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J Biol Chem, Vol. 275, Issue 11, 8032-8037, March 17, 2000

Biochemical Characterization of Endogenously Formed Eosinophilic Crystals in the Lungs of Mice^*

Lin Guo, Richard S. Johnson, and JoAnn C. L. Schuh

From Immunex Corporation, Seattle, Washington 98101

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Crystals seldom form spontaneously within tissues of mammals, except in the urinary tract or in association with eosinophil-rich diseases in humans (Charcot-Leyden crystals). Endogenously formed eosinophilic crystals have been reported in respiratory tract and other tissues of several strains of mice, but the biochemical characterization of these crystals has not been reported. In this study, eosinophilic crystal formation was examined in homozygous C57BL/6J viable motheaten mice, lung-specific surfactant apoprotein C promoter/soluble human tumor necrosis factor p75 receptor type II fusion protein transgenic mice (C57BL/6NTac × Sv/129), and CD40L-deficient mice with spontaneous Pneumocystis carinii infection. In viable motheaten but not wild type mice, rapidly developing crystals represented a major feature of the fatal lung injury induced by macrophage dysregulation. Conversely, eosinophilic crystals did not form until 4-8 months of age in transgenic and CD40L-deficient mice and were present in 10-30% of age-matched wild type controls. Mass spectrometry analysis of proteins from bronchoalveolar lavage fluid identified the crystals as Ym1, sometimes referred to as T-lymphocyte-derived eosinophil chemotactic factor. The Ym1 sequence was homologous to chitinase, and enzymatic assays indicated a 3-5-fold increase in chitinase activity compared with control mice. Intracellular and extracellular crystals associated with epithelial damage suggested that the crystals may contribute to lung inflammation through mechanical damage and enzymatic degradation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

As early as 1905 (1), eosinophilic crystals were reported microscopically in the respiratory tract (lung and trachea), the biliary tree (gall bladder and liver), and other tissues of C57BL/6, Swiss Webster, and related strains of mice (2-7). Unstained crystals are colorless, but their positive charge attracts eosin, and the crystals are brightly eosinophilic (acidophilic) in tissue sections histochemically stained with a standard hematoxylin and eosin protocol. The sharply angular eosinophilic crystals are of variable size and shape and are generally found in the cytoplasm of macrophages and multinucleated giant cells and extracellular within tissue lumina (7). In the most severe form within the lung, eosinophilic crystals have been called acidophilic macrophage pneumonia of mice (8).

In mice, eosinophilic crystals are often considered to be histologically identical to the Charcot-Leyden crystals in humans. Charcot-Leyden crystals form in association with eosinophil-rich inflammation of the lung and other tissues and are an autocrystallizing protein (lysophospholipase) of eosinophils and basophils related to carbohydrate-binding galectins (9). But unlike Charcot-Leyden crystals, eosinophilic crystals in mice are reported over a broader range of diseases without excess eosinophils, and the causative protein has not been identified. Formation of eosinophilic crystals in mice is enhanced in spontaneous diseases, such as lung tumors (3), and Pneumocystis carinii infections in deletional mutants (7); in the lungs of experimentally manipulated mice, such as models of eosinophilia (asthma and parasites), inhalation toxicity studies (particulates and tobacco smoke) (8); and in genetically altered mice, particularly those generated on a C57BL/6 or Sv/129 background (7, 10-12).

In this study, crystals from three variants and two commercial sources of the C57BL/6 mouse were analyzed. Viable motheaten mice (me^v/me^v),¹ a spontaneous mutation of C57BL/6J (Jackson Laboratories) mice, are deficient in SHP-1 protein-tyrosine phosphatase activity with resulting broad hematopoietic and immunological dysregulation including hyperactivity of alveolar macrophages (13, 14). Progressive and fatal pulmonary injury in these mice includes consistent formation of intrapulmonary eosinophilic crystals that can be retrieved by bronchoalveolar lavage (BAL). CD40L-deficient mice (15, 16) and lung-specific transgenic mice with soluble human tumor necrosis factor receptor p75 (type II) inserted under the control of the surfactant apoprotein C promoter (SPCTNFRIIFc) (17) were also utilized because eosinophilic crystals were observed consistently in the lungs, in an age-dependent fashion.

We have purified eosinophilic crystals from BAL fluids of me^v/me^v mice and identified the protein as Ym1 or T-lymphocyte-derived eosinophil chemotactic factor (ECF-L) through peptide sequencing by nanoelectrospray tandem mass spectrometry. We report here the histopathological examination, identification, and characterization of murine eosinophilic crystals and report that they are morphologically similar, but not biochemically identical, to Charcot-Leyden crystals in humans.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mice-- The C57BL/6J viable motheaten mice were initially obtained from Jackson Laboratories (Bar Harbor, ME) and propagated by heterozygous mating. Control animals included +/+ and me^v/+ littermates, and sex- and age-matched C57BL/6NTac or BALB/c mice (Taconic, Germantown, NY). The SPCTNFRIIFc transgenic mice (C57BL/6NTac × Sv/129) secrete soluble human tumor necrosis factor receptor p75 under the control of the surfactant apoprotein C promotor (17) and were paired with littermates or age-matched control mice (gift of Drs. C. Wilson and S. Smith, University of Washington). Heterozygote matings were used to generate additional mice to backcross 4-5 generations onto C57BL/6NTac mice. Lung tissue was also examined from an in-house colony of C57BL/6J × Sv/129 and C57BL/6 mice with a targeted deletion of CD40L that results in spontaneous P. carinii infection (15). All mice were housed in microisolator units and fed rodent chow and water ad libitum, and the colony was routinely monitored for murine pathogens.

Isolation of Eosinophilic Crystals-- At 6-10 weeks (me^v/me^v) or 16-88 weeks (SPCTNFRIIFc) of age, mice were humanely killed by CO₂ and exsanguinated through the abdominal vena cava. The trachea was exposed by a midline incision and cannulated with an infusion set (21 g, 3/4 in; Becton Dickinson, Franklin Lakes, NJ). The lungs were lavaged with 1-3 ml of sterile Hanks' balanced salt solution delivered by one or more tuberculin syringes. The cellularity of the 1-2 ml of recovered BAL was evaluated by examination of cytospin preparations stained with Diff-quik^® stain (Dade Diagnostics, Inc., Aguada, PR). The entire lung was collected into 10% neutral buffered formalin or Fekete's formol-acetic alcohol, processed, embedded in paraffin, and cut at 5 µm, and sections were stained by hematoxylin and eosin stain.

To purify the crystals, 3 ml of BAL fluid from me^v/me^v mice were mixed with 6 ml of diatrizoate-Ficoll (Isolymph^®, Gallard-Schlesinger Industries, Inc., Carle Place, NY) and centrifuged at 250 × G for 10 min at 4 °C. The supernatant and most of the diatrizoate-Ficoll layer were removed by aspiration. The remaining ~0.2 ml of diatrizoate-Ficoll from the bottom of the tube, which contained the crystals, were resuspended in 2.8 ml of PBS and 6 ml of diatrizoate-Ficoll, and the precipitation was repeated. The above PBS wash/diatrizoate-Ficoll precipitation step was repeated five more times. The final ~0.2 ml of crystal-containing diatrizoate-Ficoll was resuspended in 2 ml of PBS and centrifuged at 200 × g for 5 min at 4 °C, and the bottom ~0.2 ml of PBS plus crystals was preserved. Microscopically, a few cell contaminants were observed with the purified crystals. After removing the residual cell contaminants under a microscope using a small gel loading tip, the purified crystals were solubilized by boiling in SDS-PAGE sample buffer for 10 min, and the protein components were identified by SDS-PAGE and mass spectrometry analysis.

SDS-PAGE and In-gel Trypsin Digestion-- SDS-polyacrylamide gel electrophoresis was performed under reducing conditions using Tris-glycine 4-20% gradient gels (Novex, San Diego, CA). Protein bands were detected by staining with Colloidal Blue (Novex), excised from the SDS-PAGE gel, and destained by washing with mixtures of 200 mM NH₄HCO₃/acetonitrile (1:1). Proteins were reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin (Promega, Madison, WI) as described (18). Tryptic peptides were concentrated and desalted with approximately 150 nl of POROS R2 sorbent (Perseptive Biosystems, Framingham, MA) before mass spectrometric analysis.

Mass Spectrometry Methods-- Mass spectrometry analysis of tryptic peptides was performed on a Finnigan LCQ ion trap instrument (Finnigan, San Jose, CA). Ionization was achieved with a home-built nanoflow electrospray source (19). The nanospray tips were purchased from Protana (Denmark). A search of a nonredundant protein sequence data base with the collision-induced dissociation mass spectra was performed using the SEQUEST^TM data base search program (20), and matches were verified using a de novo sequence interpretation program (Lutefisk 97) (21).

Enzyme Assays-- The chitinase activity of BAL was assayed with the fluorogenic substrates 4-methylumbelliferyl (4-MU) N-acetyl--D-glucosaminide, 4-MU -D-N,N'-diacetylchitobioside, and 4-MU -D-N,N',N"-triacetylchitotrioside (all from Sigma). Assays were performed as described with minor modifications (22). Briefly, BAL fluid was incubated with a substrate in citrate/phosphate buffer (0.1 M/0.2 M), pH 5.2, at a concentration of 0.02 µM. After incubation at 37 °C for 15 min, the reaction (final volume, 110 µl) was stopped with 1 ml of 0.3 M glycine/NaOH buffer, pH 10.6, and the fluorescent 4-methylumbelliferone released was measured with a fluorimeter (excitation, 350 nm; emission, 450 nm). A 4-methylumbelliferone (Sigma) standard curve was generated and used to quantify the enzyme activity. Protein concentrations were determined using the Pierce micro-BCA protein assay kit. Chitotriosidase activity was also assayed after gel electrophoresis. Nonreducing SDS-PAGE (with 4-20% Novex gradient gels) were used to separate BAL protein components. After electrophoresis, the gel was soaked in 25% (v/v) isopropyl alcohol for 15 min, followed by rehydration in water for 20 min. Five milliliters of 4-MU--D-N,N', N"-triacetylchitotrioside in citrate/phosphate buffer (0.1 M/0.2 M, pH 5.2) at 0.2 mg/ml was added to the gel and incubated at 25 °C for 15 min. The enzymatic reactions were stopped with 1 ml of 0.3 M glycine, pH 10.6, and the activities were detected as fluorescent bands under UV light. After the in-gel enzyme assay, the gel was stained with Colloidal Coomassie (Novex).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tissue Characterization of Eosinophilic Crystals-- Eosinophilic crystals were identified in varying numbers, sizes, and shapes within the cytoplasm of alveolar macrophages and multinucleate giant cells and free within alveolar spaces in the lungs of me^v/me^v (Fig. 1A), SPCTNFRIIFc transgenic (Fig. 1B) and wild type litter mate control mice, CD40L-deficient mice (Fig. 1C), and occasionally in older C57BL/6 control mice. In younger mice, eosinophilic crystals were only found intracellularly in activated alveolar macrophages and often in isolated scattered intra-alveolar clusters in one or two lung lobes. Intracellular and extracellular crystals were found in moribund me^v/me^v mice by 5-8 weeks of age and in the SPCTNFRIIFc mice by 32 weeks of age. As the transgenic mice aged, diffuse involvement of all five lung lobes was normal, whereas age-matched control mice seldom had more than a few foci within several lung lobes.

View larger version (119K): [in this window] [in a new window]   Fig. 1.   A, B, and C, eosinophilic crystals (arrows) in lung sections of mice, hematoxylin, and eosin stain. A, stacked intracytoplasmic crystals in alveolar macrophages and multinucleated giant cells within the alveolar spaces of a moribund mev/mev. B, intracytoplasmic and free eosinophilic crystals and inflammation in the lung of a transgenic mouse (SPCTNFRIIFc). C, intracytoplasmic and free eosinophilic crystals in the lung of a CD40L-deficient mouse with acquired P. carinii pneumonia. D, an extracellular eosinophilic crystal surrounded by a neutrophil and red blood cells obtained from bronchoalveolar lavage fluid of a mev/mev mouse (Diff-Quik® stain).

In the tissue sections, many intracellular crystals appeared to be fine spicules, which were found to correspond to stacks of flat, 10-µm² crystals identified in the BAL cytospin. Crystals between 20 and 120 µm in length were also identified in BAL cytospin (Fig. 1D) as multifaceted (8-10 sides) and in tissue sections as extracellular. Large crystals were found in association with degeneration and hyperplasia of respiratory epithelium and mixed cellular inflammation, particularly in the SPCTNFRIIFc mice. In the me^v/me^v mice, the inherent progressive lung injury consisted of numerous infiltrating neutrophils, lymphocytes, and eosinophils, along with hemorrhage that generally proved fatal by 10 weeks of age. In the original line of SPCTNFRIIFc mice, eosinophilic crystals and lung pathology (Fig. 1B) was consistently identified only in mice older than 24 weeks of age, with some mortality after 48 weeks of age. With advancing age, the frequency and size of crystals, mixed inflammation, and mechanical injury induced by the crystals increased dramatically. Littermate controls were also found to have eosinophilic crystals but at a much lower frequency and generally only as intracellular crystals. Subsequently, SPCTNFRIIFc mice backcrossed onto C57BL/6Ntac mice (4-5 generations) were less frequently affected, had fewer lung lobes involved, and had less lung injury (epithelial hyperplasia and inflammation) than found in the original line (C57BL/6NTac × Sv/129). CD40L-deficient mice consistently developed spontaneous P. carinii infection typified by foamy alveolar material and proliferation of fungus-laden macrophages and multinucleated giant cells. Eosinophilic crystals were rarely found early in the disease but were an obvious intracellular and extracellular component in most moribund mice. Except for P. carinii in CD40L-deficient mice, all mice were determined to be free of other spontaneous murine pathogens or other confounding diseases that might alter the frequency of pulmonary damage and eosinophilic crystal formation.

Biochemical Characterization of Eosinophilic Crystals-- SDS-PAGE gels were used to separate the BAL fluid protein components. Several additional protein bands were observed in lanes loaded with me^v/me^v BAL fluids compared with the control BAL fluid (Fig. 2A). Protein bands from me^v/me^v BAL were excised and were identified through in-gel trypsin digest and mass spectrometry sequencing of tryptic peptides. Proteins identified as BAL components of me^v/me^v mice include transferrin, serum albumin, Ym1, actin, ferritin light chain, and hemoglobin (Fig. 2A).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Identification of eosinophilic crystals as Ym1. A, SDS-PAGE analysis comparing crude BAL from either mev/mev or control mice with purified crystals. Protein bands identified by mass spectrometry were transferrin (1), serum albumin (2), Ym1 and actin (3), Ym1 (4, 5, and 6), ferritin light chain (7), hemoglobin (8), and actin (9). The protein band from the purified crystals was identified as Ym1 (see also Table I). B, collision-induced dissociation mass spectrum obtained on the (M + 2H)2+ ions at m/z 861.2, which was identified as a tryptic peptide FGPAPFSAM*VSTPQNR (where M* represents methionine sulfoxide) from Ym1. The fragment ion assignments use the Biemann nomenclature (27); calculated nominal masses for each ion are shown, and ions observed in the spectrum are underlined. y12+2 indicates a doubly charged y12 ion.

Through repeated Ficoll precipitation/PBS washing procedures, eosinophilic crystals were further purified. By SDS-PAGE, a single protein band with apparent molecular mass of ~40 kDa was observed (Fig. 2A). Using mass spectrometry, sequences of 11 tryptic peptides from this protein band were obtained and shown to encompass 125 amino acids of the Ym1 sequence (Table I). Besides peptides from Ym1, autolysis products of trypsin were the only other peptides found in the sample. Therefore, Ym1 was identified as the sole protein component of the eosinophilic crystal. The ~40-kDa protein band from the unpurified me^v/me^v BAL contained two proteins, Ym1 and actin (Fig. 2A), whereas a ~40-kDa protein band from the control BAL was shown to only contain actin (Fig. 2A). This further supports the notion that Ym1 is a unique protein component of me^v/me^v BAL. Besides the 40-kDa protein band, several lower molecular weight protein bands from me^v/me^v BAL also contained fragments of Ym1 (Fig. 2A). Unlike the 40-kDa Ym1, these bands were not present in the purified crystals (Fig. 2A), thus indicating that degraded Ym1 is not a component of the crystal.

The 40-kDa Ym1 protein was also found in BAL of SPCTNFRIIFc transgenic mice, although the protein was in much lower quantity compared with Ym1 in BAL from me^v/me^v mice (data not shown). Compared with me^v/me^v mice, the majority of the crystals in the lung sections of the SPCTNFRIIFc transgenic mice were intracellular in alveolar macrophages and thus not accessible by BAL.

The Ym1 Sequence-- The Ym1 cDNA sequence, which encodes a 399-amino acid secreted protein transiently expressed by activated murine peritoneal macrophages, was submitted directly to the GenBank^TM data base (accession no. M94584) in 1992. Later on, a second cDNA called ECF-L with a somewhat different sequence from Ym1 was also deposited with the data base (GenBank^TM accession no. D87757). Comparing the Ym1 and ECF-L sequences, several minor differences exist in the cDNA sequences. Of the Ym1 tryptic peptides sequenced in this study (Table I), we identified a 16-amino acid peptide, FGPAPFSAMVSTPQNR (Fig. 2B), which indicated that nucleotides 328-330 encode a proline (as in Ym1) rather than serine (as in ECF-L) (codon ccg instead of tcg; GenBank^TM accession no. M94584). Another tryptic peptide obtained from me^v/me^v BAL, IPELSQSLDYIQVMTYDLHDPK (data not shown), was consistent with the 9-amino acid sequence between nucleotides 595 and 624 given in the ECF-L sequence, but due to a reading frameshift in the Ym1 cDNA, a different 9- amino acid sequence was predicted. In addition, the sequence of a nontryptic peptide (YQLMCYYTSWAK) (Table I) revealed the N terminus of the Ym1 protein after the secretion signal was removed.

Sequence homology searches indicated that Ym1 belongs to a family of mammalian proteins that resemble bacterial and plant chitinases. Chitin is a linear polymer of -1,4-N-acetyl-D-glucosamine, and chitinase breaks down the -1,4 linkage between the carbohydrate monomers. Furthermore, protein sequence regions that are required for the biological activities of bacterial chitinases (23) were found to be conserved in Ym1.

Ym-1 and Enzymatic Activity-- Since Ym1 has extensive homology with chitinase, the enzymatic activity of BAL fluid from me^v/me^v mice to hydrolyze an artificial chitotrioside substrate (chitotriosidase activity) was assayed. Compared with control BAL fluids, me^v/me^v BAL fluids had 3-5-fold increases in chitotriosidase activities (Fig. 3A). The detection of chitotriosidase activity after gel electrophoresis demonstrated that the enzymatic activity in BAL co-localized with the Ym1 protein (Fig. 3B). When the purified eosinophilic crystals were solubilized in the SDS-PAGE sample buffer, the enzymatic activities were also detected by the in-gel chitotriosidase assay (data not shown). Although the possibility cannot be ruled out completely that the enzymatic activities in the BAL from me^v/me^v mice were due to trace amount of unknown enzyme contaminants at this time, our data indicated that Ym1 is probably the protein component in the BAL that contributes to the chitotriosidase activity.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Chitinase assays. A, chitotriosidase assays of BAL fluid from two different mev/mev mice. The control BAL fluids were from littermate controls of normal C57BL/6 mice. The chitotriosidase activities were expressed as nmol of 4-MU -D-N,N',N"-triacetylchitotrioside cleaved per µg of BAL protein in 1 h. B, detection of chitotriosidase activity after SDS-PAGE. BAL protein components from a mev/mev and a control mouse were separated by nonreducing SDS-PAGE, and the fluorescent image of the gel after in-gel enzymatic assays (right panel) was aligned with the Coomassie-stained image (left panel). C, chitinase assay of mev/mev BAL using 4-MU N-acetyl--D-glucosaminide (-D gl), 4-MU N-acetyl--D-glucosaminide (-D gl), 4-MU -D-N,N'-diacetylchitobioside (-D dc), or 4-MU -D-N,N'N"-triacetylchitotrioside (-D tc) as substrate. Data points presented are the average of three assay points (mean ± S.D., n = 3).

Further enzymatic assays were performed using 4-MU N-acetyl--D-glucosaminide, 4-MU N-acetyl--D-glucosaminide, or 4-MU -D-N,N'-diacetylchitobioside as substrate (Fig. 3B). These data indicated that the glycosylhydrolase activity in BAL of me^v/me^v mice only cleaves the -D-linkage of N-acetyl glucosamine, not the -D-linkage.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Eosinophilic crystals are seldom observed in the absence of preexisting pulmonary disease that induces inflammation or activation of endogenous alveolar macrophages. Our studies indicate that macrophages are the minimum and in some cases the only cell found in association with eosinophilic crystal formation. Other investigators have suggested that these eosinophilic crystals in the mouse lung may represent phagocytosed eosinophilic proteins (8). However, even in the presence of active inflammation with eosinophils, macrophages or multinucleated giant cells containing crystals are proportionally the most frequent cell type observed in mice. The possibility that previously infiltrated eosinophils have been phagocytosed cannot be completely eliminated, but the disproportionately low number of eosinophils in the face of active disease with large numbers of crystals would also suggest that the protein base for the crystals is not the eosinophil. Eosinophilic crystals have been described as an integral part of the eosinophilic infiltration and dependent on interleukin-5 in Cryptococcus neoformans infection on a C57BL/6 background. However, the investigators used BALB/c mice as controls, a mouse that does not appear to form eosinophilic crystals (24). Furthermore, interleukin-5-deficient mice lack a significant eosinophilia in response to Toxocara canis infestation in C57BL/6 mice yet have eosinophilic crystals at a similar level to wild type C57BL/6 mice (25).

With the increasing use of deletional mutant and transgenic mice, eosinophilic crystals are being identified, sometimes erroneously, as part of the phenotype of the gene deletion or overexpression itself, particularly when lung-specific expression is used or pulmonary injury is present. Experimental manipulation of genetically altered mice may also enhance crystal formation. Cryptogenic background lesions that occur sporadically are age-dependent and can be enhanced in young animals under experimental manipulations need to be considered in evaluating pulmonary findings in mice. It is probably not coincidental that in addition to eosinophilic crystals, C57BL/6 mice also develop alveolar histiocytosis, a proliferation of alveolar macrophages that may also contain a few intracytoplasmic eosinophilic crystals (26). Eosinophilic crystals in SPCTNFRIIFc mice are an example of cryptogenic enhancement of eosinophilic crystal formation in C57BL/6 mice. Other mechanisms for the enhancement of eosinophilic crystals include modification of a genetic factor from Sv/129 mice, because our backcrossed C57BL/6 line had a much lower frequency of crystals. Mice with similar severity of crystal formation secrete different levels of human TNFRII into BAL and serum, and mice transgenic for TNF under the SPC promotor also have crystals (10), indicating that manipulation of cytokines and their receptors are probably not directly involved. Furthermore, mice transgenic for interleukin-13 inserted under a Clara cell promotor also exhibit an age-dependent formation of crystals predominately in one but not both lines generated (28). If interleukin-13 induced an eosinophilia that provided a protein of eosinophil origin, then both lines would be expected to produce crystals. Reports of eosinophilic crystals in some but not all genetically altered mice with manipulation of either the surfactant C and Clara cell promotors would argue against a direct effect of the promotors. An indirect insertional event in the different transgenic lines could be the modifier that explains differing results between lines and promotors. Furthermore, eosinophilic crystals are not specific to transgenic mice. Mice with immunodeficiencies that result from targeted deletions such as CD40L and T cell receptors  and  develop crystals subsequent to P. carinii infection, and crystals are found in older severe combined immunodeficient mice (scid/scid) and in aged immunocompetent mice in our colonies (29). Age, spontaneous infections, and experimental manipulation modulate eosinophilic crystal formation in the lungs of mice. Therefore, mice that are unable to respond appropriately to opportunistic infections or environmental antigens and the age-dependent acquisition of crystals in other mice indicate that eosinophilic crystals are a secondary phenomenon of lung pathology in certain strains of mice. Care must be taken in ascribing a pulmonary phenotype to genetically altered mice. Eosinophilic crystals in the lungs of mice should be considered to be a confounding lesion rather than a primary phenotype until proven otherwise.

A recent study of the genomic structure of Ym1 gene revealed that several genes named Ym2, Ym3, and Ym4, which are highly homologous to Ym1, are clustered at the Ym1 locus. Expression of these genes is localized in different tissues, and their functions are unknown in the mouse (30). We have observed that the primary site of eosinophilic crystal accumulation is within the respiratory tract of mice. This site is consistent with Ym1 gene expression (30).

Ym1 is also known as ECF-L, a T-lymphocyte-derived eosinophil chemotactic factor. The production of ECF-L was associated with the eosinophil-rich granuloma formation in mice infected with Schistosoma japonicum or T. canis (31, 32). Chitin is not a normal component of mammalian tissue but occurs in other organisms including parasites, fungi, and bacteria. Therefore, since Ym1 has homology to chitinases and we have shown in vitro chitinase activity associated with Ym1, an anti-chitin activity against parasites may play a role in eosinophil chemotactic activity during parasitic infestations. We also routinely see eosinophilic crystal formation secondary to P. carinii fungal infections in mice (29), further suggesting that anti-chitin activity by Ym1 may be part of the normal host defense mechanism against chitin-bearing microorganisms.

Ym1 may also have biological functions that are not related to eosinophil chemotactic activity. Other proteins in the mammalian chitinase-like protein family include a human chitotriosidase that is elevated in the serum of patients with Gaucher's disease (22) and two secreted glycoproteins of cultured human chondrocytes (YKL-39 and YKL-40) (33, 34), one of which (YKL-40) is elevated in joints of patients with rheumatoid arthritis (34). Polysaccharides with similar structure form various glycosaminoglycans that are abundant in the extracellular compartment. Could Ym1 and related proteins be involved in binding extracellular polysaccharide and/or in degradation of glycosaminoglycans?

One of the other overproduced proteins that we found in the BAL of me^v/me^v mice was transferrin. Transferrin was not considered to be a component of the eosinophilic crystal because it was not enriched in partially purified crystals (Fig. 2A). In a previous study, a protein closely related to transferrin, lactoferrin, was found to co-localize and co-mobilize with another chitinase, YKL-40, found in granules of human neutrophils (35). Transferrin and lactoferrin are iron-binding proteins that are nonspecifically increased during the acute phase reaction of infections. Therefore, it would not be unexpected to find chitinase-like proteins in association with the overproduction of such acute phase reactants, particularly in the context of pulmonary injury of the me^v/me^v mice. Alveolar macrophages from pulmonary interstitial disease have increased hydrolytic enzymatic activity including -D-N-acetyl glucosaminidase (36). The progressive lung disease in me^v/me^v mice is similar to that observed in patients with chronic interstitial lung disease; therefore, proliferating alveolar macrophages may be a source of enzymatically active Ym1 in mice.

Why and under what conditions do crystals form? What are the biological functions of Ym1? Is Ym1 production regulated by cytokines? Are the eosinophilic crystals in the biliary tree and other tissues of mice also Ym1? These are some of the questions that remain for further study. Since eosinophilic crystals are usually found at the site of pulmonary inflammation involving macrophages, further studies of the biological activity of Ym1 in mice may reveal new therapeutic avenues.

	ACKNOWLEDGEMENTS

We thank R. Hall, K. Harrington, R. Koelling, and A. Wallace for technical assistance.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom all correspondence should be addressed: Immunex Corp., 51 University St., Seattle, WA 98101. Tel.: 206-389-4361; Fax: 206-233-9733; E-mail: schuhj@immunex.com.

	ABBREVIATIONS

The abbreviations used are: me^v/me^v, viable motheaten mice (SHP-1 protein-tyrosine phosphatase deficiency); BAL, bronchoalveolar lavage; SPCTNFRIIFc, transgenic mice secreting soluble human tumor necrosis factor receptor p75 fusion protein under the surfactant apoprotein C promotor; ECF-L, eosinophil chemotactic factor; 4-MU, 4-methylumbelliferyl; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				M. G. Nair, I. J. Gallagher, M. D. Taylor, P. Loke, P. S. Coulson, R. A. Wilson, R. M. Maizels, and J. E. Allen Chitinase and Fizz Family Members Are a Generalized Feature of Nematode Infection with Selective Upregulation of Ym1 and Fizz1 by Antigen-Presenting Cells Infect. Immun., January 1, 2005; 73(1): 385 - 394. [Abstract] [Full Text] [PDF]

				J. M. Hickman-Davis, J. Gibbs-Erwin, J. R. Lindsey, and S. Matalon Role of Surfactant Protein-A in Nitric Oxide Production and Mycoplasma Killing in Congenic C57BL/6 Mice Am. J. Respir. Cell Mol. Biol., March 1, 2004; 30(3): 319 - 325. [Abstract] [Full Text] [PDF]

				J. S. Welch, L. Escoubet-Lozach, D. B. Sykes, K. Liddiard, D. R. Greaves, and C. K. Glass TH2 Cytokines and Allergic Challenge Induce Ym1 Expression in Macrophages by a STAT6-dependent Mechanism J. Biol. Chem., November 1, 2002; 277(45): 42821 - 42829. [Abstract] [Full Text] [PDF]

				M. Ernst, M. Inglese, G. M. Scholz, K. W. Harder, F. J. Clay, S. Bozinovski, P. Waring, R. Darwiche, T. Kay, P. Sly, et al. Constitutive Activation of the Src Family Kinase Hck Results in Spontaneous Pulmonary Inflammation and an Enhanced Innate Immune Response J. Exp. Med., September 2, 2002; 196(5): 589 - 604. [Abstract] [Full Text] [PDF]

				S.-I. Hung, A. C. Chang, I. Kato, and N.-C. A. Chang Transient expression of Ym1, a heparin-binding lectin, during developmental hematopoiesis and inflammation J. Leukoc. Biol., July 1, 2002; 72(1): 72 - 82. [Abstract] [Full Text] [PDF]

				R. J. Homer, T. Zheng, G. Chupp, S. He, Z. Zhu, Q. Chen, B. Ma, R. D. Hite, L. I. Gobran, S. A. Rooney, et al. Pulmonary type II cell hypertrophy and pulmonary lipoproteinosis are features of chronic IL-13 exposure Am J Physiol Lung Cell Mol Physiol, July 1, 2002; 283(1): L52 - L59. [Abstract] [Full Text] [PDF]

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				M. Harbord, M. Novelli, B. Canas, D. Power, C. Davis, J. Godovac-Zimmermann, J. Roes, and A. W. Segal Ym1 Is a Neutrophil Granule Protein That Crystallizes in p47phox-deficient Mice J. Biol. Chem., February 8, 2002; 277(7): 5468 - 5475. [Abstract] [Full Text] [PDF]

				D. C. Webb, A. N. J. McKenzie, and P. S. Foster Expression of the Ym2 Lectin-binding Protein Is Dependent on Interleukin (IL)-4 and IL-13 Signal Transduction. IDENTIFICATION OF A NOVEL ALLERGY-ASSOCIATED PROTEIN J. Biol. Chem., November 2, 2001; 276(45): 41969 - 41976. [Abstract] [Full Text] [PDF]

				F. H. Falcone, P'n. Loke, X. Zang, A. S. MacDonald, R. M. Maizels, and J. E. Allen A Brugia malayi Homolog of Macrophage Migration Inhibitory Factor Reveals an Important Link Between Macrophages and Eosinophil Recruitment During Nematode Infection J. Immunol., November 1, 2001; 167(9): 5348 - 5354. [Abstract] [Full Text] [PDF]

				A. Pande, J. Pande, N. Asherie, A. Lomakin, O. Ogun, J. King, and G. B. Benedek Crystal cataracts: Human genetic cataract caused by protein crystallization PNAS, May 22, 2001; 98(11): 6116 - 6120. [Abstract] [Full Text] [PDF]

				M. Feldmesser, Y. Kress, and A. Casadevall Intracellular Crystal Formation as a Mechanism of Cytotoxicity in Murine Pulmonary Cryptococcus neoformans Infection Infect. Immun., April 1, 2001; 69(4): 2723 - 2727. [Abstract] [Full Text] [PDF]

				J. P. Mizgerd, M. R. Spieker, and C. M. Doerschuk Early Response Cytokines and Innate Immunity: Essential Roles for TNF Receptor 1 and Type I IL-1 Receptor During Escherichia coli Pneumonia in Mice J. Immunol., March 15, 2001; 166(6): 4042 - 4048. [Abstract] [Full Text] [PDF]