Introduction

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Haptoglobin (Hp) is an 2-acid glycoprotein with a strong hemoglobin-binding capacity. It is produced mainly by the liver and secreted into the circulation. As a major positive acute-phase reactant, Hp increases in plasma during inflammation, infection, trauma, tissue damage, and malignant proliferation. In addition to transporting hemoglobin to the liver, thus facilitating hemoglobin catabolism and preventing tissue injury, several functions have been assigned to Hp, including antioxidant activity (1) and angiogenesis (2). Recent studies suggest that another important biologic function of Hp is its involvement in the host defense response to infection and inflammation by acting as a natural antagonist for receptor-ligand activation of the immune system (reviewed in References 3 and 4).

Several studies have demonstrated the interactions of Hp with leukocytes. Hp has a negative effect on phytohemagglutinin-induced lymphoblast transformation (5), and it also inhibits different forms of lectin-induced lymphocyte transformations (6). The -chain of Hp was shown to bind CD22 (7), a B-cell adhesion glycoprotein that mediates B-cell interactions with other cells. A specific binding of Hp with granulocytes and monocytes has also been reported. Native Hp blocks the human neutrophil response to a variety of agonists with defined receptors (8). Hp predominantly binds cells that express Mac-1 (CD11b/CD18) receptor (9), which belongs to the integrin family and is involved in cell-cell and cell-matrix interactions, such as binding to fibrinogen and to the cell-surface molecule intercellular adhesion molecule-1. In addition to its interaction with leukocytes, Hp can also exert an anti-inflammatory action by inhibiting inflammatory mediators, including prostaglandin synthase and cathepsin (10 and 11).

Consistent with its roles in modulating immune responses, Hp levels were found to be significantly decreased in a substantial portion of patients with asthma and/or rhinitis (12). Conversely, increased Hp values together with increased acute-phase reactants are associated with a more severe disease state, suggesting more inflammatory activity in these patients. In another study, wheezing was reported to be significantly related to low levels of Hp, and bronchial hyperresponsiveness related to high levels of Hp (13).

In a previous study (14), we identified the lung as a major site of extrahepatic synthesis of Hp in mice and baboons. A high level of Hp messenger RNA (mRNA) was detected specifically in airway epithelial cells. Expression of the Hp gene in mouse and baboon lung is regulated during development and inflammation, suggesting protective roles of Hp against infection and in the repair of injured tissues. As a first step toward understanding the biologic functions of Hp in human lung and Hp's roles in airway diseases, we have investigated the pulmonary expression of human Hp gene in humans and in transgenic mice. We have found that Hp gene is expressed in diseased or inflamed lung, but not in normal human lung. Interestingly, the cell type-specific expression of Hp gene in the lung of human is different from that in mouse and baboon, but is maintained in that of the transgenic mouse carrying human Hp gene. Our study suggests that Hp may play important roles in a variety of lung inflammatory diseases in humans.

	Materials and Methods

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Collection of Lung Tissues and Lavage Cells

Surgical specimens of human lung tissues were obtained, quick-frozen in Tissue-Tek OCT compound, and stored at 80°C until used for the preparation of cryosections. Lung lavage cells were collected from healthy volunteers. These individuals were prescreened using the following criteria: age between 18 and 40 yr, nonsmokers for at least 5 yr before study, no history of allergies or respiratory diseases, and not presently on any medication prescribed by a physician. Using a standard protocol (15), the volunteers underwent bronchoscopy with lavage. The pooled lavage fluid was centrifuged at 600 × g for 10 min at 4°C to separate cells from supernatant. The cells were resuspended in Hanks' balanced salt solution, and, using a cytospin, 2 × 10⁵ cells were then pelleted onto slides. The slides were fixed with 4% paraformaldehyde and stored at 80°C until used for in situ hybridization experiments. To collect lavage cells from mice, the animals were anesthetized, killed, and tracheally lavaged with 1.0 ml normal saline. The lavage procedure was repeated twice. The combined lavage fluid was used to collect cells for in situ hybridization as described earlier. In some experiments, mouse lungs were intratracheally instilled with 4% paraformaldehyde at a constant pressure of 20 cm water, immersed in the same fixative for 16 h, emblocked, and embedded in paraffin using standard histologic procedures. In other experiments, lung tissue was immediately stored in liquid nitrogen for the isolation of RNA.

Production of Transgenic Mice

A 9-kb human genomic DNA fragment (XbaI-HpaI), which contains the entire Hp 2 gene plus 1 kb of the 5' and 1.5 kb of the 3' flanking regions (16), was introduced into fertilized mouse eggs. Transgenic mice were developed in a background of CB6F1 by using the techniques of Gordon and Ruddle (17), following the procedure described previously (18). Mice carrying the human Hp gene were identified by polymerase chain reaction using a pair of primers specific for the human Hp gene. This was confirmed by Southern blot analysis. Two human Hp transgenic mouse lines obtained were then bred to C57BL/6.

Detection and Quantitative Analysis of Hp Protein

The serum level of human Hp protein in transgenic mice was analyzed by rocket immunoelectrophoresis according to the procedure described by Laurell (19), and the peak heights of the rocket immunoprecipitates of the samples were compared. Goat antihuman haptoglobin antiserum was obtained from United States Biochemical Corp. (Cleveland, OH). Production of human Hp protein in transgenic mice was also confirmed by polyacrylamide gel electrophoresis (PAGE) after the plasma was mixed with hemoglobin derived from red blood-cell lysate as previously described (20).

Isolation of RNA and Northern Blot Analysis

Total RNA was extracted from untreated mice or mice treated with 6 µg lipopolysaccharide (LPS) (Escherichia coli serotype 055:B5; Sigma, St. Louis, MO) per gram of body weight for 24 h. This was performed using TRIZOL reagent (Life Technologies, Rockville, MD) according to the protocol provided by the vendor. Northern blotting was conducted by using formaldehyde agarose gel electrophoretic separation and capillary transfer of RNA onto the nylon membrane (Micron Separation, Inc., Westborough, MA). Human Hp complementary DNA (cDNA) insert (21) was [³²P]- labeled by a random priming procedure and was used as a hybridization probe. Hybridization and washing of the blots were performed at high stringency conditions to reduce cross hybridization between human and mouse Hp sequences. Quantitative analysis of hybridization signals was conducted using a PhosphorImager analyzer (Molecular Dynamics, Sunnyvale, CA). Amounts of ribosomal RNA in each gel slot were used as gel loading controls.

Tissue In Situ Hybridization

Lung cryosections or paraffin sections of 5 µm thickness were prepared. Processing and hybridization of lung sections were performed as previously described by Zeller and Rogers (22). [³⁵S]- labeled single-stranded RNA probes were synthesized using the Riboprobe System (Promega Co., Madison, WI) according to the procedure supplied by the vendor. The two plasmid DNA templates used for riboprobe synthesis contained a 1.6-kb human Hp cDNA insert (21) and a 1.1-kb mouse Hp cDNA insert (23), respectively. After hybridization, sections were treated with 20 µg/ ml ribonuclease A at 37°C for 30 min and washed at high stringency (50% formamide, 2 × saline sodium citrate and 0.1% -mercaptoethanol at 55°C). For autoradiography, slides were coated with film emulsion (Kodak NTB-2) and exposed at 4 to 8°C for 3 to 5 d. Slides were then developed with Kodak D19 developer and stained with hematoxylin and eosin.

	Results

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Expression of Hp in Human Lung

In humans, Hp is produced mainly in the liver and secreted into circulation. In a previous study we found that the lung is a major extrahepatic site for Hp synthesis in mouse and baboon (14). In addition, studies by others indicate that Hp may play a role in inflammatory diseases of the lung in the human (12, 13). We therefore conducted in situ hybridization experiments to determine whether the Hp gene is expressed in the human lung. Lung biopsy specimens from patients with diagnoses of either malignant or fibrotic diseases were examined. The hybridization signal to Hp mRNA was found in lung tissues derived from six out of eight patients studied. Interestingly, Hp gene expression was consistently detected in or near regions of focal interstitial and/or intraalveolar infiltrates of inflammatory cells (Figures 1A and 1B), but not in the adjacent normal-appearing lung tissue. Hp mRNA was localized in several cell types in the inflamed lung, including alveolar macrophages (Figure 1C) and eosinophils (Figure 2). Most impressive were the high levels of hybridization signals seen in eosinophils, which were often present along the basement membrane of the airways (Figure 2). In a higher magnification of the micrographs, eosinophils can be identified by their characteristic bilobed nucleus and eosin-stained cytoplasmic granules. Unlike what was observed in mice and baboons (14), Hp mRNA was not detected in airway epithelial cells (Figure 2) in either inflamed or normal-appearing human lung tissues. We also conducted in situ hybridization experiments on alveolar macrophages recovered from lavage fluids of normal healthy volunteers (age 20 to 28 yr). Hp mRNA was not detected in the alveolar macrophages from any of the six normal healthy individuals studied (data not shown).

View larger version (105K): [in this window] [in a new window]   Figure 1.   Detection of Hp mRNA in human lung by in situ hybridization. A high level of Hp mRNA was detected in many lung cells at or adjacent to inflammatory sites in the lung. Thickening in the interstitium of alveolar walls with inflammatory cells was evident (thin arrows) and can be compared with normal-appearing walls (open arrows) (A). The hybridization signals for Hp mRNA (arrowheads in B) are clearly demonstrated in the darkfield micrograph of the upper portion of A. Alveolar macrophages with hybridization signals (arrowheads in C) were among the cells that express the Hp gene in inflamed lungs. Original magnifications: A and B, ×122; C, ×488.

View larger version (114K): [in this window] [in a new window]   Figure 2.   Localization of Hp mRNA in human eosinophils. High levels of Hp mRNA were detected in eosinophils (arrowheads) present along the basement membrane of airways (A, C, and D) and in the peripheral region (B) in an inflamed human lung. Eosinophils can be identified by their characteristic bilobed nucleus and eosin-stained cytoplasmic granules. The Hp gene is also expressed in human circulating eosinophils (arrowheads in E and F). Note that other cells, such as neutrophils (small arrows in B, E, and F) and airway epithelial cells (large arrow in A and D), are devoid of hybridization signals. A, C, and E are darkfield, and B, D, and F are brightfield pictures. Original magnifications: A, ×122; B-F, ×488.

Detection of Hp mRNA in Circulating Eosinophils

To determine whether the Hp gene is expressed in circulating eosinophils, buffy coat smears were prepared from normal healthy donors and hybridized to the human Hp antisense RNA probe. As shown in Figures 2E and 2F, Hp mRNA is present in eosinophils but not in any other blood cell. To determine the abundance of eosinophils in these individuals, differential cell counts were conducted in several fields of over 200 cells on each specimen. In all specimens examined, eosinophils represented 2 to 5% of the total white cells, which is within the normal range expected in healthy individuals. We therefore conclude that Hp gene is expressed constitutively in circulating eosinophils.

Regulated Expression of Human Hp Gene in Transgenic Mice

To study the expression and regulation of human Hp gene in the lung, we produced transgenic mice carrying a 9-kb human Hp genomic DNA that codes for type 2 human Hp. This 9-kb DNA fragment contains all exons and introns of Hp type 2 gene plus 1 kb of the 5' flanking sequence and 1.5 kb of the 3' flanking region. As shown in Figure 3A, human type 2 Hp protein, which can be distinguished from the endogenous mouse type 1 Hp, can be detected in the transgenic mouse serum. Similar to what was observed in humans, the level of human Hp protein in transgenic mouse serum (Figure 3B) appeared to be modulated by inflammatory stimuli such as endotoxin (LPS). The human Hp mRNA level in the transgenic mouse liver also increased after the animals were treated with LPS (Figure 3C). Northern blot analysis revealed the production of human Hp mRNA in the lung of both transgenic mouse lines generated (Figure 3D). Coinciding with the hepatic response, the pulmonary expression of the human Hp transgene was also augmented upon LPS treatment (Figure 3D). In transgenic mice, human Hp transgene was also expressed in the female reproductive tract and adipose tissues (data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 3.   Hepatic and pulmonary expression of the human Hp gene in transgenic mice. In A, PAGE was performed to confirm the presence of the human Hp protein in transgenic mouse serum. Sera from a human and from several wild-type mice were included for comparison. In B, rocket immunoelectrophoresis was performed to analyze the human Hp protein level in the serum of transgenic mice (lanes 1-6 and 9-12) treated with either endotoxin (LPS) or saline. Serum samples from a wild-type mouse treated with LPS (lanes 7 and 8) were included as controls. Serum was collected from each animal before (odd-numbered lanes) and 24 h after (even-numbered lanes) treatment. In C and D, human Hp mRNA levels in the liver (C) and in the lung (D) of transgenic (T) and wild-type (W) mice were compared in saline- or LPS-injected animals. V16I and V16II are two independent transgenic mouse lines. Northern blot analysis was conducted with 10 µg of total RNA from each tissue and hybridized with a human Hp cDNA probe. A low level of cross hybridization between the human Hp cDNA probe and mouse Hp mRNA can be seen in wild-type mice.

Cell Type-Specific Expression of Human Hp Gene in Transgenic Mouse Lung

The cell type-specific expression of human Hp gene in transgenic mouse lung was studied using the technique of in situ hybridization. In both transgenic mouse lines studied, the 9-kb human Hp 2 gene was expressed constitutively at a high level in alveolar macrophages (Figure 4). In situ hybridization was performed on tissue sections of inflated lung specimens (Figure 4A), and on cells recovered by bronchoalveolar lavage (Figure 4B). Hybridization signals to human Hp mRNA were detected in a majority of the lavage cells (Figure 4B), which contained > 90% of alveolar macrophages. The human transgene is expressed in alveolar macrophages but not in airway epithelial cells (Figures 4A, 4B, 4D, and 4F), and the endogenous mouse Hp gene is expressed in airway epithelial cells but not in alveolar macrophages (Figures 4C and 4E). In this experiment, two lung sections from each mouse lung were hybridized to human Hp and mouse Hp antisense RNA probes, respectively. Both hybridization and washing were conducted under high-stringency conditions. Therefore, very little cross hybridization between human and mouse Hp gene sequences was detected. The cell type-specific expression of human Hp transgene and mouse endogenous Hp gene appears to be consistent in the lungs derived from adult (Figures 4C and 4D) and newborn (Figures 4E and 4F) transgenic mice.

View larger version (60K): [in this window] [in a new window]   View larger version (74K): [in this window] [in a new window]   View larger version (83K): [in this window] [in a new window]   View larger version (83K): [in this window] [in a new window]   View larger version (98K): [in this window] [in a new window]   View larger version (86K): [in this window] [in a new window]   Figure 4.   Cell type-specific expression of human Hp gene and mouse endogenous Hp gene in the transgenic mouse lung. In situ hybridization was conducted on adult (A, C, and D) and newborn (E and F ) mouse lung sections, and a cytospin preparation of lavage cells from an adult mouse (B). The tissue or cell preparation was hybridized to either an antisense human Hp RNA probe (A, B, D, and F ) or an antisense mouse Hp RNA probe (C and E). Human Hp mRNA is detected in alveolar macrophages (arrowheads) but not in the airway epithelium (arrows), and the endogenous mouse Hp gene is expressed in airway epithelial cells but not in alveolar macrophages (C and E). In B, human Hp mRNA can be detected in a majority of the lavage cells, which are predominantly alveolar macrophages. Original magnifications: A and B, ×488; C, D, and F, ×244; E, ×122.

Eosinophils in Transgenic Mice Express the Human Hp Gene

As shown earlier, the Hp gene is expressed in eosinophils in inflamed lung as well as in circulation in the human. To determine whether the human Hp gene is expressed in eosinophils in transgenic mice, white blood cells isolated from the mice were used to prepare the blood-cell smears. Hybridization signals to human Hp mRNA were detected in eosinophils (Figure 5) but not other blood cells (data not shown). No hybridization signal was found when the white-cell smear preparation was hybridized to the mouse antisense Hp RNA probe (data not shown). We concluded that the human Hp transgene, but not the endogenous mouse Hp gene, is expressed in eosinophils in transgenic mice.

View larger version (87K): [in this window] [in a new window]   Figure 5.   Detection of human Hp mRNA in eosinophils from a transgenic mouse. Leukocytes isolated from transgenic mouse blood were hybridized with an antisense human Hp RNA probe. Hybridization signals were detected in eosinophils (arrowheads). Original magnifications: ×488.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Extravasation of erythrocytes into the lower respiratory tract occurs in numerous injuries, including bronchitis, cystic fibrosis, lung cancer, pneumonia, and diffuse alveolar hemorrhage. Free iron released from hemoglobin after he-molysis can catalyze the formation of reactive oxygen species and lead to oxidative damage in lung tissues. Both free hemoglobin and erythrocytes have been shown to induce lung injury in experimental animal models (24, 25; and Ghio and colleagues, unpublished data). Nevertheless, clearance in the lung is very efficient, and little free hemoglobin remains after large exposures (Ghio and colleagues, unpublished data). In the present study, we found that the Hp gene is expressed at a high level in diseased lung tissues but not in normal human lung. Because the Hp-hemoglobin complex can be removed efficiently by alveolar macrophages, Hp synthesized by alveolar macrophages at the site of inflammation could contribute significantly to the clearance of hemoglobin and thus protect the lower respiratory tract against hemoglobin-mediated oxidative damage.

We found that the Hp gene is also expressed at a high level in eosinophils in inflamed lung tissues. Both Hp-synthesizing eosinophils and alveolar macrophages were detected in lung tissues from some patients. However, in lung specimens from other patients, only Hp-synthesizing alveolar macrophages or eosinophils were detected. Although eosinophils and alveolar macrophages can be increased in various lung diseases, eosinophilia usually suggests an allergic disorder. Our finding that Hp is produced not only in alveolar macrophages but also in eosinophils suggests that Hp could be involved in a variety of lung inflammatory diseases, including respiratory allergy and asthma.

Although a number of studies have suggested a role of Hp in modulating immune response, little is known about the regulation and expression of the Hp gene in cells involved in immune reactions. Low levels of serum Hp have been associated with respiratory allergy and bronchial asthma (12, 13, 26). The precise role of Hp in immune response is not known. Hp has been shown to decrease the reactivity of lymphocytes and neutrophils toward a variety of stimuli, and may act as a natural antagonist for receptor-ligand activation of the immune system (8). It binds to lymphocytes and granulocytes via specific receptors, in particular the integrin family Mac-1 (CD11b/CD18) receptor, and can regulate Mac-1-dependent cell function in vivo (9). Therefore, a change in the concentration of Hp at the site of inflammation can modulate the immune reactivity of the inflammatory cells.

Expression of Hp gene is controlled mainly at the transcriptional level. During the acute-phase reaction, the level of Hp in circulation reflects the change of the Hp mRNA level in the liver. However, phagocytes such as neutrophils and monocytes that do not synthesize Hp de novo can take up exogenous Hp (27). Hp stored in the cytoplasmic granules can be released upon stimulation by tumor necrosis factor (TNF)-. The induction of Hp release in neutrophils appears to be mediated by the interaction between TNF- and a distinct cellular receptor p55 (28). Apparently, inflammatory stimuli and/or cytokines can affect not only the synthesis but also the release of Hp from its storage sites. Interestingly, children with a late asthmatic response had a decreased serum level of Hp after an allergen challenge, followed by an increase in serum Hp levels after 24 h (29). The authors explained these findings by suggesting that Hp might be infused into airways during the inflammatory phase of the late asthmatic response, followed by an enhanced Hp synthesis. The profile of serum Hp (decreased in the late asthmatic response, increased 24 h later) appeared to be analogous to the eosinophil counts in peripheral blood after the allergen challenge test (30). The concentrations of Hp in lung tissues during the course of an allergic response have not been studied. Our work suggests that the synthesis and secretion of Hp by eosinophils be investigated at different stages of the inflammatory process. The potential effects of Hp on the physiologic features of eosinophils are of particular interest.

We have reported that Hp is synthesized in the airway epithelial lining in both mouse and baboon (14). However, in the present study Hp mRNA was not detected in the airway epithelium in any of the human lung specimens examined. The physiologic significance of Hp gene expression in different loci in the lung among different species is currently unknown. The expression of the human Hp gene appears to maintain its cell-type specificity in the transgenic mouse background. In transgenic mice carrying a 9-kb human Hp genomic DNA fragment, the human Hp gene is expressed in alveolar macrophages but not in airway epithelium, whereas the endogenous mouse Hp gene is expressed in airway epithelial cells and not in alveolar macrophages. In humans, Hp mRNA can be found only in inflamed or diseased lung tissues, not in normal lung tissue or in alveolar macrophages isolated from healthy volunteers. However, the 9-kb human Hp transgene is expressed constitutively in alveolar macrophages in transgenic mice. Apparently, derepression of the Hp gene occurs when the 9-kb Hp DNA is removed from its natural chromosomal location. Alternatively, the trans-acting factor(s) required for the activation of the Hp gene might be present in murine alveolar macrophages. Therefore, activation of the human Hp gene in alveolar macrophages by inflammatory stimuli or other modulators is no longer needed in the transgenic mice. Interestingly, the cell type-specific expression of the human Hp gene in transgenic mice is also maintained in eosinophils. We have detected human Hp mRNA but not mouse Hp mRNA in eosinophils derived from peripheral blood of transgenic mice. Comparable to the response in humans, inflammatory stimuli can augment the expression of the human Hp transgene in both the liver and lung of the transgenic mice.

Hp has been shown to have many biologic functions. We have recently found that the human Hp protein produced in the transgenic mouse lung is functional and that overexpression of Hp attenuates blood-induced lung injury (Ghio and coworkers, unpublished observation). After an intratracheal instillation of blood, transgenic mice were more efficient than wild-type mice in removing hemoglobin. The preservation of cell type-specific expression of human Hp gene in alveolar macrophages and eosinophils makes the transgenic mouse line a promising system for studying the functions of Hp in vivo in different lung diseases, especially allergen-induced airway inflammation and asthma.