysis. The values were normalized to HPRT, and fold changes were generated by comparing with WT unchallenged mice. Greater than 2.5-fold change in expression was considered significant. A, Fold change in mRNA expression for chitinase-like genes in WT, IL-10/12 KO, IL-10/4 KO, IL-10 KO mice. B, Fold change in mRNA expression for chitinase-like genes in WT and IL-13KO mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The classification of IL-10/IL-4 KO and IL-10/IL-12 KO into Th1- and Th2-polarized mice was confirmed by a variety of methods. The cytokine profiles reflect Th1- and Th2-mediated responses, with elevated IL-4, IL-13, and IL-5 in the 10/12 KO and elevated IFN-{gamma} in the 10/4 KO. Furthermore, IFN-{gamma} is induced slightly in WT mice, with virtually no induction in the IL-10/12 KO. The microarray study compounded this data. Genes reflective of IFN-{gamma} activation were significantly up-regulated in the Th1-polarized mice. Chemokines and their receptors associated with a Th1 response, e.g., RANTES and IFN-inducible protein-10, were increased, attracting neutrophils, monocytes, and lymphocytes (18, 19). In contrast, the chemokines expressed by 10/12 KO mice, including MCP-2, MCP-3, and MCP-5, as well as TARC, C10, eotaxin, and eotaxin 2, are increased in mice with enhanced IL-13 expression and mice lacking IFN-{gamma} (18, 20). Likewise, MIP-1{gamma} has been associated with a Th2 phenotype (18). Furthermore, TARC and eotaxin have both been observed in the bronchoalveolar lavage fluid of asthmatic patients (21, 22). Together, these chemokines recruit activated Th2 cells, eosinophils, and monocytes. Indeed, several markers of eosinophil activation such as eosinophil-associated ribonucleases are up-regulated in mice exhibiting Th2 responses (23, 24).

Two major groups of genes emerged in the Th1-polarized mice: those involved in the acute-phase reaction and those in apoptosis (Table II, group 4). Consistent with the enhanced acute-phase response, mice with a polarized Th1 response show marked weight loss relative to infected wild-type mice upon infection with a variety of pathogens, including S. mansoni and Plasmodium berghei (25, 26, 27). In addition to cachexia, mice infected with P. berghei in a Th1 environment suffered extensive coagulative necrosis of the liver despite lower parasitemia, and IL-10/4 KO mice infected with S. mansoni had significantly more hepatotoxicity, reflected by elevated aminotransferase levels (26, 27). Similarly, rIL-12 administration to mice infected with Candida albicans resulted in enhanced renal necrosis and accelerated mortality by an IFN-{gamma}-mediated mechanism (28). Furthermore, mice deficient in IL-10, resulting in enhanced Th1 responses, succumbed to infection with an avirulent strain of Toxoplasma gondii, with markedly enhanced liver and lung necrosis (29, 30). Numerous studies have also implicated ubiquitin in the proteolysis that accompanies apoptosis (31, 32, 33, 34). Although no gross necrosis was observed in the lung sections during this limited time period, the apparent activation of the cell cycle could be the organism’s attempt to compensate for cells lost due to tissue damage.

The predominant theme among genes up-regulated in Th2-polarized mice is wound healing. Indeed, the massive up-regulation of collagens and enzymes involved in collagen synthesis reflects tissue repair. Epithelial repair involves the deposition of a fibrin matrix, epithelial cell flattening along the basement membrane, and migration from the wound edge, followed by epithelial cell proliferation and myofibroblast differentiation (37). Squamous epithelial cell proliferation and differentiation are indicated by the presence of small proline-rich proteins (SPRRs), which form the cornified envelope underlying the plasma membrane of epithelial cells. In fact, squamous differentiation of the tracheobronchial epithelial cells is only seen during injury and repair, suggesting the presence of these processes (38). Although the SPRR1 family has been observed in the lung, the SPRR2 family has not. In addition, this is the first time an association between a Th2-type inflammatory response and SPRR2 has been made. Beneath the epithelium, myofibroblasts are then activated by growth factors such as IL-4, IL-13, or TGF-{beta} to synthesize collagens (39). Healthy lung consists of 90% collagen types I and III, while collagen type IV, and to a lesser extent, type V, are the main components of the alveolar and capillary basement membranes (40). Recently, type XVIII collagen has also been localized to the alveolar basement membrane (41, 42). Not surprisingly, then, the collagens up-regulated following egg exposure include types I, III, V, and XVIII, but not type IV. Interestingly, the subepithelial fibrosis manifested in asthma consists of collagens III and V, while the basal lamina, consisting of type IV, remains unchanged (43, 44). Also, the ratio of collagens to each other is important. The collagen I induction, normalized for changes in MMPs and TIMP expression, is much greater than that of type III, which would lead to the formation of larger fibrils compared with naive mice (45). Likewise, fiber size increases with higher collagen I:V ratios, yet the ratio of normalized I:V in both WT and 10/12 KO remains relatively unchanged or slightly increased throughout the time course (46). Together, the increased normalized I:III with little change in I:V suggests the formation of larger collagen fibrils in these mice. In addition, we observed the up-regulation of two collagens, XIV and XV, not previously identified in the lung. Collagen XIV, also known as undulin, binds to glycosaminoglycans and is frequently associated with collagen fibrils (47, 48). Interestingly, collagen XV has been observed in the alveolar basement membranes of fetal mice, decreasing dramatically several days before birth, implicating it in lung development (49). However, because the mRNA encodes for procollagen, further processing and assembly must occur before collagen deposition (50). Nonetheless, collagen XIV may stabilize the fibrils formed by collagens I and III, with V, XV, and XVIII repairing the alveolar basement membrane and XV playing a crucial role in the new developing alveolus structure.

Moreover, the specific collagen expression patterns depend upon the cytokines present. Without IL-10, regardless of the presence of IL-12, collagen XIV mRNA is up-regulated, suggesting that IL-10 suppresses its expression. Yet, in IL-10/4 KO mice, collagen XIV is not induced, indicating IL-4 may stimulate or IFN-{gamma} may suppress its expression. Alternatively, both IL-10 and IFN-{gamma} could be suppressing collagen XIV mRNA synthesis. Likewise, IL-4 may induce or IFN-{gamma} may suppress collagen XVIII as it is dramatically increased in the Th2-polarized mice, while the expression in IL-13 KO mice resembles WT mice. IL-13 seems to be necessary for collagens I, III, and V because they show little induction in its absence. Interestingly, IL-13 has been implicated in the pathogenesis of asthma in a number of studies, and increased deposition of collagens I, III, and V has been observed in the lungs of asthmatic patients (51, 52, 53, 54). Thus, IL-13 stimulates the precise collagens responsible for the pulmonary fibrosis seen in asthma patients.

MMPs have been associated with inflammatory responses in a wide variety of diseases. MMP-9, which can be expressed by T cells, facilitates inflammatory cell infiltration, including T cells and eosinophils, into tissues (55, 56, 57). MMP-9 also assists the migration of bronchial cells to areas of alveolar injury (58, 59). Likewise, MMP-12, expressed predominantly by macrophages, is required for macrophage and possibly neutrophil tissue penetration (60, 61). Consequently, the significant up-regulation could reflect the inflammatory cells’ synthesis of these MMPs to enter the lung. In contrast, given the high TIMP-1:MMP ratio and the fact that inflammatory cells produce many of these MMPs, the increased MMP levels could merely reflect the presence of the cells themselves and not necessarily tissue degradation. Thus, the marked up-regulation in MMP-12 seen at day 14 in WT and IL-10/12 KO mice could reflect massive infiltration of macrophages rather than elastin degradation, especially because protease activity may be modulated at this time point by TIMP-1.

Both Th1 and Th2 cytokines can affect MMP and TIMP expression. We observed an increase in MMP-3 expression in the WT and 10/4 KO mice and a decrease in the 10/12 KO mice. This finding is consistent with previous studies showing that IL-1{beta} and TNF-{alpha} up-regulate MMP-3 while IL-13 decreases it (62, 63, 64, 65, 66). Furthermore, expression of both MMP-9 and MMP-12 has been attributed to a Th2 environment, particularly TGF-{beta} and IL-13, which could explain their induction in WT and 10/12 KO mice (67, 68, 69, 70, 71). That TIMP-1 is increased by all these cytokines as well as by IL-10 most likely explains why it is increased in all three strains (68, 69, 72, 73, 74, 75, 76, 77). However, the absence of TIMP-1 induction in IL-13 KO mice demonstrates that IL-13 is necessary for its expression in the lung.

Extracellular matrix turnover is hypothesized to depend on MMP:TIMP ratios. Higher MMP expression leads to more degradation, while higher TIMP levels result in inhibition of degradation, allowing collagen to be deposited. In fact, hepatic fibrosis, usual interstitial pneumonia, and fibrosis in left ventricular remodeling have all been attributed to increased TIMP:MMP ratios (78, 79, 80). Likewise, decreased TIMP:MMP ratios have been shown to mediate the antifibrotic effects of treatments such as IFN-{alpha} and polyenylphosphatidylcholine on hepatic fibrosis (81, 82). Strikingly, the ratios we observed exactly parallel the observed granuloma size in the polarized mice. That is, the ratios are highest in Th2-polarized mice, lowest in Th1-polarized mice, and intermediate in the WT mice (Fig. 7), suggesting that this ratio, rather than expression of individual collagens, MMPs, and TIMPs, determines the size and character of the granuloma that develops. Together, these conclusions nicely demonstrate how pathogenic processes can be better understood by performing global gene expression profiling in whole tissues.

Transcripts for several macrophage phenotypic markers were also distinct between the WT, IL-10/IL-12 KO, and IL-10/IL-4 KO strains. The presence of macrophage C-type lectin and the absence of arginase expression in the 10/4 KO mice are consistent with the predominance of classically activated macrophages. In fact, a Th1 environment has already been shown to drive the classical activation of macrophages (83). Inducible NO synthase, one of the major products of classically activated macrophages, plays a key role in macrophage cytotoxicity by facilitating the production of NO (84). The up-regulation of arginase-1, Ym1, and FIZZ1 indicates that alternatively activated macrophages dominate the response in WT and Th2-polarized mice (85). Arg-1 may promote the development of fibrosis when L-arginine is metabolized to proline in the cell (86). Although collagen/MMP:TIMP ratios peak at day 4 postchallenge, suggesting this is the peak time of collagen mRNA production, the actual manufacture of collagen may take longer. The continuing high expression of arginase-1, Ym1, and FIZZ1 at day 14 most likely reflects the continuing presence and activity of a large population of alternatively activated macrophages. The high expression of MMP-12, or macrophage metalloelastase, at day 14 also supports this conclusion. If a large number of alternatively activated macrophages are present and synthesizing arginase, then a large amount of proline would be available to fibroblasts for collagen synthesis.

The chitinase-like family of enzymes has recently been recognized in mammals. Although Ym1 demonstrates no chitinase activity, it appears to bind saccharides with a free amine group, including N-glucosamine, and their oligosaccharides, as well as heparin (90). Interestingly, S. mansoni eggs, although they do not contain chitin, do express a number of oligosaccharides with amine groups (91). Thus, Ym1 and the other induced chitinases could play a role in egg degradation. Alternatively, leukocyte homing receptors bind to the same substrates, so Ym1 may compete with leukocytes for binding to the extracellular matrix and thereby modulate inflammatory cell infiltration. However, the evidence is growing that the chitinase-like family may be involved in wound healing. Acidic chitinase localizes within macrophages, and Ym1 and FIZZ1 have specifically been associated with alternatively activated macrophages (85, 93, 94, 95). Importantly, all chitinase-like family members are secreted proteins whose peak expression level correlates with inflammation, development, tissue remodeling, and/or wound healing (90). Finally, expression of Ym2, closely related to Ym1, depends upon IL-4R{alpha} signal transduction (94, 95). We show that in the absence of IL-13, Ym1, FIZZ1, and acidic chitinase mRNA are significantly reduced, and their expression correlates with a Th2-polarized phenotype (96, 97). The dramatic induction of these genes between days 8 and 14 indicates they may be involved in tissue repair or may simply reflect an increase in the alternatively activated macrophage population.

Although the Th1- and Th2-polarized mice develop distinct expression profiles, the WT mice display an intermediate phenotype. S. mansoni egg-induced pulmonary granuloma formation has been characterized by an initial mixed Th1- and Th2-type inflammation, followed by a polarized Th2 response. As early as day 1 postchallenge, IL-6, IL-1{beta}, and IFN-{gamma} are induced in WT mice, with subsequent induction of IL-2, IL-4, IL-5, and IL-10 by 3 days postchallenge (9, 98). IFN-{gamma} production falls dramatically by 7–10 days postchallenge, while the other cytokines remain elevated (98). The early presence of IFN-{gamma} has been shown to be crucial in controlling granuloma size (11). Our microarray studies nicely illustrate this early mixed response with simultaneous induction of Th1- and Th2-type genes at day 4, followed by only Th2-type genes at days 8 and 14 (Fig. 3, A and B). Furthermore, the microarray studies suggest several possible mechanisms for Th1-mediated granuloma modulation. First, as noted above, WT and Th1-polarized mice, but not Th2-polarized mice, elaborate genes responsible for neutrophil and NK cell-mediated cytotoxicity, inducing apoptosis of the target cells. The decreased eosinophilia observed in the WT mice relative to Th2-polarized mice could reflect this cytotoxic control of inflammatory cells. Second, the induction of IFN-{gamma} could inhibit production of both MMPs and TIMPs, thereby decreasing extracellular matrix turnover. Support for this comes from IL-10 KO mice, which have increased IFN-{gamma} production and decreased production of most MMPs and TIMP-1 relative to WT mice. Thus, although Th2-type cytokines dominate in WT mice, the Th1 effect results in fewer inflammatory cells and less fibrosis-inducing IL-13, as well as IFN-{gamma} present to antagonize the IL-13-mediated induction of both MMPs and TIMP-1.

In conclusion, we have demonstrated that the Th1 response to i.v. S. mansoni eggs is characterized by expression of genes crucial for the development of cytotoxicity, while the Th2 response directs tissue remodeling and wound healing. We have demonstrated that IL-13 is essential for production of collagens I, III, and V. In addition, we developed a novel approach to illustrate interactions between collagens, MMPs, and TIMPs. These data showed excess production of collagens I, IV, and V relative to their degradation, suggesting these collagens are crucial for repairing the lung tissue and responsible for much of the tissue remodeling observed in Th2-polarized responses. Moreover, we discovered elevations of Ym1, FIZZ1, and acidic chitinase mRNA only during a Th2 response, and that IL-13 stimulates their synthesis by alternatively activated macrophages. In summary, in this study, we used mice with polarized immune responses to define the pathways by which Th1- and Th2-mediated inflammation cause such divergent pathology in the lung. Furthermore, we extended the characterization of the Th2 response by eliminating IL-13 and demonstrate that it serves an important and nonredundant role in Th2-mediated inflammation by regulating many key elements of tissue remodeling and repair.


   Acknowledgments
 
We thank Dr. Fred Lewis (Biomedical Research Institute, Rockville, MD) for providing the schistosome eggs and other parasite materials. We are also grateful to Dr. Mike Wilson and the members of the NIAID microarray facility for their excellent advice and guidance.


   Footnotes
 
1 N.G.S. is a Howard Hughes Medical Institute-National Institutes of Health Research Scholar. Back

2 Address correspondence and reprint requests to Dr. Thomas A. Wynn, LPD/NIAID, National Institutes of Health, 50 South Drive, Room 6153/MSC 8003, Bethesda, MD 20892. E-mail address: twynn{at}niaid.nih.gov Back

3 Abbreviations used in this paper: KO, knockout; HPRT, hypoxanthine phosphoribosyltransferase; MCP, monocyte chemotactic protein; MIP, macrophage-inflammatory protein; MMP, matrix metalloproteinase; SPRR, small proline-rich protein; TARC, thymus- and activation-regulated chemokine; TIMP, tissue inhibitor of MMP; WT, wild type. Back

Received for publication April 18, 2003. Accepted for publication July 30, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. American Thoracic Society. 2000. Idiopathic pulmonary fibrosis: diagnosis and treatment: international consensus statement: American Thoracic Society (ATS), and the European Respiratory Society (ERS). Am. J. Respir. Crit. Care Med. 161:646.[Free Full Text]
  2. Majumdar, S., D. Li, T. Ansari, P. Pantelidis, C. M. Black, M. Gizycki, R. M. du Bois, P. K. Jeffery. 1999. Different cytokine profiles in cryptogenic fibrosing alveolitis and fibrosing alveolitis associated with systemic sclerosis: a quantitative study of open lung biopsies. Eur. Respir. J. 14:251.[Abstract]
  3. Costabel, U., J. Guzman. 2001. Bronchoalveolar lavage in interstitial lung disease. Curr. Opin. Pulm. Med. 7:255.[Medline]
  4. Westermann, W., R. Schobl, E. P. Rieber, K. H. Frank. 1999. Th2 cells as effectors in postirradiation pulmonary damage preceding fibrosis in the rat. Int. J. Radiat. Biol. 75:629.[Medline]
  5. National Asthma Control Program. Center for Disease Control. www.cdc.gov/nceh/airpollution/Asthma/
  6. Robinson, D. S., Q. Hamid, S. Ying, A. Tsicopoulos, J. Barkans, A. M. Bentley, C. J. Corrigan, S. R. Durham, A. B. Kay. 1992. Predominant Th2-like bronchoalveolar T lymphocyte population in atopic asthma. N. Engl. J. Med. 326:298.[Abstract]
  7. Cohn, L., R. J. Homer, A. Marinov, J. Rankin, K. Bottomly. 1997. Induction of airway mucus production by T helper 2 (Th2) cells: a critical role for interleukin 4 in cell recruitment but not mucus production. J. Exp. Med. 186:1737.[Abstract/Free Full Text]
  8. Robinson, D., Q. Hamid, A. Bentley, S. Ying, A. B. Kay, S. R. Durham. 1993. Activation of CD4+ T cells, increased TH2-type cytokine mRNA expression, and eosinophil recruitment in bronchoalveolar lavage after allergen inhalation challenge in patients with atopic asthma. J. Allergy Clin. Immunol. 92:313.[Medline]
  9. Wynn, T. A., I. Eltoum, A. W. Cheever, F. A. Lewis, W. C. Gause, A. Sher. 1993. Analysis of cytokine mRNA expression during primary granuloma formation induced by eggs of Schistosoma mansoni. J. Immunol. 151:1430.[Abstract]
  10. Chensue, S. W., P. D. Terebuh, K. S. Warmington, S. D. Hershey, H. L. Evanoff, S. L. Kunkel, G. I. Higashi. 1992. Role of interleukin-4 and {gamma}-interferon in Schistosoma mansoni egg-induced hypersensitivity granuloma formation: orchestration, relative contribution and relationship to macrophage function. J. Immunol. 148:900.[Abstract]
  11. Wynn, T. A., I. Eltoum, I. P. Oswald, A. W. Cheever, A. Sher. 1994. Endogenous interleukin 12 (IL-12) regulates granuloma formation induced by eggs of Schistosoma mansoni and exogenous IL-12 both inhibits and prophylactically immunizes against egg pathology. J. Exp. Med. 179:1551.[Abstract/Free Full Text]
  12. Flores Villanueva, P. O., H. Reiser, M. J. Stadecker. 1994. Regulation of T helper cell responses in experimental murine schistosomiasis by IL-10: effect on expression of B7 and B7-2 costimulatory molecules by macrophages. J. Immunol. 153:5190.[Abstract]
  13. Gazzinelli, R. T., S. Hieny, T. A. Wynn, S. Wolf, A. Sher. 1993. Interleukin 12 is required for the T-lymphocyte-independent induction of interferon {gamma} by an intracellular parasite and induces resistance in T-cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115.[Abstract/Free Full Text]
  14. Mosmann, T. R.. 1994. Properties and functions of interleukin-10. Adv. Immunol. 56:1.[Medline]
  15. Wynn, T. A., R. Morawetz, T. Scharton-Kersten, S. Hieny, H. C. r. Morse, R. Kuhn, W. Muller, A. W. Cheever, A. Sher. 1997. Analysis of granuloma formation in double cytokine-deficient mice reveals a central role for IL-10 in polarizing both T helper cell 1- and T helper cell 2-type cytokine responses in vivo. J. Immunol. 159:5014.[Abstract]
  16. Hoffmann, K. F., S. L. James, A. W. Cheever, T. A. Wynn. 1999. Studies with double cytokine-deficient mice reveal that highly polarized Th1- and Th2-type cytokine and antibody responses contribute equally to vaccine-induced immunity to Schistosoma mansoni. J. Immunol. 163:927.[Abstract/Free Full Text]
  17. Abbas, A. K., K. M. Murphy, A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature 383:787.[Medline]
  18. Park, M. K., K. F. Hoffmann, A. W. Cheever, D. Amichay, T. A. Wynn, J. M. Farber. 2001. Patterns of chemokine expression in models of Schistosoma mansoni inflammation and infection reveal relationships between type 1 and type 2 responses and chemokines in vivo. Infect. Immun. 69:6755.[Abstract/Free Full Text]
  19. Chensue, S. W., K. S. Warmington, E. J. Allenspach, B. Lu, C. Gerard, S. L. Kunkel, N. W. Lukacs. 1999. Differential expression and cross-regulatory function of RANTES during mycobacterial (type 1) and schistosomal (type 2) antigen-elicited granulomatous inflammation. J. Immunol. 163:165.[Abstract/Free Full Text]
  20. Zhu, Z., B. Ma, T. Zheng, R. J. Homer, C. G. Lee, I. F. Charo, P. Noble, J. A. Elias. 2002. IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13-induced inflammation and remodeling. J. Immunol. 168:2953.[Abstract/Free Full Text]
  21. Sekiya, T., M. Miyamasu, M. Imanishi, H. Yamada, T. Nakajima, M. Yamaguchi, T. Fujisawa, R. Pawankar, Y. Sano, K. Ohta, et al 2000. Inducible expression of a Th2-type CC chemokine thymus- and activation-regulated chemokine by human bronchial epithelial cells. J. Immunol. 165:2205.[Abstract/Free Full Text]
  22. Lamkhioued, B., E. A. Garcia-Zepeda, S. Abi-Younes, H. Nakamura, S. Jedrzkiewicz, L. Wagner, P. M. Renzi, Z. Allakhverdi, C. Lilly, Q. Hamid, A. D. Luster. 2000. Monocyte chemoattractant protein (MCP)-4 expression in the airways of patients with asthma: induction in epithelial cells and mononuclear cells by proinflammatory cytokines. Am. J. Respir. Crit. Care Med. 162:723.[Abstract/Free Full Text]
  23. Venge, P., J. Bystrom. 1998. Eosinophil cationic protein (ECP). Int. J. Biochem. Cell Biol. 30:433.[Medline]
  24. Batten, D., K. D. Dyer, J. B. Domachowske, H. F. Rosenberg. 1997. Molecular cloning of four novel murine ribonuclease genes: unusual expansion within the ribonuclease A gene family. Nucleic Acids Res. 25:4235.[Abstract/Free Full Text]
  25. Hirunpetcharat, C., F. Finkelman, I. A. Clark, M. F. Good. 1999. Malaria parasite-specific Th1-like T cells simultaneously reduce parasitemia and promote disease. Parasite Immunol. 21:319.[Medline]
  26. Tan, R. S., A. U. Kara, C. Feng, Y. Asano, R. Sinniah. 2000. Differential interleukin-10 expression in interferon regulatory factor-1 deficient mice during Plasmodium berghei blood-stage infection. Parasite Immunol. 22:425.[Medline]
  27. Hoffmann, K. F., T. C. McCarty, D. H. Segal, M. Chiaramonte, M. Hesse, E. M. Davis, A. W. Cheever, P. S. Meltzer, H. C. Morse, III, T. A. Wynn. 2001. Disease fingerprinting with cDNA microarrays reveals distinct gene expression profiles in lethal type 1 and type 2 cytokine-mediated inflammatory reactions. FASEB J. 15:2545.[Abstract/Free Full Text]
  28. Lavigne, L. M., L. R. Schopf, C. L. Chung, R. Maylor, J. P. Sypek. 1998. The role of recombinant murine IL-12 and IFN-{gamma} in the pathogenesis of a murine systemic Candida albicans infection. J. Immunol. 160:284.[Abstract/Free Full Text]
  29. Gazzinelli, R. T., M. Wysocka, S. Hieny, T. Scharton-Kersten, A. Cheever, R. Kuhn, W. Muller, G. Trinchieri, A. Sher. 1996. In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-{gamma} and TNF-{alpha}. J. Immunol. 157:798.[Abstract]
  30. Scharton-Kersten, T. M., T. A. Wynn, E. Y. Denkers, S. Bala, E. Grunvald, S. Hieny, R. T. Gazzinelli, A. Sher. 1996. In the absence of endogenous IFN-{gamma}, mice develop unimpaired IL-12 responses to Toxoplasma gondii while failing to control acute infection. J. Immunol. 157:4045.[Abstract]
  31. Delic, J., M. Morange, H. Magdelenat. 1993. Ubiquitin pathway involvement in human lymphocyte {gamma}-irradiation-induced apoptosis. Mol. Cell. Biol. 13:4875.[Abstract/Free Full Text]
  32. Haas, A. L., O. Baboshina, B. Williams, L. M. Schwartz. 1995. Coordinated induction of the ubiquitin conjugation pathway accompanies the developmentally programmed death of insect skeletal muscle. J. Biol. Chem. 270:9407.[Abstract/Free Full Text]
  33. Jones, M. E., M. F. Haire, P. M. Kloetzel, D. L. Mykles, L. M. Schwartz. 1995. Changes in the structure and function of the multicatalytic proteinase (proteasome) during programmed cell death in the intersegmental muscles of the hawkmoth, Manduca sexta. Dev. Biol. 169:436.[Medline]
  34. Jesenberger, V., S. Jentsch. 2002. Deadly encounter: ubiquitin meets apoptosis. Nat. Rev. Mol. Cell Biol. 3:112.[Medline]
  35. Chuang, P. I., S. Morefield, C. Y. Liu, S. Chen, J. M. Harlan, D. M. Willerford. 2002. Perturbation of B-cell development in mice overexpressing the Bcl-2 homolog A1. Blood 99:3350.[Abstract/Free Full Text]
  36. Orlofsky, A., L. M. Weiss, N. Kawachi, M. B. Prystowsky. 2002. Deficiency in the anti-apoptotic protein A1-a results in a diminished acute inflammatory response. J. Immunol. 168:1840.[Abstract/Free Full Text]
  37. Vignola, A. M., S. La Grutta, G. Chiappara, A. Benkeder, V. Bellia, G. Bonsignore. 2002. Cellular network in airways inflammation and remodelling. Paediatr. Respir. Rev. 3:41.[Medline]
  38. Deng, J., R. Pan, R. Wu. 2000. Distinct roles for amino- and carboxyl-terminal sequences of SPRR1 protein in the formation of cross-linked envelopes of conducting airway epithelial cells. J. Biol. Chem. 275:5739.[Abstract/Free Full Text]
  39. Davies, D. E.. 2001. The bronchial epithelium: translating gene and environment interactions in asthma. Curr. Opin. Allergy Clin. Immunol. 1:67.[Medline]
  40. Marshall, R. P., R. J. McAnulty, G. J. Laurent. 1997. The pathogenesis of pulmonary fibrosis: is there a fibrosis gene?. Int. J. Biochem. Cell Biol. 29:107.[Medline]
  41. Tomono, Y., I. Naito, K. Ando, T. Yonezawa, Y. Sado, S. Hirakawa, J. Arata, T. Okigaki, Y. Ninomiya. 2002. Epitope-defined monoclonal antibodies against multiplexin collagens demonstrate that type XV and XVIII collagens are expressed in specialized basement membranes. Cell Struct. Funct. 27:9.[Medline]
  42. Saarela, J., M. Rehn, A. Oikarinen, H. Autio-Harmainen, T. Pihlajaniemi. 1998. The short and long forms of type XVIII collagen show clear tissue specificities in their expression and location in basement membrane zones in humans. Am. J. Pathol. 153:611.[Abstract/Free Full Text]
  43. Hoshino, M., Y. Nakamura, J. Sim, J. Shimojo, S. Isogai. 1998. Bronchial subepithelial fibrosis and expression of matrix metalloproteinase-9 in asthmatic airway inflammation. J. Allergy Clin. Immunol. 102:783.[Medline]
  44. Knight, D.. 2001. Epithelium-fibroblast interactions in response to airway inflammation. Immunol. Cell Biol. 79:160.[Medline]
  45. Lapiere, C. M., B. Nusgens, G. E. Pierard. 1977. Interaction between collagen type I and type III in conditioning bundles organization. Connect. Tissue Res. 5:21.[Medline]
  46. Adachi, E., T. Hayashi. 1986. In vitro formation of hybrid fibrils of type V collagen and type I collagen: limited growth of type I collagen into thick fibrils by type V collagen. Connect. Tissue Res. 14:257.[Medline]
  47. Giry-Lozinguez, C., E. Aubert-Foucher, F. Penin, G. Deleage, B. Dublet, M. van der Rest. 1998. Identification and characterization of a heparin binding site within the NC1 domain of chicken collagen XIV. Matrix Biol. 17:145.[Medline]
  48. Kannus, P., L. Jozsa, T. A. Jarvinen, T. L. Jarvinen, M. Kvist, A. Natri, M. Jarvinen. 1998. Location and distribution of non-collagenous matrix proteins in musculoskeletal tissues of rat. Histochem. J. 30:799.[Medline]
  49. Muona, A., L. Eklund, T. Vaisanen, T. Pihlajaniemi. 2002. Developmentally regulated expression of type XV collagen correlates with abnormalities in Col15a1-/- mice. Matrix Biol. 21:89.[Medline]
  50. Ghosh, A. K.. 2002. Factors involved in the regulation of type I collagen gene expression: implication in fibrosis. Exp. Biol. Med. 227:301.[Abstract/Free Full Text]
  51. Huang, S. K., H. Q. Xiao, J. Kleine-Tebbe, G. Paciotti, D. G. Marsh, L. M. Lichtenstein, M. C. Liu. 1995. IL-13 expression at the sites of allergen challenge in patients with asthma. J. Immunol. 155:2688.[Abstract]
  52. Kroegel, C., P. Julius, H. Matthys, J. C. Virchow, Jr, W. Luttmann. 1996. Endobronchial secretion of interleukin-13 following local allergen challenge in atopic asthma: relationship to interleukin-4 and eosinophil counts. Eur. Respir. J. 9:899.[Abstract]
  53. Naseer, T., E. M. Minshall, D. Y. Leung, S. Laberge, P. Ernst, R. J. Martin, Q. Hamid. 1997. Expression of IL-12 and IL-13 mRNA in asthma and their modulation in response to steroid therapy. Am. J. Respir. Crit. Care Med. 155:845.[Abstract]
  54. Roche, W. R., R. Beasley, J. H. Williams, S. T. Holgate. 1989. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1:520.[Medline]
  55. Leppert, D., E. Waubant, R. Galardy, N. W. Bunnett, S. L. Hauser. 1995. T cell gelatinases mediate basement membrane transmigration in vitro. J. Immunol. 154:4379.[Abstract]
  56. Faveeuw, C., G. Preece, A. Ager. 2001. Transendothelial migration of lymphocytes across high endothelial venules into lymph nodes is affected by metalloproteinases. Blood 98:688.[Abstract/Free Full Text]
  57. Okada, S., H. Kita, T. J. George, G. J. Gleich, K. M. Leiferman. 1997. Migration of eosinophils through basement membrane components in vitro: role of matrix metalloproteinase-9. Am. J. Respir. Cell Mol. Biol. 17:519.[Abstract/Free Full Text]
  58. Betsuyaku, T., Y. Fukuda, W. C. Parks, J. M. Shipley, R. M. Senior. 2000. Gelatinase B is required for alveolar bronchiolization after intratracheal bleomycin. Am. J. Pathol. 157:525.[Abstract/Free Full Text]
  59. Legrand, C., C. Gilles, J. M. Zahm, M. Polette, A. C. Buisson, H. Kaplan, P. Birembaut, J. M. Tournier. 1999. Airway epithelial cell migration dynamics: MMP-9 role in cell-extracellular matrix remodeling. J. Cell Biol. 146:517.[Abstract/Free Full Text]
  60. Shipley, J. M., R. L. Wesselschmidt, D. K. Kobayashi, T. J. Ley, S. D. Shapiro. 1996. Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice. Proc. Natl. Acad. Sci. USA 93:3942.[Abstract/Free Full Text]
  61. Warner, R. L., C. S. Lewis, L. Beltran, E. M. Younkin, J. Varani, K. J. Johnson. 2001. The role of metalloelastase in immune complex-induced acute lung injury. Am. J. Pathol. 158:2139.[Abstract/Free Full Text]
  62. Kusano, K., C. Miyaura, M. Inada, T. Tamura, A. Ito, H. Nagase, K. Kam