TUMOR CELLS TRANSFECTED WITH THE β-CHEMOKINE GENE CCL16 (LEC) ARE MORE VULNERABLE TO INFECTION

Brancone ML, Conti P. Tumor cells transfected with the β-chemokine gene CCL16 (LEC) are more vulnerable to infection. International Journal of Infection. 2025;9(3):87-96.

M.L. Brancone¹ and P. Conti^2*

¹ U.O.C. Pathology Department, Mazzini Hospital, Teramo, Italy;
² Postgraduate Medical School, University of Chieti-Pescara, Chieti, Italy.

*Correspondence to:
Professor Pio Conti,
Postgraduate Medical School,
University of Chieti-Pescara,
66100 Chieti, Italy.
e-mail: pioconti@yahoo.it

Received: 20 June, 2025 Accepted: 07 November, 2025

ISSN 3103-6678 [online] Copyright 2025 © by Biolife-publisher This publication and/or article is for individual use only and may not be further reproduced without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties. Disclosure: all authors report no conflicts of interest relevant to this article.

ABSTRACT

This study targets the chemokine CCL16 and its gene, LEC, which belongs to the β-chemokine family, also known as CC chemokines. The specific location of the LEC gene has not yet been precisely determined. In macrophages, LEC is encoded by two mRNAs, is influenced by IL-10, and plays an interesting biological role during the early stages of the inflammatory process. Recombinant LEC protein has demonstrated a dose-dependent chemoattractive capacity towards non-activated human monocytes and the human THP-1 monocyte line. Here we report that subcutaneous injection of cells obtained from a murine mammary adenocarcinoma (TSA) engineered to produce LECs leads to the development of an inflammatory response, resulting in hyperplasia of the lymph nodes draining the inoculation area. Our results demonstrate that LEC is a chemokine with high proinflammatory activity and LEC production is upregulated by IL-10.

KEYWORDS: CCL16, LEC, chemokine, tumor cell, gene, IL-10, inflammation

INTRODUCTION

Transfection is a useful method in gene therapy that introduces exogenous genetic material (transgene) into recipient cells (1). Transfection is used to introduce foreign DNA or RNA into cells, allowing for the expression of specific genes (2). It can be transient or stable if the transfected DNA is kept in the cytoplasm for a limited period or integrated into the cellular genome. However, transfected tumor cells can be more susceptible to infections for various reasons (3). Transfection can modify the expression of genes involved in the immune response, and the transfected tumor cells may become more susceptible to viral, bacterial or fungal infections (4). Some transfections are designed to knock down or knock out specific genes, including those that might be important in immune responses.

There are 3 subclasses of chemokines: a-chemokines (or CXC chemokines), b-chemokines (or CC chemokines), and CX3C chemokines. CXC chemokines have the first 2 cysteine ​​residues separated by a different amino acid residue (5), while CC chemokines have the first 2 cysteine ​​residues adjacent. CX3C chemokines are encoded by a single gene located on chromosome 16q13 (6) (Table I).

Table I. Organization of Human CXC and CC genes.

At least 27 distinct members of the CC subgroup have been reported in mammals, with CC chemokine ligands (CCL) designated from 1 to 28 (CCL10 is equivalent to CCL9). The CC chemokine group includes MIP-1α, MIP-1β, MCP-1, RANTES, C10, and I309. CC chemokines are primarily represented by MCP-1, which acts on monocytes, lymphocytes, and eosinophils by binding to the CCR2 or CCR3 receptor (7). Based on the chemokine class, chemokines are distinguished between CXC2, 3, 4, and 5 receptors, which bind CXC chemokines, and CCR1 and CCR9 receptors, which bind C-C chemokines. XCR1 receptors bind leptin (a C-C chemokine), while CX3CR1 receptors bind frattaquine or neurotactin (8) (Table II, Fig.1).

Table II. Summary of the known chemokine receptors and some of their known human ligands.

Fig. 1. Endothelial cells, monocytes, fibroblasts, and dendritic cells produce cytokines and chemokines that influence the activity of immune cells (Th1, Th2, basophils, and natural killer cells) and vice versa.

CCL16 is a ligand of the C-C chemokine family that is involved in immune and inflammatory responses (9). CCL16 can attract different types of immune cells (including monocytes and lymphocytes) to sites of infection, where they mount an immune response against the microorganisms (10). CCL16 binds to specific receptors on immune cells, guiding them to the site of infection. During the infectious phase, CCL16 expression can increase, allowing the recruitment of immune cells to fight pathogens and inflammation. However, it is important to understand the mechanism by which CCL16 intervenes in infections. This could contribute to the development of new therapeutic strategies against infectious and inflammatory processes.

Our study aimed to analyse the role of the chemokine CCL16, which has been reported as an unmapped LEC gene that resides in a chemokine-encoded gene cluster located on chromosome 17 (17q11.2) and is considered to be a chemoattractant for lymphocytes and monocytes. The protein encoded by the LEC gene is composed of a 120-amino acid chain with a 20-amino acid primer and a carboxy-terminal end containing a putative N-linked glycosylation site (11).

CCL16 is encoded by two mRNAs; one is 1.5 Kb long and is detected in activated monocytes and stabilized (or upregulated) by the interleukin IL-10, while the other is 0.5 Kb long and whose expression is unaffected by IL-10 (12). The effect of IL-10 in enhancing LEC mRNA transcription is unique, as IL-10 generally downregulates the expression of all other cytokines. This suggests that LEC plays an interesting biological role during the early stages of the inflammatory process. In some studies, a virtually ubiquitous transcription of the 0.5 Kb LEC mRNA has been found (13).

Some biological characteristics of LEC have been tested in vitro and mirror the properties of other chemokines of the C-C subclass. In particular, the recombinant protein CCL16 has demonstrated a dose-dependent chemoattractive capacity towards non-activated human monocytes and the human monocyte cell line THP-1, with a peak response at a concentration of 1 μg/ml (14). The observed activity is certainly chemotactic and not chemokinetic. Furthermore, CCL16 induces a flux of Ca²⁺ ions in THP-1 monocytes. Ca²⁺ ion flux in response to CCL16 is observed at chemokine concentrations as low as 10^-9 mol/L and is dose-dependent, with a maximal response of 10^-6 mol/L (15). Ca²⁺ ion flux is reduced by prior incubation of THP-1 monocytes with RANTES.

In this study, we demonstrate how subcutaneous injection of cells derived from a murine mammary adenocarcinoma (TSA) engineered to produce LECs leads to the development of a complex inflammatory response that causes hyperplasia of the lymph nodes draining the injection site.

MATERIALS AND METHODS

Tumor Cells

The TSA cell line was derived from an aggressive, poorly immunogenic, moderately differentiated mammary adenocarcinoma that arose spontaneously in female BALB/c mice. Parental TSA-pc cells express major histocompatibility complex class I (MHC-I) molecules, but not MHC class II (MHC-II) molecules, and secrete G- and GM-CSF, TGF-β, basic fibroblast growth factor (bFGF), and VEGF, but not LEC. TSA-pc cells were grown to a cell density of 6 × 10¹⁻¹/100 mM in the culture dish and were transfected with the LEC gene using Lipofectamine reagent (Life Technologies, Rockville, MD). The clone derived from the TSA-LEC transfection, seeded at a concentration of 1 x 10¹⁻¹/ml, was cultured at 60-70 ng of LEC/1 ml for 48 hours.

Mice

7-week-old BALB-cAnCr female mice (Charles River Laboratories, Calco, Italy) were treated by injecting 0.2 ml of a cell suspension containing 1 x 10⁶ TSA-pc or TSA-LEC cells into the left flank.

Morphological analysis

For histological evaluation, tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin-eosin or Giemsa. For electron microscopic analysis, samples were fixed in glutaraldehyde in 2.5% cacodylate buffer, fixed in osmium tetroxide, and embedded in EPON 812. Ultrathin sections were then stained with uranyl acetate-lead citrate. For immunohistochemical investigation, acetone-fixed cryostat sections were incubated for 30 minutes with antibodies to CD4, CD8-a (Sera Laboratories International Ltd, West Sussex, UK), MAC-1 (anti CD11b/CD18) anti-Mac-3, Ia (Behringer Mannheim, Milan Italy), polymorphonuclear leukocytes (RB6-8C5) anti-endothelial cells (mEc-13,324 or anti-CD31 (provided by Istituto Negri Nord, Italy), dendritic cells (DCs) (NLDC 145, DEC 205, Cederlane Laboratories, Ontario, Canada), anti-TNF, anti-IFNg (provided by Dr. S Landolfo, University of Turin Italy), anti-MCP-1, anti-CD11b (integrin (Mach-1 alpha chain) and anti-CD61 (beta-3 integrin chain), collagen type 4 (Chemicon, Temecula, California), anti-lamin (Becton Dickinson, Bedford, Massachusetts), anti-VEGF, anti-FGFb and anti-PDGF (Santa Cruz Biotechnology, Inc., Santa Cruz, California), anti-IP-10, anti-Exodus-2/SLC (secondary lymphoid tissue chemokine) and anti-RANTES (Peprotech, Inc., Rocky Hill, NJ), anti-MIG (R&D Systems GmbH, Wiesbaden, Nordenstadt, Germany) and anti-MIP-2.

After washing, the sections were stimulated for 30 minutes with biotinylated goat anti-rat, goat anti-hamster, goat anti-rabbit, or horse anti-goat immunoglobulins. Unbound antibodies were removed by repeated washing, and the sections were then incubated with an avidin/biotin alkaline phosphatase complex. Quantitative studies were then performed in a blinded fashion on three or more samples from different animals, examining ten random fields for each sample. Antibody-positive CD31 microvessels and cells were counted under a microscope (10 fields with a 40x objective, 0.18 mm< per field). The expression of cytokines and adhesion molecules on cryostat sections tested with the corresponding antibody was classified as absent (-), weak (+/-), moderate (+), diffuse (++), and strong (+++).

Chemokine production

To obtain macrophages, the peritoneal cavity of BALB-c mice was washed several times with 5 ml of RPMI 1640 containing 10 U/ml heparin. Differential cell counts, performed on the supernatant treated with Diff Quick, showed that the cell population obtained with repeated washings consisted of approximately 55–65% macrophages. To increase macrophage counts, 1 ml of this suspension, containing 8 x 10⁶ cells, was plated in 24 wells and incubated at 37°C for 2 hours. Non-adherent cells were removed by vigorous washing with RPMI. Adherent cells (macrophages) were incubated in a final volume of 1 ml of RPMI with 10% FBS, in the presence or absence of 1, 10, and 100 ng/ml of recombinant LEC (Peprotech). The plates were incubated for 72 hours at 37°C, and the amount of MCP-1, MIP-2, MIP-1a, and RANTES released was determined by ELISA using commercial kits (R&D System Inc., Minneapolis, MN). The results shown here are representative of at least four independent experiments.

Antibody screening

7-30 days after the first injection of TSA-LEC, sera were collected from groups of 5 mice. Normal sera were instead collected from 5 untreated animals. TSA-pc cells from in vitro cultures were washed twice with cold HBSS with 2% BSA and 0.05% sodium azide and tested against immune serum or normal serum in a 1:10 dilution with HBSS-azide-BSA. Subsequently, the following antibodies were used: FITC-labelled goat anti-mouse immunoglobulin (Technogenetics, Milan, Italy), FITC-labeled rat anti-mouse immunoglobulins of the G1, G2a Ig, and G3 classes. All steps ended with a 30-minute incubation at 4°C and were separated by 2 washes with cold HBSS-azide BSA. The labelled cells were then analyzed with a FACS analysis cytofluorimeter (Becton Dickinson, Mountain View, CA). In each experiment, 10⁴ cells were analyzed.

RESULTS

Histology and immunohistochemistry

Seven days after inoculation, numerous macrophages, lymphocytes, granulocytes, and DCs were observed in BALB/c mice, both among the TSA-LEC cell aggregates and at the periphery of the resulting mass (Fig.2b, Table III). Giemsa staining and immunohistochemistry with anti-TNF antibodies demonstrated the presence of basophils and mast cells with numerous metachromatic granules in the cytoplasm. This inflammatory infiltrate was associated with the marked expression of adhesion molecules (ICAM-1, ELAM-1, and VUCAM-1) on the endothelium of numerous vessels (Fig.2d,2e). The numerous capillaries formed an extensive network between the TSA-LEC cells (Fig.2b, Table III). The lamina, an essential component of the basement membrane, was arranged linearly and without interruptions around some capillaries, but appeared inhomogeneous and fibrillar around micro vessels originating from sprouting. Staining with antibodies against type 4 collagen showed a distribution pattern very similar to that found for the lamina. These characteristics were very frequent in the growth area of TSA-LEC cells, while they were only occasionally present in the tumor mass consisting of TSA-pc (Fig.2a).

The expression of the proangiogenic factors VEGF and FGFb and the antiangiogenic chemokines MIG and IP10 was almost superimposable in the growth areas of TSA-pc and TSA-LEC. In the latter, a different expression of PDGF was observed near the areas rich in macrophages and endothelial cells (Table III). In the LEC-secreting cell inoculation area, locally recruited leukocytes showed a massive expression of pro-inflammatory cytokines, primarily TNF and IFN-γ. The chemokine RANTES was sparsely present, while MIP-2 and MCP-1 were highly expressed (Fig.2b, Table III).

Histological and immunohistochemical analysis demonstrated hyperplasia of both areas (cortical and paracortical) and of the lymph node draining the LEC-secreting cell area, while the marginal sinus appeared dilated and filled with mononuclear cells. In the cortical area, several secondary follicles with large germinal centers were observed. Hyperplasia of the lymph node draining the TSA-pc area was much less pronounced and mainly confined to the paracortical area. A particular characteristic was the massive infiltrate of DCs among the T lymphocytes in the paracortical area of the lymph node draining the TSA-LEC inoculation area (Fig.2a,2c). In this lymph node, the expression of Exodus 2/SLC in the high endothelial venules (HEVs) and surrounding cells was much more evident than in the lymph node draining the TSA-pc inoculum. MCP-1 expression was strong and localized mainly in the mantle zone and the luminous zone of the germinal centers of the lymph nodes draining the TSA-LEC cells.

Fig. 2. Figures (2a) and (2c) show dendritic cell (DC) infiltration among lymphocytes in the paracortical area of ​​the lymph node draining the TSA-LEC inoculation site. Exodus 2/SLC expression in the high endothelial venules (HEVs) and surrounding cells is much more pronounced than in the lymph node draining the TSA-pc inoculum. MCP-1 is mainly localized in the mantle zone and the luminous zone of the germinal centers of the lymph nodes draining the TSA-LEC cells. (2b) TSA-LEC cell aggregates containing macrophages, lymphocytes, granulocytes, and DCs. (2d,2e) Inflammatory infiltrate associated with the marked expression of adhesion molecules on the endothelium of numerous vessels.

Table III. Histochemical and immunohistochemical analysis of the TSA-pc and TSA-LEC cell injection area 7 days after challenge.

1. Cell and microvessel counts were performed at x 400 in a 0,180 mm² field on 10 randomly chosen field/sample. Results are means +/- DS of positive cells or vessels/field evaluated on cryostat sections by immunohistochemistry and/or Giemsa-stained sections (basophils/mast cells).
2. The expression of angiogenic factor and chemokines was classed as absent (-), scarcely (+/-), moderately (+), frequently (++), or strongly (+++) present on cryostat sections tested with corresponding Abs.

Values significantly different (p < 0,005) from corresponding value in TSA-pc.

Ultrastructural examination

The immunohistochemical demonstration of a rich cellular infiltrate in the growth area of TSA-LEC was supported by ultrastructural images showing the presence of DCs and macrophages in proximity to or in direct contact with the numerous lymphocytes present among the cellular aggregates of TSA-LEC. The basophilic and mast cell infiltrate was present in foci. Signs of degranulation were also characteristic. The newly formed capillaries in TSA-LEC consisted of endothelial cells covered with rolling or adherent lymphocytes. Their lumen was sometimes interrupted or obstructed by thrombi composed of platelets and leukocyte aggregates. Fragmentation of the lamina was frequent and associated with neoangiogenesis, recognizable by the presence of numerous buds.

Chemokine production by recombinant LEC-stimulated macrophages in vitro

Numerous reactive cells, including macrophages, showed expression of various chemokines. Confirmation of these immunohistochemical findings was obtained by assessing the presence of RANTES, MCP-1, MIP-1a, and MIP-2 in the supernatant of peritoneal macrophages after 72-hour culture with or without recombinant (rLEC) supplementation at 1, 10, and 100 ng/ml. The secretion of RANTES, MCP-1, and MCP-2 increased 6.35-fold and 1.2- to 1.7-fold, respectively, in the presence of rLEC, while the secretion of MIP-1a was unchanged.

Anti-TSA-pc antibody production

The presence of specific anti-TSA-pc antibodies was assessed by flow cytometry in the sera of normal mice and mice inoculated with TSA-LEC. Anti-TSA-pc antibodies were present as early as 7 days after inoculation of TSA-LEC cells. At 30 days post-inoculation, the antibody level was even higher (98% positive cells). These antibodies belonged to the IgG1 and IgG2 immunoglobulin subclasses.

DISCUSSION

Using an experimental model in which engineered tumor cells act as micropumps that ensure the massive presence of a given factor in their growth microenvironment, it is possible to study the activity of cytokines and chemokines in vivo (16). The activity of the LEC gene has not yet been well defined (14). The use of TSA cells engineered with the LEC gene is able to release high amounts of this chemokine and has led to the conclusion that LEC has a strong pro-inflammatory activity that does not appear to be directly related to its chemotactic activity as demonstrated in vitro on DCs and the myeloid cell line THP-1 (17). The chemotactic activity demonstrated in vitro occurred only at high concentrations of 1 mg per ml and cannot be considered representative of in vivo activity.

Our studies suggest that the recruitment of such diverse leukocyte subpopulations into the cellular microenvironment of TSA-LECs results from the cascade of other factors, all induced by LECs. However, the recruitment of monocyte-macrophages appears to play a major role. Human monocyte-macrophages express CCR1 and CCR8, identified as two receptors for LECs (17). Similar receptors also appear to be expressed by murine monocyte-macrophages, as they respond to LECs by producing RANTES, MCP-1, and MCP-2, as demonstrated in vitro and in vivo (18).

MCP-1 is the major chemotactic activating factor for monocytes-macrophages, mast cells, and basophils (19). It promotes the recruitment of T lymphocytes and DCs (20). Indeed, the latter cell populations are more representative in the response after TSA-LEC inoculation. Ultrastructural data have also demonstrated that basophils actively participate in the inflammatory process induced by TSA-LEC by releasing their granules (21). MIP-2, also known as GRO/KC, is the murine functional analogue of IL-8 and is chemotactic for neutrophils and T lymphocytes (22). Chemokines produced by macrophages can recruit T lymphocytes and DCs to the site of TSA-LEC injection. Macrophages and granulocytes are also attracted to the site of TSA-LEC injection into nu/nu nude mice.

The chemotaxis exerted by LEC secretion is mediated by LEC-induced production of MCP-1 and MIP-2 on macrophages themselves, while it does not appear to depend on the expression of other pro-inflammatory cytokines or endothelial adhesion molecules (23). Chemokines produced by macrophages can recruit T lymphocytes and DCs to the TSA-LEC injection site. Macrophages and granulocytes are also attracted to the site by TSA-LEC cells injected into nu/nu nude mice. The chemotaxis exerted by LEC secretion is mediated by LEC-induced production of MCP-1 and MIP-2 on macrophages themselves, while it does not appear to depend on the expression of other pro-inflammatory cytokines or endothelial adhesion molecules. Both of these, detected in the TSA-LEC growth site in normal mice, are induced by T lymphocytes, which play a primary role in the cascade of LEC-induced inflammatory events (24).

LEC-activated inflammation is associated with angiogenesis, as demonstrated by vascular sprouting and increased micro vessel numbers in the TSA-LEC-induced microenvironment (25). Immunohistochemical data indicate that the angiogenic mediators TNF, PDGF, and MIP-2 are expressed to a greater extent than IP10 and MIG. Comparison with the TSA-pc model could lead to an underestimation of the angiogenic potential of TSA-LEC cells, since TSA-pc cells produce VEGF and FGFβ (26). The LEC-induced reaction is accompanied by hyperplasia of the lymph node draining the inoculation area, which shows a marked expression of Exodus 2/SLC in the paracortical area and in the numerous high endothelial venules, whose volume is densely populated by lymphoid cells.

Physiologically, Exodus 2/SLC is involved in the migration of DCs from the skin to the draining lymph nodes. It may therefore be active on DCs at an intermediate stage of maturation, immediately after antigen uptake on their way to secondary lymphoid organs. Exodus-2/SLC is also chemotactic for T and B cells, and for natural killer (NK) cells (27). Numerous secondary follicles characterized by extensive cortical germinal centers represent the morphological background of the significant humoral response (28). MCP-1 expression in the mantle zone and germinal centers indicates an ongoing B cell response directed at TH2 lymphocytes (29). However, the presence of IgG2a and IgG2b antibodies in serum suggests that the reaction triggered by LEC is not only TH2-type, but also TH1-type (30). However, the fact that the antibody response was so immediate and intense after TSA-LEC inoculation highlights that the interrelationships between antigen-presenting cells (in particular DCs) and lymphocytes are particularly facilitated. This is particularly true in the inoculation area, where macrophages are directly or indirectly attracted and where DCs and T lymphocytes are induced into a close interaction regulated by LECs, through a stabilization of the bonds between the CCR1 and CCR8 receptors (31).

The cytokine cascade found in untreated animals, but absent in nu-nu mice, indicates that these interactions result in intense CCR1-mediated T cell activation (32). The increased adhesion of antigen-presenting cells and T cells does not prevent their drainage towards the local lymph node, where the intense traffic found at the level of the marginal sinus and high endothelial venules, and the hyperplasia of the cortical and paracortical areas, strengthen the influx of immune cells capable of triggering an efficient immune response through the production of specific antibodies.

Tumor cells transfected with a β-chemokine gene (such as CCL16) undergo changes in the microenvironment that can affect their survival. β-chemokines can modulate T-cell-mediated immunity against pathogenic microorganisms, particularly viruses. When a tumor cell expresses CCL16, it attracts immune cells into the tumor, which recognize and destroy the tumor cell. This can create a vulnerable environment for pathogens to more easily express their infectious power. Therefore, tumor cells transfected with β-chemokine genes become more visible and susceptible to attack by the immune system, which can indirectly influence infections and antiviral responses.

CONCLUSIONS

In this work, it is important to emphasize that our results demonstrate that LEC is a chemokine with high pro-inflammatory activity and LEC production has been reported to be upregulated by IL-10, which is an immune inhibitor. These findings suggest that alterations in LEC production may be a key feature of some human inflammatory disorders characterized by the presence of elevated IL-10 levels, such as inflammatory bowel disease, microbial infections, and rheumatoid arthritis.

Conflict of interest

The authors declare that they have no conflict of interest.

Funding sources

Not applicable.

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