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(Journal of Leukocyte Biology. 2001;70:585-591.)
© 2001 by Society for Leukocyte Biology

Leukocyte elastase in murine and human non-Hodgkin lymphomas

Pascal De Noncourt*, Olivier Robledo*, Tommy Alain{dagger}, Anna E. Kossakowska{dagger}, Stefan J. Urbanski{dagger}, Edouard F. Potworowski* and Yves St-Pierre*

* Human Health Research Center, INRS-Institut Armand-Frappier, University of Quebec, Laval, Québec, Canada; and
{dagger} Department of Pathology, University of Calgary and Calgary Laboratory Services, Calgary, Alberta, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular proteases play a crucial role in the invasive behavior of normal and transformed leukocytes. Thus far, however, most of the attention has been focused on members of the family of matrix metalloproteinases. In this work, we show that lymphoma cells can express leukocyte elastase (LE) and recruit the enzyme at their surface via ICAM-1. The expression of LE by lymphoma cells was augmented significantly by stimulation with IL-6 and IL-13, both of which also induced the expression of MMP-9. Although LE and IL-13 transcripts were detected in several non-Hodgkin’s lymphomas, immunohistochemical analysis of lymphoma tissues also showed that LE was strongly expressed in infiltrating leukocytes. Given the spectrum of key molecules that can be cleaved by LE and that LE and MMP-9 are involved in the invasive behavior of normal or transformed leukocytes, our results raise the hypothesis that LE plays a crucial role in the multistep processes of inflammation and lymphoma metastasis.

Key Words: ICAM-1 • MMP-9 • cytokines • B cells • IL-6 • IL-13


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The phenomenon of metastasis requires extensive phenotypic changes within the tumor cells at the primary site. They must detach from the primary tumor, enter the blood circulation, resist hemodynamic shearstress of the blood circulation (home to the target organ), and successfully extravasate. All tumor cells do not necessarily undergo uncontrolled division in order to form secondary tumors once they have homed to a target site: one of the major rate-limiting steps in metastasis is the ability of the extravasated tumor cells to find an appropriate "nest" with favorable growth conditions. (This process is referred to herein as nidification.) Only these cells, which have successfully completed nidification, will undergo massive proliferation and form a secondary tumor. Nidification is facilitated when tumor or peritumoral cells produce specific proteases, which degrade structural proteins of the extracellular matrix. In fact, among the key candidate genes known to confer metastatic behavior to tumor cells are those that encode for members of the matrix metalloproteinase (MMP) family of enzymes [1 ]. In human high-grade non-Hodgkin’s lymphomas (NHL), for instance, there is a correlation between the level of expression of MMP-9 and the clinical aggressiveness of malignant lymphomas [2 ]. Moreover, overexpression of MMP-9 by gene transfer in lymphoma increases their tumorigenicity [3 ].

The expression of MMP by tumor cells at late stages of metastases is thought to occur during contact with the vascular endothelium via cell-adhesion molecules [4 5 6 ]. Our recent finding that the vascular endothelial cell-adhesion molecule intercellular adhesion molecule-1 (ICAM-1) controls the production of MMP-9 in lymphoma cells [4 ] and that ICAM-1-deficient mice are resistant to the spread of lymphoma [7 ] indicates a strong relationship between adhesion molecules and extracellular proteases. However, the MMPs are not the only extracellular proteases that could be involved in lymphomas. Cathepsins and leukocyte elastase (LE), two extracellular proteases expressed in normal leukocytes, are among the key candidates that could modulate the spread of lymphoma cells. Although the pathophysiological role of these enzymes, most notably LE, has been well documented in pulmonary disorders such as emphysema and cystic fibrosis [8 ], any participation of LE in cancer has never been established.

Several studies support the hypothesis that LE could be involved in metastases, most notably during the spread of lymphoma cells to peripheral organs. Not only can LE cleave cell-surface ICAM-1 [9 ], it can also directly influence the architecture of target organs by degrading components of the extracellular matrix. Kossakowska et al. [10 ] have demonstrated that cells isolated from aggressive NHL, but not those isolated from hyperplastic tonsils, had an increased ability to penetrate a physical barrier constituted of elastin. LE could also indirectly affect remodeling of the extracellular matrix at late stages of metastasis by activating MMP-9 [11 ] or by degrading TIMP-1 complexed with active MMP-9 [12 ]. LE can also degrade interleukin (IL)-2 to yield IL-2 polypeptides that inhibit IL-2-induced release of lymphoid cells from laminin, collagen IV, and fibronectin [13 ]. Moreover, the impact of LE in lymphomas could go well beyond its ability to enhance the migration of tumor cells through the extracellular matrix; apart from cleaving ICAM-1, LE has also been shown to cleave many regulators of the anti-tumoral response, such as immunoglobulin G (IgG), CD4, and CD8 [14 ]. However, the fact that aggressive lymphomas express high levels of MMP-9, an enzyme with an intrinsic elastolytic activity [15 ], implies that the ability of lymphoma cells to penetrate through the elastin barrier may not necessarily be mediated through the secretion of LE. Thus, in the present work, we sought to determine if LE is expressed in lymphoma cells and tissues and how this expression is regulated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
C57BL/6-ICAM-1-deficient mice (C57BL/6tm1jcgr) carrying a deletionnal mutation in the fourth exons of the ICAM-1 gene [16 ] were bred in our animal facility and maintained under specific, pathogen-free conditions and in accordance with institutional guidelines. These mice do not express the common form of ICAM-1 but do express a limited repertoire of minor isoforms generated by alternative splicing [17 ].

Reagents and antibodies (Abs)
Purified, human LE (HLE) was obtained from Calbiochem (La Jolla, CA). Sputum samples of patients with cystic fibrosis containing high concentrations of HLE were also used as a source of HLE for some experiments. The samples were prepared as described previously [9 ]. The rat anti-mouse ICAM-1 monoclonal antibody (mAb) YN-1 was purified from hybridoma culture supernatants by chromatography on protein G-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ) using standard protocols. Although the epitope recognized by this antibody is yet unknown, it is likely located in or nearby the first domain of ICAM-1, because this antibody can inhibit lymphocyte function-associated antigen-1 (LFA-1)-mediated intercellular adhesion [18 ]. Detection of YN-1 binding was carried out using a phycoerythrin (PE)-conjugated, donkey anti-rat antibody (Jackson ImmunoResearch Laboratory, Mississsauga, Ontario, Canada). The biotinylated 3E2 anti-mouse ICAM-1 mAb was obtained from Pharmingen (San Diego, CA). Mouse monoclonal IgG1 specific for HLE was obtained from Calbiochem. Biotinylated anti-mouse IgG (Fc-specific) was obtained from Sigma Chemical Co. (St. Louis, MO). Streptavidin (SA)-PE was obtained from Beckman-Coulter (Ville St-Laurent, Quebec, Canada). Recombinant cytokines IL-3, IL-5, IL-6, IL-11, and IL-13 were obtained from R&D Systems (Minneapolis, MN). pBK-RSV vector was obtained from Stratagene (La Jolla, CA).

Culture of cell lines
The mouse T lymphoma cell lines 267 and 718 were established in our laboratory from radiation-induced T-cell lymphomas in C57BL/Ka- and ICAM-1-deficient mice, respectively, as described previously [3 ]. Consequently, the 267 cells expressed the common form of ICAM-1 on its surface, and the 718 cells only expressed a low but detectable level of ICAM-1 isoforms ([3 ] and unpublished results). Both cell lines expressed surface T-cell receptors, CD90, CD102 (ICAM-2), and CD31. The human Burkitt’s lymphoma Raji cell line was obtained from the American Type Culture Collection (Manassas, VA). All cells were maintained in culture using RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 10 mM HEPES, and antibiotics (complete medium).

Transfection of ICAM-1 in 718 lymphoma cells
To restore the expression of ICAM-1 in 718 cells, the cDNA encoding the common form of murine ICAM-1 was subcloned into the SR{alpha} eukaryotic expression vector (kindly provided by Dr. François Denis, INRS-Institut Armand-Frappier, Quebec, Canada). The 718 lymphoma cells were transfected using Superfect (Stratagene) according to the manufacturer’s instructions. Stable cell lines expressing distinct ICAM-1 levels were established using puromycin selection. The ICAM-1 expression was assessed by flow cytometry using the biotinylated 3E2 anti-ICAM-1 mAb and SA-PE.

Binding of LE to the surface of lymphoma cells
Aliquots of 3–4 x 105 lymphoma cells were transferred to 96-well plates and washed twice with serum-free RPMI medium. The cells were then incubated at 37°C with the indicated concentrations of purified recombinant HLE or dilutions of LE-containing human sputum. After washing with phosphate-buffered saline (PBS), LE binding was measured by flow cytometry using LE-specific antibodies, biotinylated anti-mouse IgG, and SA-PE. Controls included cells incubated sequentially with LE, with the anti-mouse IgG, and with SA-PE.

Flow cytometry
Cells were stained at 4°C and washed with PBS containing 0.5% bovine serum albumin (BSA) and 0.2% sodium azide (PBA). Prior to staining, cells were incubated with 10 µg/ml human IgG (Sigma) for 20 min at 4°C to block nonspecific binding. Fluorochrome- or biotin-labeled mAbs were then added at appropriate concentrations and incubated for another 20 min. Cells were then washed four times with PBA. For indirect staining with SA-Red 670 (Gibco-BRL, Grand Island, NY), cells were washed three times following the reaction with the first mAb and then incubated 20 min on ice with the fluorescent conjugate. Results shown are representative of at least three independent experiments. Flow cytometric analyses were performed on 1–5 x 105 events using a Coulter XL-MCLTM flow cytometer (Coulter Electronics, Hialeah, FL) and recorded on a logarithmic scale.

Stimulation of Raji cells
Raji cells were cultured at 107/ml in serum-free RPMI medium for 12 h in the presence or absence of phorbol 12-myristate 13-acetate (PMA; Sigma), 50 nM; db-cAMP (Sigma), 1 mM; or with recombinant cytokines, i.e., IL-3, 10 ng/ml; IL-5, 10 ng/ml; IL-6, 100 ng/ml; IL-11, 10 ng/ml; and IL-13, 100 ng/ml. Cells were then centrifuged, and the pellets were frozen for RNA extraction.

Lymphoma specimen collection
NHL tissue samples in excess of that needed for diagnostic purposes were received in the Department of Pathology at Foothills Hospital (Calgary, Alberta, Canada), snap-frozen in liquid nitrogen, and kept at -70°C. The cases were classified according to the REAL Working Formulation [19 ]. All except Case 11 were of B-cell origin. Case 11 represents a large T-cell lymphoma. Cases 6, 8, 13, 14, 16, and 18–32 were large B-cell, aggressive lymphomas. Cases 1 and 10 were small lymphocytic, indolent lymphomas. Cases 2 and 3 were low-grade, follicular lymphomas. Case 4 was a mantle cell lymphoma. Cases 12 and 15 represented angioimmunoblastic lymphadenopathy, which are not lymphomas. RNA extraction and reverse transcription (RT) were carried out as described previously using the acid guanidium thiocyanate-phenol-chloroform-extraction method and the Superscript RT RNAse H-RT (Gibco-BRL), as described previously [10 ].

RNA isolation and analysis
Unless otherwise indicated, total cellular RNA was isolated using the Trizol reagent (Gibco-BRL) according to the manufacturer’s instructions. First-strand cDNA was prepared from total cellular RNA using the Superscript RT RNAse H-RT. After RT, polymerase chain reaction (PCR) amplification was carried out using the PCR core kit (Boehringer Mannheim, Laval, Quebec, Canada) with specific primers for LE, human MMP-9 [10 ], IL-13 [20 ], ß-actin (Stratagene), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; kindly provided by Dr. Daniel Oth, INRS-Institut Armand-Frappier; see Table 1 ). The design of human, LE-specific primer pairs was carried out using DNA sequences obtained from Genbank (National Center for Biotechnology Information, Bethesda, MD) and was chosen to have approximately 50% GC content. The sense and anti-sense primers were located in exons III and V, respectively. Thirty cycles of amplification were performed in an MJ Research Thermal Cycler (model PTC-100TM). Each cycle of amplification consisted of a denaturating step at 94°C for 1 min, a primer annealing step at 60°C for 2 min, and a strand-elongation step at 72°C for 1 min. The amplification for each gene was in the linear curve. The reaction mixture (5–15 µl) was size-separated on a 1.5% agarose gel, and specifically amplified products were detected by ethidium-bromide staining and ultraviolet transillumination. Loading was equalized to the internal control mRNA (GAPDH or ß-actin). Semi-quantitative analysis was carried out using a computerized densitometric imager (model GS-670, BioRad, Mississauga, Ontario, Canada).


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  		Table 1. Sequence of the Oligoprimers Used for the Detection of MMP, LE, and IL-13 in NHL

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Binding of HLE to the surface of lymphoma cells
Because freshly isolated lymphoma cells have an intrinsic elastolytic activity [10 ], and LE has been shown to bind cell-surface receptors on leukocytes via the integrin receptor CD11b/CD18 [21 ], we first sought to determine whether LE could bind to the membrane of T lymphoma cells, although these lymphoma cells do not express detectable levels of this integrin ([22 ] and unpublished results). For this purpose, 267 lymphoma cells were incubated with various concentrations of purified human recombinant LE, and the binding was measured by flow cytometry using specific anti-HLE mAbs. Our results showed that LE can bind to the surface of lymphoma cells in a dose-dependent pattern (Fig. 1A ). The binding was time-dependent (Fig. 1B) and was found to be reversible (Fig. 1C) , having reached a peak within 30 min. Moreover, the kinetics of de-binding, which occurs following the cleavage of ICAM-1 [9 ], correlated with the increase proteolytic activity in the supernatant, indicating that LE released from the cell surface remained proteolytically active. Similar results were obtained using natural LE using sputum of patients with cystic fibrosis (unpublished results). Thus, all subsequent binding assays were performed at 37°C for 30 min using recombinant LE.



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  		Figure 1. Binding of LE to lymphoma cells. (A) Dose-dependent binding of recombinant HLE to lymphoma cells. Lymphoma cells (267) were incubated with the indicated concentrations of purified LE at 37°C for 30 min, and binding of LE was detected by flow cytometry using antibodies to LE. (B) Time-dependent binding of LE to 267 lymphoma cells. Lymphoma cells were incubated with 5 µg recombinant LE for the indicated periods of time, and binding of LE was detected by flow cytometry using antibodies to LE. (C) Release of active recombinant LE in the supernatant after binding to lymphoma cells. Lymphoma cells were incubated with 5 µg recombinant LE for the indicated periods of time. LE activity ({blacksquare}) in the supernatants was measured by fluorescent-activated substrate conversion. Binding of LE ({blacktriangleup}) was measured as described. Data are representative of at least three separate experiments.

 
Binding LE to the surface of lymphoma cells correlates with ICAM-1 levels
Considering the fact that LE has been shown to cleave ICAM-1 [9 ] and that high concentrations of soluble ICAM-1 are found in patients with malignant lymphomas [23 ], it became pertinent to determine whether the level of expression of ICAM-1 could influence the binding of LE at the surface of lymphoma cells. To test this hypothesis, we compared the binding of LE on ICAM-1-deficient and ICAM-1-expressing lymphoma cells. The ICAM-1-deficient lymphoma 718 cell line was generated from a radiation-induced thymic lymphoma isolated from C57BL/6-ICAM-1-deficient mice (C57BL/6tm1jcgr) [16 ], and the 718-derived clones expressing different levels of the common form of ICAM-1 were obtained upon gene transfer of the cDNA encoding the common form of murine ICAM-1. Our results showed that binding of LE was increased significantly on the surface of clone 70, expressing high levels of ICAM-1 (Fig. 2 ). Binding LE on clone 36, expressing intermediate levels of ICAM-1, was also higher than that observed on the 718 parental cell line, indicating that increased binding of LE correlated with the expression of ICAM-1 on the cell surface of lymphoma cells. Pre-incubation of lymphoma cells with saturating concentrations of murine ICAM-1-specific mAbs YN-1, capable of inhibiting LFA-1 binding to ICAM-1 [18 ], did not decrease LE binding (unpublished results). These results suggest that the capacity of lymphoma cells to recruit LE on their surface is dependent on their level of expression of ICAM-1.



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  		Figure 2. Correlation between LE binding and ICAM-1 expression in lymphoma cells. ICAM-1-deficient 718 cells were transfected with cDNA encoding murine ICAM-1. Stable clones expressing different levels of ICAM-1 (lower panel) were then characterized for their ability to bind LE (upper panel). Binding was carried out at 37°C for 30 min using recombinant LE and determined by flow cytometry using anti-LE antibodies. Results represent the mean fluorescent intensity (MFI) measured on 5000 cells. Results are representative of at least two independent experiments.

 
LE expression by lymphoma cells
The above results showed that ICAM-1 expressed at the surface of lymphoma cells could recruit LE secreted in the extracellular space. Although expression of LE had almost exclusively been associated with cells of the monocytic lineage, we next investigated whether LE could be expressed in lymphoma cells as well and whether cytokines, previously shown to modulate the expression of MMPs in lymphoma cells [10 ], could modulate LE expression. Using the Raji lymphoma cells, we found no, or only very weak, expression of LE in resting lymphoma cells. However, exposure of lymphoma cells to IL-6, and IL-13 even more, strongly induced the expression of LE (Fig. 3 ). The up-regulation of LE expression was concomitant with that of MMP-9, suggesting that LE and MMP-9 are transcriptionally associated. This hypothesis was supported by the fact that IL-6 also up-regulated LE and MMP-9, although the increase in the latter case was more modest; induction of MMP-9 and LE was induced by PMA as well as by cAMP, both activators of protein kinase (PK)C and PKA enzymes, respectively. Taken together, these results showed that lymphoma cells can express LE upon stimulation with cytokines and that this up-regulation was concomitant with that of MMP-9.



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  		Figure 3. Up-regulation of LE in Raji cells upon exposure to IL-6 and IL-13. Raji cells were stimulated with various cytokines, PMA (50 nm), or db-cAMP (1 mM) for 8 h. LE and MMP-9 mRNA expression was then measured by RT-PCR. Results are representative of at least three independent experiments.

 
Expression of IL-13 and LE in human lymphoma tissues
Because IL-13 was a strong inducer of LE and MMP-9 and are expressed at high levels in human lymphomas, we proceeded to determine whether IL-13 was expressed in human NHL. Previous studies have indeed shown that IL-13 expression was found in cases of Burkitt’s lymphomas [24 ]. Thus, we analyzed by RT-PCR the expression of IL-13 in NHL tissues and found evidence of its expression at different levels in the majority of NHL cases of B- or T-cell origin (Fig. 4 ). The strongest expression of IL-13 was found in a peripheral, large T-cell lymphoma (Case 11).



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  		Figure 4. Expression of IL-13 and LE in NHL lymphoma tissues. Semi-quantitative analysis of the expression of human IL-13 and LE was measured by RT-PCR of RNA extracted from lymphoma tissues collected from 20 different cases of NHL. Results are representative of three independent experiments.

 
In the next series of experiments, we sought to determine whether expression of LE could be found within lymphoma tissues. To date, no studies have demonstrated such expression of LE in lymphoma tissues. For this purpose, we performed RT-PCR analyses on cDNA generated from RNA extracted from different cases of human NHL. Among the panel we tested, we found that LE was expressed in five cases (Fig. 4) , all of which were high-grade NHL, although not all aggressive NHL expressed LE. Case 11 had the highest expression level of LE; this case was a peripheral T-cell NHL, large cell type. The other positive cases expressing intermediate LE levels were aggressive, high-grade lymphomas of B-cell origin.

We next investigated the expression of LE at the protein level in lymphoma tissue. Figure 5A shows a low power view of a large cell, peripheral T-cell NHL, where LE expression was found to be present within the tumor. The blue area represents a residual, normal lymphoid follicle where no staining of LE could be detected. Figure 5B shows high magnification of the cells expressing HLE. Small (reactive) lymphocytes do not appear to express HLE.



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  		Figure 5. HLE expression in peripheral, large T-cell NHL. (A) Section of a lymph node shows partial replacement by peripheral, large T-cell NHL. Anti-HLE antibodies bind to macrophages and polymorphonuclear (pmn) leukocytes within the tumor. Central area represents residual lymphoid follicle and shows no evidence of HLE expression. Original magnification x40. (B) Section from the tissue adjacent to the NHL described in A, stained with anti-HLE antibodies. Macrophages and pmn outside the tumor express HLE. Original magnification x100.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown here that LE exhibits specific, saturable, and reversible binding to the surface of lymphoma cells; transfection of ICAM-1 into lymphoma cells increases their ability to bind LE; evidence of LE expression was found in aggressive, human malignant NHL of T- and B-cell origin; LE expression can be induced in lymphoma cells upon exposure to cytokines—most notably IL-13; induction of LE is concomitant to that of MMP-9; IL-13 is expressed in many subtypes of NHL; and evidence of LE expression can be found at mRNA and protein levels in human NHL tissues.

To our knowledge, the present study is the first to directly address the expression of LE in tumors. The importance of extracellular proteases, such MMPs, has been well-recognized at different stages of tumor development or during the spread of tumor cells to the periphery. A first indication that elastolytic activity plays a role in lymphoma was demonstrated in a previous study showing that lymphoma cells had the ability to penetrate through an elastin-rich matrix in vitro, suggesting that lymphoma cells possess an intrinsic elastolytic activity [10 ]. We propose that this elastolytic activity results from the up-regulation of LE expression by the lymphoma cells. Our in vitro data clearly established that lymphoma cells have the ability to express LE upon exposure to specific cytokines, such as IL-6 and IL-13, which we found to be expressed in NHL. Alternatively, lymphoma cells may use their ability to recruit, via ICAM-1, soluble forms of LE released in the extracellular space by peritumoral cells, most notably, those of the monocytic lineage, such as macrophages or lymphocytes. Indeed, there is increasing evidence that peritumoral cells contribute significantly to the local production of extracellular proteases, thereby favoring tumor growth [25 26 27 ]. Our in vivo data showing that LE was expressed by infiltrating leukocytic cells surrounding the lymphoid tumor are consistent with this possibility. Notwithstanding these observations, we believe that the biological impact of the presence of LE within the tumor is independent of its cellular origin. The concept that extracellular proteases play an important part in the remodeling of the tissue architecture, regardless of their cellular origin, has been well-demonstrated by the studies of the late Paul Basset [25 ].

We found that IL-13 increased the mRNA levels of LE and MMP-9 genes in Raji cells. Such a concomitant expression of both of these proteases was also observed upon exposure to IL-6, suggesting that LE and MMP-9 genes could share common, transcriptional, and regulatory elements in their promoters. One candidate is PU.1, a transcription factor of the ets family present in B cells and macrophages that have been shown to regulate the expression of LE and MMP-9 in monocytes and lymphocytes [28 , 29 ]. PU.1 involvement is supported by the fact that IL-13-mediated signals in B lymphocytes, such as those leading to the Ig heavy-chain class switching to IgE, involve PU.1 in cooperation with STAT6 [30 ]. However, there is also an argument against this possibility, namely the recent observation that PU.1 is part of a silencing sequence located in the promoter of gelatinase A [31 ]. This enzyme, a member of the gelatinase subfamily of MMPs with structural and functional homology with MMP-9, has also been implicated indirectly in the invasive behavior of lymphomas [10 ]. Furthermore, the fact that intracellular signals mediated through the IL-6 receptor seem to be intact in PU.1-deficient mice also suggests that other pathways may be involved in the activation of LE by these cytokines. A second candidate is the GABP, a heteromeric, transcription-factor complex that consists of GABP alpha, an ets factor, and GABP beta, a Notch-related protein. The binding of the GABP protein to the LE promoter ets site strongly activates its expression alone and in cooperation with the transcription factors c-Myb and C/EBP [32 ]. Future experiments using reporter constructs containing promoter elements of MMP-9 and LE genes will be needed to answer this question.

We found no direct correlation between the expression of IL-13 and that of LE. This was expected in cases where LE expression was not accompanied by IL-13 expression, because LE could also be up-regulated upon stimulation by other cytokines often expressed in malignant lymphomas, such as IL-6 [10 ]. In cases where we found no detectable LE expression in the presence of IL-13, we could envisage that up-regulation of LE by IL-13 was inefficient because of inappropriate numbers of functional, cell-surface receptors at that stage of the tumor development, as discussed above. However, IL-13 could induce the secretion of LE by peritumoral cells, which can be recruited at the surface of lymphoma cells via their receptors, most notably ICAM-1. If so, it is likely that the expression of elastolytic activity would not be restricted to lymphomas but could also be implicated in other types of cancer associated with a density of ICAM-1 adhesion molecules at their surface, such as melanoma [33 ]. Although ICAM-1 has been shown to act as an accessory molecule in the killing of tumor cells by cytolytic T lymphocytes (CTL) or natural killing (NK) [34 35 36 ], our data could provide an alternative explanation for the association of ICAM-1 with poor prognosis in stage I tumors and for inhibition of experimental metastasis by melanomas by interfering with its expression [34 ].

Our study is the first to show the up-regulation of LE expression upon exposure to IL-13 or IL-6. It is interesting that there has been an increasing number of studies pointing out a possible role of IL-13 in tumor development, most notably lymphomas. Using cDNA microarrays, Kapp et al. [24 ] found that IL-13 was highly expressed in Hodgkin’s disease (HD)-derived cell lines. Although the authors concluded that the expression of IL-13 seemed to be restricted to HD lymphomas, our results clearly show that IL-13 can also be expressed in NHL cases, including T-cell lymphomas. The same study by Kapp et al. showed that in situ hybridization of lymph-node tissue from HD patients demonstrated that elevated levels of IL-13 were expressed specifically by Hodgkin’s/Reed-Sternberg (H/RS) tumor cells and that neutralizing antibodies to IL-13 inhibited the proliferation of these cells, indicating that IL-13 could have multiple role in lymphomas. Moreover, Riemann et al. [37 ] had shown previously that IL-13 up-regulated protein expression as well as enzymatic activity of aminopeptidase N and dipeptidylpeptidase IV, two transmembrane type II molecules thought to be involved in metastasis. Experimental studies using gene-transfer strategies or genetically engineered mouse models should help to determine the molecular mechanisms controlled via IL-13 that contribute to the aggressiveness of lymphoma cells.

We have clearly established that LE can be expressed by lymphoma cells or can be recruited to its surface via ICAM-1 when found in its soluble form. The exact binding site(s) on ICAM-1 is unknown. The common form of ICAM-1 is a large molecule containing five Ig-like domains. That YN-1 antibody did not interfere with binding of LE on ICAM-1 is not surprising; in fact, it is likely that LE binds to at least two distinct binding sites, because cleavage of purified ICAM-1 generates several fragments [9 ]. The molecular mechanism by which LE could modulate the development of lymphoma is as yet unknown but is likely to involve local extracellular matrix remodeling and/or protection from the anti-tumoral response. Although constitutive expression of high levels of LE in murine lymphoma cells is difficult to obtain, experiments using gene transfection could help to resolve that question (P. De Noncourt, Y. St-Pierre, unpublished observations). However, so far our experiments using lymphomas transfected with a cDNA encoding LE have shown that transfection of LE in lymphoma cells did not have any significant effect on their growth after their intrathymic injection (unpublished results), as we had previously observed in the case of MMP-9 transfection [3 ]. This would suggest that the biological role of LE in lymphomas could be distinct from that of MMP-9 and that the elastolytic activity of MMP-9 is different from that of LE. This would be consistent with our previous observations showing that only LE, not MMP-9, can cleave ICAM-1 even at high concentrations [9 ]. Because LE can bind to ICAM-1 and cleave several key receptors involved in the recognition of tumor cells, it will be important to determine whether increased binding of LE on lymphoma cells can alter their recognition by anti-tumoral cells. The use of genetically engineered mouse models with altered LE gene expression will help to better understand its role in lymphomagenesis.

The localization of LE in lymphoma cells stimulated with IL-6 or IL-13 is yet unknown. We hypothesize that LE can be stored in an endosomal compartment, associated with the plasma membrane, or secreted. Whether this localization depends on the stimulus will require an extensive investigation using confocal microscopy. Moreover, phylogenetic studies have shown that LE is a member of a separate branch (Class 6) of serine proteases that is expressed by cells of the immune system and that clearly differs from that of well-known digestive enzymes such as trypsin, chymotrypsin, and pancreatic elastase [38 ]. Cytogenetic-positioning studies have shown that Class 6 enzymes are disseminated in three main, multi-gene clusters. As for the LE gene, it is colocalized with azurocidin and proteinase 3 within an approximately 50-kb cluster on human chromosome 19, sharing strong homology in terms of exon-intron organization and transcriptional orientation [39 ]. Because all three genes are often expressed coordinately, it will be interesting to test the role of other members of the Class 6 enzymes in the development of lymphoid and myeloid neoplasia.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Medical Research Council of Canada (E. F. P. and Y. S-P.) and the Fonds pour la Formation de Chercheur et d’Aide à la Recherhe (FCAR; Y. S-P.). Y. S-P. is a Fonds de la recherche en Santé du Québec scholar. P. D. N. is supported by a studentship from La Fondation Armand-Frappier. We thank Ms. Doris Legault, Claire Beauchemin, and M. Marcel Desrosiers for their excellent assistance and Dr. André Cantin (University of Sherbrooke) for providing sputum samples.


   FOOTNOTES
 
Correspondence: Yves St-Pierre, Ph.D., Human Health Research Center, INRS-Institut Armand-Frappier, 531 Boul. Des Prairies, Laval, Québec, Canada H7V 1B7. E-mail: yves.st-pierre{at}inrs-iaf.uquebec

Received January 13, 2001; revised April 23, 2001; accepted May 23, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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