ARTICLE |
Correspondence to: Jeannette Bourguignon, INSERM, Unité 295, Faculté de Médecine-Pharmacie de Rouen, 22 Boulevard Gambetta, F-76183 Rouen Cedex, France.
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Summary |
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The inter--trypsin inhibitor (ITI) family is a group of plasma proteins built up from heavy (HC1, HC2, HC3) and light (bikunin) chains synthesized in the liver. In this study we determined the distribution of ITI constitutive chains in normal and cancerous lung tissues using polyclonal antibodies. In normal lung tissue, H2, H3, and bikunin chains were found in polymorphonuclear cells, whereas H1 and bikunin proteins were found in mast cells. Bikunin was further observed in bronchoepithelial mucous cells. In lung carcinoma, similar findings were obtained on infiltrating polymorphonuclear and mast cells surrounding the tumor islets. Highly differentiated cancerous cells displayed strong intracytoplasmic staining with H1 and bikunin antiserum in both adenocarcinoma and squamous cell carcinoma. Moreover, weak but frequent H2 expression was observed in adenocarcinoma cells, whereas no H3-related protein could be detected in cancer cells. Local lung ITI expression was confirmed by RT-PCR. Although the respective role of inflammatory and tumor cells in ITI chain synthesis cannot be presently clarified, these results show that heavy chains as well as bikunin are involved in malignant transformation of lung tissue. (J Histochem Cytochem 47:16251632, 1999)
Key Words:
intra--trypsin inhibitor, ITI heavy chains, bikunin, immunohistochemistry, RT-PCR, normal lung tissue, lung adenocarcinoma, squamous cell lung carcinoma
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Introduction |
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The inter--TRYPSIN INHIBITOR (ITI) family is a group of proteins assembled in different ways from two main precursors: heavy and light chains. The major form found in human plasma is the inter-
-trypsin inhibitor, which contains two homologous heavy chains, HC1 and HC2, bound to the light chain called bikunin. Another member is the pre-
-trypsin inhibitor (P
I), composed of bikunin and HC3 (
1-microglobulin, a separate entity (
I and in ITI, the constitutive polypeptides are covalently linked via a chondroitin sulfate chain carried by bikunin (
To date, the study of ITI immunoreactivity in human tissues has been performed only with antisera directed against the whole ITI molecule or bikunin, and showed a localization of ITI-related proteins and bikunin in mast cells and connective tissues of the bladder (
Several functions have been proposed for the heavy and bikunin chains. Bikunin has potent inhibitory effects on the activities of trypsin, chymotrypsin, plasmin, and human leukocyte elastase (
In this study we used antibodies specific for each chain to examine the localization of ITI heavy H1, H2, H3, and bikunin chains in human specimens from normal lung and from adenocarcinoma and squamous cell lung carcinoma. Total RNAs extracted from lung tissues were also examined by RT-PCR to assess the expression of these proteins in the lung.
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Materials and Methods |
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Tissues
Blocks of human lung tumors were retrieved from the pathology files of the Rouen University Hospital. The specimens involved adenocarcinoma (n = 3) and squamous cell carcinoma (n = 4), the corresponding normal tissues being sampled in distant histologically normal areas. The samples were fixed with 4% paraformaldehyde in PBS for 24 hr at 4C, dehydrated, and embedded in paraffin.
Antibodies
Polyclonal antiserum against bikunin was produced in rabbits by immunization with proteins purified from normal human urine (
Immunohistochemistry
Paraffin-embedded tissues were cut into 4-µm sections, placed on silanized slides, then treated with xylene to remove the paraffin and rehydrated through graded alcohol. After washing with PBS containing 0.05% Tween-20, the sections were placed in a humidified chamber and preincubated with normal human or goat serum for 30 min at room temperature (RT) to block nonspecific binding. The sections were then incubated with the primary antibodies (crude antisera) diluted 1:20 with Tris-buffered saline (TBS: 0.1 mol/liter, pH 8.2; Dako, Trappes, France) for 1 hr at RT, followed by a second-step incubation with alkaline phosphatase-labeled goat anti-rabbit immunoglobulins (Dako) diluted 1:40 for 30 min at RT. After washing in TBS, the alkaline phosphatase activity was detected with the Fast Red substrate system (Dako), containing naphthol phosphate, Fast Red, and levamisole in TBS. Optimal labeling was checked under the microscope during the final reaction step. Sections were finally washed under tapwater, counterstained with hematoxylin (Dako), and mounted with Faramount aqueous medium (Dako). Control experiments for staining specificity included replacement of primary antibody with preimmune rabbit serum or TBS alone. To visualize tissue mast cells, the adjacent serial sections were stained with 0.1% Alcian blue (Sigma; St Louis, MO) in acetic acid, pH 2.4, for 20 min at RT.
Microscopy
Immunostaining was studied using an Axiophot microscope (Zeiss) equipped with a 3CCD camera (Sony) and then analyzed with SAMBA 2005 (Système d'Analyse Microphotométrique à Balayage Automatique, Alcatel; TITN, Grenoble, France), an image processing system (
Reverse Transcriptase Polymerase Chain Reaction
Total RNAs were extracted from liquid nitrogen-frozen normal and tumor lung tissue using the guanidinium isothiocyanate procedure followed by centrifugation through a 5.7 M CsCl cushion as described (
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DNA Sequence analysis
Purified PCR products were cloned into the SmaI site of pUC18 vector. DNA sequencing was performed using the dideoxy chain termination method (
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Results |
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Immunohistochemical Staining of Normal Lung Tissue
The distribution of ITI constitutive chains was studied on serial paraffin sections from tumor-free regions of lung tissue. The sections of lung tissue contained large segmental bronchi as well as smaller subsegmental bronchi, alveolar walls, and medium-sized pulmonary arteries. Expression of bikunin was observed in the apical pole of some mucous cells of the bronchiolar epithelium and in the underlying connective tissues (Figure 1E and Figure 1F). Strong immunohistochemical staining for H2, H3, and weaker staining for bikunin were observed in the inflammatory granulocyte cells scattered in the interstitial connective tissue (Figure 1C1E and Figure 1H) as well as in the lumen of blood vessels (not shown). The negative reaction of these cells with nonimmune rabbit serum (Figure 1A) or anti-H1 antibody (Figure 1B) showed the specificity of this finding. In contrast, H1 immunoreactivity was found to be associated with mast cells (Figure 1B), identified by their positive Alcian blue staining in adjacent sections (not shown). Mast cells also stained positive with bikunin (Figure 1E and Figure 1G). No signal could be observed with either anti-H antiserum in the bronchial epithelium or in alveolar walls.
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Immunohistochemical Staining of Lung Cancer Tissues
As a first step towards determining how ITI chain expression is altered during the malignant process, we examined multiple lung tissue sections from three adenocarcinomas and four squamous cell carcinomas. The polymorphonuclear cells infiltrating the tumor stroma were strongly stained with H2 and H3 antisera (Figure 2F and Figure 2H). No immunostaining for H2 and H3 was found in the tumor cells of squamous cell carcinomas. In contrast, highly differentiated cancer cells were weakly stained with H1 (Figure 2D). The mast cells were strongly stained with H1 antibody in one of the squamous cell carcinomas studied (not shown). The strongest staining was observed with bikunin in highly differentiated cells (Figure 2I2K) from squamous cell carcinomas and adenocarcinomas. Detectable H2, H1, and bikunin immunostaining was observed in adenocarcinomas, with some variability in the staining intensity: strong with bikunin (Figure 2I) and H1 (Figure 2C), weak with H2 (Figure 2E). For H3, all adenocarcinoma cells were negative (Figure 2G). The staining covered the whole cytoplasm for H1 and bikunin but was predominantly apical for H2. Positive staining was not observed in a control section incubated with normal rabbit serum or TBS alone (Figure 2A and Figure 2B).
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RT-PCR
To determine whether the immunohistochemical staining observed in lung tissue sections corresponded to simple adsorption of proteins or to local synthesis by lung tissues, total lung RNAs extracted from the corresponding normal and tumor tissues were analyzed using RT-PCR. The PCR products obtained with tumor (Figure 3, Lanes 3) and normal human lung tissue (Figure 3, Lanes 4) displayed sizes similar to those observed with HepG2 RNAs (Figure 3, Lanes 1), corresponding to the expected values, i.e., 138 bp (H1), 127 bp (H2), 318 bp (H3), and 294 bp (bikunin). Sequence analysis of the amplified products confirmed that they consisted of authentic H1, H2, H3, and bikunin cDNAs. A semiquantitative comparison of ITI expression in HepG2 cells (Figure 3, Lanes 1) and nontumor lung (Figure 3, Lanes 4) or lung cancer tissues (Figure 3, Lanes 3) showed equivalent levels of H1 and H3 mRNA transcripts but a lower level of H2 and bikunin in lung compared to hepatoma cells.
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Discussion |
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The presence of bikunin was previously reported in a variety of human and rodent tissues, i.e., brain, liver, kidney, intestine, and pancreas (
Taking advantage of specially designed antisera, we could clarify the ITI immunoreactivity of mast cells described by
In the present study we demonstrate for the first time a strong immunostaining of polymorphonuclear cells for H2, H3 and, to a lesser degree, for bikunin. The question of whether this immunostaining is a widespread phenomenon or results from local lung activation of polymorphonuclear cells cannot be clarified at this stage. However, preliminary experiments from our laboratory using peripheral blood cell smears also showed immunostaining with H2, H3, and bikunin antisera in some leukocytes, suggesting a constitutional expression, whereas no labeling could be observed with H1 antiserum (unpublished data).
In non-small-cell lung cancer tissue, we found positive immunostaining for bikunin in stromal cells, as shown by
In a previous work,
The present study has established a local synthesis of bikunin, H1, H2, and H3 chains in human normal and tumorous lung. It should be noted that in plasma, ITI is only present as HC1/HC2/ bikunin or PI (HC3/bikunin). The presence of H2, H3, and bikunin chains in polymorphonuclear cells suggests that a local synthesis of ITI-like protein (HC2/bikunin) may take place in the lung. Such a tissue-specific expression of HC2/bikunin has been described in hepatoma cells (
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Acknowledgments |
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Supported by INSERM grant PARMERCA9611.
We wish to thank Ms E. Abdelouhab for preparation of tissue sections, Mr M. Fraterno for SAMBA analysis, Ms N. Porchet for secretarial assistance, and Ms A. Brickwood for careful reading of the manuscript.
Received for publication June 10, 1999; accepted July 13, 1999.
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