The Neutrophil Granule Protein Cathepsin G Activates Murine T Lymphocytes and Upregulates Antigen-Specific Ig Production in Mice

Biochemical and Biophysical Research Communications 282, 971–976 (2001) doi:10.1006/bbrc.2001.4676, available online at http://www.idealibrary.com on

The Neutrophil Granule Protein Cathepsin G Activates Murine T Lymphocytes and Upregulates Antigen-Specific Ig Production in Mice Kenji Tani,* William J. Murphy,† Oleg Chertov,† Joost J. Oppenheim,* and Ji Ming Wang* ,1 *Laboratory of Molecular Immunoregulation and †Intramural Research Support Program, SAIC Frederick, National Cancer Institute–Frederick Cancer Research and Development Center, Frederick, Maryland 21702

Received February 23, 2001

Cathepsin G is a neutrophil granule derived antimicrobial chymotrypsin-like enzyme. Our previous study showed that cathepsin G induces chemotactic migration of human phagocytic leukocytes and increases random migration of T lymphocytes. In this study, we investigated the capacity of cathepsin G to activate T lymphocytes and to modulate antigen-specific humoral responses in mice. We found that cathepsin G is mitogenic for and induces production of IFN-␥ by murine T cells in vitro. Injection of cathepsin G in BALB/c mice immunized with keyhole limpet hemocyanin (KLH) adsorbed to aluminum hydroxide resulted in a significantly increased production of KLH-specific IgG1 and IgG2a antibodies. There was a dosedependent increase in KLH-specific proliferation of lymphocytes from draining lymph nodes from mice treated with KLH and cathepsin G when compared with those treated with KLH alone. Subsequent analysis of IFN-␥ and IL-4 release following in vitro restimulation of draining lymph node lymphocytes obtained from KLH-immunized mice suggested that cathepsin G augments KLH-specific Ig antibody production via activation of T cells, presumably involving both Th1 and Th2 pathways. Thus, neutrophil granule cathepsin G, in addition to its capacity to kill microbes By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. The experiments were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources, National Research Council, Department of Health, Education, and Welfare Publication 78-23 (National Institutes of Health, Bethesda, MD). Abbreviations used: KLH, keyhole limpet hemocyanin; PMSF, phenylmethanesulphonyl fluoride. 1 To whom correspondence and reprint requests should be addressed at Laboratory of Molecular Immunoregulation, Building 560, Room 21-89A, National Cancer Institute–Frederick Cancer Research and Development Center, Frederick, MD 21702-1201. Fax: 1-301-846-7042. E-mail: [email protected].

and to enhance leukocyte motility, activates T lymphocytes and modulates humoral immunity. Key Words: cathepsin G; T cell; antibody; adjuvant.

The secretion of antibiotic proteins including proteases by neutrophils is thought to play a major role in resistance to the early stage infection by killing microbes (1, 2). Following phagocytosis of microbial agents or other particulate substances, these proteins are released from the granules into phagocytic vacuoles and also into the extracellular milieu (3). Depleting rat neutrophils reduced subsequent development of chronic delayed-type hypersensitivity reaction (4, 5). Similarly, it has been reported that suppression of neutrophil migration to inflammatory sites by infusion of anti-IL-8 antibody decreased not only acute inflammatory responses (6 – 8) but also delayed-type hypersensitivity responses (9). IL-8, a chemokine with potent chemotactic and activating effects on human neutrophils, induced neutrophil accumulation at the injection site followed by T cell infiltration in SCID mice administered with human T cells, suggesting that neutrophils could release chemoattractants that mediate T cell accumulation at sites of inflammation (10). We have purified and identified several neutrophil-derived T cell chemoattractants such as defensins and azurocidin/CAP37 (11). These results suggest that neutrophil-derived proteins may play a role in the communication between cell types involved in innate or natural resistance and those responsible for adaptive immunity such as T and B cells. Cathepsin G is an antimicrobial chymotrypsin-like enzyme that is found in the azurophil granules of human neutrophils, monocytes and spleen cells (12, 13) and comprises 18% of the azurophil granule protein (1). The gene for cathepsin G-related serine protease is also expressed in murine bone marrow myeloid cells (14). Previous reports have shown that human cathepsin G

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selectively activates murine B cells (15) and stimulates increased DNA synthesis in both human T and B lymphocytes (16). Yamazaki and Aoki showed that cathepsin G exhibits specific binding to human lymphocytes (17). In addition, we have observed that human cathepsin G is a potent chemoattractant for human phagocytic leukocytes and increases random migration of T lymphocytes in vitro (18). Subcutaneous injection of cathepsin G in mice induced significant infiltration of monocytes, neutrophils and T cells (18). These results suggest that cathepsin G may play a role in the regulation of innate host defense and T cell-dependent immunological reactions. In this study we examined the capacity of cathepsin G to activate T lymphocytes and to act as an immune adjuvant in mice. Here we report that cathepsin G enhanced antigen-specific antibody production by mice immunized with KLH, in association with T cell activation and increased cytokine production. MATERIALS AND METHODS Reagents and animals. Human cathepsin G (lot B15225) and KLH were purchased from Calbiochem (La Jolla, CA). Chromogenic Limulus Amebocyte Lysate assays (Whittaker Bioproducts) indicated less than 0.125 endotoxin unit per 50 ␮g of cathepsin G protein. LPS (Escherichia coli; 055:B5) was purchased from Sigma Chemical Co. (St. Louis, MO). Phenylmethanesulphonyl fluoride (PMSF) was purchased from Boehringer Mannheim (Indianapolis, IN). Female BALB/c mice were obtained from the Animal Production Area (National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD), and were used at 8 to 12 weeks of age. In vitro proliferation and IFN-␥ production by normal mouse spleen cells. Single cell suspension prepared from spleens of normal BALB/c mice were processed with T cell enrichment columns (R & D Systems, Minneapolis, MN) to purify CD3 ⫹ T cells. This procedure typically yields greater than 90% pure CD3 ⫹ T cells with less than 2% B lymphocytes as verified by immunofluorescence flow cytometry. Spleen cells or purified T cells (5 ⫻ 10 5 per well in triplicates) from normal BALB/c mice were incubated with various concentrations of cathepsin G in serum-free RPMI 1640 medium in 96-well plates at 37°C in 5% CO 2. After 2 h, FBS was added to a concentration of 10%. After further incubation for 3 days, cell proliferation was measured by colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT; Sigma) (19). Actual cell numbers were counted in parallel experiments. For cytokine production, the cells were incubated with cathepsin G in serum-free RPMI1640 medium for 24 h, and the culture supernatants were collected and measured for IFN-␥ and IL-4 with specific ELISA kits (sources of kits: IFN-␥, Genzyme, Cambridge, MA; IL-4, Endogen, Boston, MA). Sensitivity limit for IFN-␥ and IL-4 detection was 27.0 and 5.8 pg/ml, respectively. Inactivation of enzymatic activity of cathepsin G. The enzymatic activity of cathepsin G was attenuated by mixing 20 ␮l 0.1 M PMSF in isopropanol and 20 ␮g cathepsin G in 400 ␮l PBS. After 30 min incubation at 22°C, the protein preparation was desalted on a reverse phase column RP-4 (15 ⫻ 3.2 mm; NewGuard, Brownlee). Inactivated cathepsin G was recovered using a linear gradient from 0 to 90% of buffer B (0.05% trifluoroacetic acid in acetonitrile) at a flow rate of 1 ml/min and lyophilized. The treatment of cathepsin G by PMSF resulted in a reduction of the original enzymatic activity by 97%, as determined by a microtiter assay using the thiobenzylester substrate Succ-Phe-Leu-Phe-S-Bzl (Sigma) (20).

Immunization. Mice were immunized s.c. in the base of their tails once on Day 1 with 50 ␮g KLH adsorbed to aluminum hydroxide dissolved in 0.1 ml PBS. Mice were additionally injected s.c. at the same site with various concentrations of cathepsin G dissolved in 0.1 ml PBS on Days 1, 2, 3, 4, and 5. Control mice were injected s.c. with PBS alone or cathepsin G boiled for 20 min. Sera and inguinal lymph nodes were collected 10 days after KLH injection. Measurement of KLH-specific antibodies. KLH-specific antibodies in sera were measured by ELISA (21). Briefly, 96-well flatbottomed plates were coated with 10 ␮g/ml of KLH in carbonate buffer (0.1 M NaHCO 3, pH 9.6) for 2 h, and washed 3 times with PBS containing 0.05% Triton X-100 (washing buffer). The plates were then incubated with a blocking buffer (5% dry milk in PBS) for 1 h to saturate nonspecific binding sites. Serial fivefold dilutions of the sera in PBS containing 2% BSA (Sigma) were added for 1 h followed by a 30-min incubation with isotype-specific biotinylated anti-mouse antisera (The Binding Site, San Diego, CA). After washing 5 times, streptavidin-peroxidase (The Binding Site) in PBS containing 2% BSA was added for 30 min. The plates were then washed 7 times, the substrate ABTS (Kirkegaard & Perry Lab, Inc, Gaithersburg, MD) was added, and the absorbance was determined at 405 nm with a reference wavelength of 490 nm. Proliferation and cytokine secretion of cells from immunized mice. For cell proliferation, cells from draining lymph nodes in immunized mice (5 ⫻ 10 5 lymph node cells/well in triplicates) were incubated with KLH in a final volume of 0.1 ml RPMI1640 supplemented with 10% FBS in 96-well plates at 37°C. After 5 days, the cell proliferation was measured by MTT assay. For cytokine production, cells from draining lymph nodes in immunized mice were cultured at 37°C in 24-well plates at a density of 2 ⫻ 10 6/ml in RPMI1640 containing 10% FBS. KLH at 50 ␮g/ml was added to the cells. After 48 h, the supernatants were harvested, and the concentration of cytokines was measured with ELISA. Statistical analysis. All experiments were performed at least three to four times and each experimental group consisted of three mice. The results from representative experiments were shown. The statistical significance of the experimental parameters between test and control groups was determined by Student’s t test. Statistically significant difference was defined at the P ⬍ 0.05.

RESULTS Cathepsin G Stimulates Murine T Cell Proliferation in Vitro The mitogenic activity of cathepsin G was examined by using normal murine splenocytes. Cathepsin G stimulated the proliferation of spleen cells in a dosedependent manner with maximal effect at 1–5 ␮g/ml (Fig. 1A). The mitogenic activity of cathepsin G was dependent on its enzymatic activity, since pretreatment of cathepsin G with PMSF ablated its effect on splenocyte proliferation (Fig. 1B). In the same concentration range, cathepsin G also stimulated the proliferation of purified T cells (Fig. 1C). The concentrations of cathepsin G used in these experiments did not affect cell viability as determined by trypan blue exclusion assay and quantitation of viable cells showed that 1–5 ␮g/ml of cathepsin G resulted in 2.5-fold increase in the numbers of spleen cells and T cells. These results suggest that cathepsin G is a mitogenic agent for T lymphocytes.

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FIG. 1. Induction of proliferation of T lymphocytes from normal mouse spleen. Spleen cells (A) or purified T cells (C) from normal BALB/c mice were incubated with different concentrations of cathepsin G and cell proliferation was measured after 3 days by colorimetric MTT assays. Cathepsin G treated with PMSF was also tested for its mitogenic effect on splenocytes (B). The mean values (⫾SD) of triplicate cultures are shown as the percent increase over the value of control cultures with medium. * indicates statistically significant differences (P ⬍ 0.01) relative to cells treated with medium alone.

Cathepsin G Stimulates Production of IFN-␥ by Murine T Cells

Cathepsin G Augments KLH-Induced Proliferation of Lymph Node Cells from Immunized Mice

We next examined the levels of IFN-␥ in culture supernatants of cathepsin G stimulated spleen cells and T cells from normal mice. As shown in Table 1, cathepsin G at concentrations ranging form 10-500 ng/ml increased IFN-␥ production by normal spleen cells and purified T cells. No IL-4 was detected in the supernatant of murine spleen cells or T cells cultured in the presence or absence of cathepsin G.

In vitro KLH-induced proliferation of lymph node cells from mice previously immunized with KLH was assayed by colorimetric MTT assays (Fig. 3). Significant enhancement of KLH-induced proliferation of lymph node cells from mice previously injected 5 times with 50 or 500 ng per day of cathepsin G was detected relative to those received KLH alone. Immunofluorescence flow cytometry showed that there was no significant difference in subpopulations of lymph node cells between mice treated with KLH

Cathepsin G Upregulates Production of KLH-Specific Antibodies Immunization with protein adsorbed to aluminum hydroxide typically induces T cell-dependent immune responses and favors a Th2-like immune response. Such an approach has been used to examine antigenspecific cellular and humoral responses and the adjuvant activity of IL-12 (22–25). We immunized mice with KLH adsorbed to aluminum hydroxide and various concentrations of cathepsin G. After 10 days, KLHspecific Ig subclasses in mouse sera were measured by ELISA. Serum IgG1, IgG2a, and IgG2b antibodies against KLH were detected in mice treated with KLH alone (Fig. 2). The administration of cathepsin G significantly upregulated the synthesis of KLH-specific IgG1 and IgG2a antibodies, whereas KLH-specific IgG2b levels were unaffected. In contrast, boiled or PMSF-treated cathepsin G did not induce any increases in the production of anbodies, suggesting that the effect of cathepsin G is associated with its enzymatic activity and is not due to endotoxin contamination.

TABLE 1

The Effect of Cathepsin G on IFN-␥ Production by Normal Murine Spleen Cells and Purified T Lymphocytes a IFN-␥ (pg/ml) Cathepsin G (ng/ml) 0 1 10 100 500

Spleen cells b

ND ND 133 ⫾ 35 c 237 ⫾ 17 c 113 ⫾ 15 c

T lymphocytes ND ND ND 90 ⫾ 14 c 114 ⫾ 24 c

a Spleen cells were obtained from normal BALB/c mice. T lymphocytes were purified from spleen cells by negative selection using T cell enrichment columns. Spleen cells (2 ⫻ 10 6/ml) and T cells (1 ⫻ 10 6/ml) were incubated with cathepsin G for 24 h and IFN-␥ was measured in the supernatants by ELISA. Results are mean values (⫾SD) from triplicate cultures. b ND, not detected at the sensitivity limit of 27 ng/ml. c P ⬍ 0.001 compared to cells incubated in the absence of cathepsin G.

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FIG. 2. KLH-specific antibodies in sera of mice treated with cathepsin G. Mice were immunized s.c. with 50 ␮g KLH on Day 1, and treated s.c. with various concentrations of cathepsin G daily from Day 1 to 5. On Day 10, KLH-specific antibodies in sera were measured by ELISA. Boiled cathepsin G (500 ng) or PBS alone were used as controls. Results are mean values (⫾SD) from three mice. * indicates statistically significant difference (P ⬍ 0.05) relative to mice treated with KLH alone.

plus cathepsin G and the mice treated with KLH alone (CD4⫹ cells, 42– 47%; CD8⫹ cells, 14 –17%; B220⫹/IgM⫹ cells; 24 –30%).

Cathepsin G Upregulates Cytokine Production by Lymph Node Cells from KLH-Immunized Mice Cytokines released by activated T cells are required for the development of antibody responses. In mice, IFN-␥, a Th1 cytokine, enhances the production of IgG2a, whereas IL-4, a Th2 cytokine, upregulates IgG1 (26 –28). We examined the effect of cathepsin G on cytokine production by ex vivo KLH-activated lymph node cells from immunized mice. As shown in Table 2, the administration of cathepsin G significantly enhanced the ability of lymph node cells from immunized

TABLE 2

The Effect of Cathepsin G on KLH-Induced Cytokine Release by Lymph Node Cells from Immunized Mice a In vivo treatment KLH ⫹ PBS KLH ⫹ cathepsin G (5 ng) KLH ⫹ cathepsin G (50 ng) KLH ⫹ cathepsin G (500 ng) KLH ⫹ boiled cathepsin G (500 ng) FIG. 3. The effect of cathepsin G on proliferation of antigen re-stimulated lymph node cells from immunized mice. Single cells from draining lymph nodes of mice previously immunized with 50 ␮g KLH along with cathepsin G were incubated in vitro with KLH for 5 days. Cell proliferation was measured by colorimetric MTT assays. Open circles, control; closed circles, 5 ng cathepsin G; closed triangles, 50 ng cathepsin G; closed squares, 500 ng cathepsin G. The results are expressed as percent increase over the value of medium control cultures and are mean values (⫾SD) from three mice. * indicates statistically significant difference (P ⬍ 0.05) relative to cells treated with medium alone.

IFN-␥ (pg/ml)

IL-4 (pg/ml)

47.0 ⫾ 21.6 159.3 ⫾ 38.7 b 224.3 ⫾ 22.4 c 289.3 ⫾ 42.7 c

231.3 ⫾ 33.1 500.7 ⫾ 61.5 c 860.0 ⫾ 146.1 c 651.0 ⫾ 126.6 c

32.7 ⫾ 9.8

285.7 ⫾ 26.1

Mice were immunized s.c. with 50 ␮g KLH in combination with various concentrations of cathepsin G. After 10 days, single cells prepared from draining lymph nodes were incubated with 50 ␮g/ml KLH in vitro 48 h, then the culture supernatants were harvested and measured for INF-␥ and IL-4 by ELISA. Boiled cathepsin G was used as a control. Results are mean values (⫾SD) from three mice. b Significantly increased cytokine production compared to cells from mice treated with KLH ⫹ PBS (P ⬍ 0.05). c P ⬍ 0.01 compared with cells from mice treated with KLH ⫹ PBS.

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mice to secrete IFN-␥ upon in vitro restimulation with KLH. In addition, in vitro treatment with KLH significantly enhanced secretion of IL-4 by lymph node lymphocytes in mice treated with KLH and cathepsin G when compared with those treated with KLH alone. No increase in cytokine production was observed in lymph node cells from mice treated with boiled cathepsin G. These results indicate that cathepsin G enhanced the ability of lymph node cells from immunized mice to secrete IFN-␥ and IL-4 following antigen restimulation. DISCUSSION Human neutrophil granules contain proteins that participate in innate host defense and adapted immune responses. We previously observed that the human neutrophil granule ␣-defensins and azurocidin/ CAP37 are T cell chemoattractants in vitro and in vivo in mice (11). Human ␣-defensins also promoted specific antibody response in mice and activates murine T lymphocytes (29). We additionally observed that another neutrophil granule protein, cathepsin G, is a potent chemoattractant for human monocytes and neutrophils and increases the random migration of human T lymphocytes (18). Since cathepsin G activates T cells, we hypothesized that this neutrophil granule protein may also serve as a neutrophil-derived signal that modulates T cell-dependent immune responses. This hypothesis was tested for the capacity of cathepsin G to promote in vivo antibody responses to KLH in mice, since mice also produce cathepsin G and its gene was detected in myeloid cells of murine bone marrow (14). Our results clearly showed that human cathepsin G is mitogenic for murine T cells in agreement with previous reports that human cathepsin G can increase DNA synthesis in both human T and B cells and bind to human T cells, B cells and NK cells (16, 17). We also detected increased IFN-␥ production by normal murine T cells after stimulation with cathepsin G. Our study demonstrated that cathepsin G upregulates antigen-specific Ig production in mice, suggesting that this neutrophil granule protein may act as an immune adjuvant in addition to possessing antibacterial activity. Cathepsin G increased serum KLHspecific IgG1 and IgG2a subclasses in vivo. In vitro re-stimulation of lymph node cells from immunized mice with KLH showed that cathepsin G increased KLH-specific lymphoproliferative responses and induced a marked increase in IFN-␥ production. IFN-␥, a Th1 cytokine, is known to be associated with cellmediated immunity and the preferential induction of IgG2a (24, 27). IFN-␥ is presumably responsible for cathepsin G-enhanced KLH-specific IgG2a response in the immunized mice. Cathepsin G also increased KLHspecific production of the Th2 cytokine, IL-4. This may account for KLH-specific IgG1 production because IL-4

is known to be critical for the expansion of Th2 responses characterized by increased synthesis of IgG1 (30, 31). Our results suggest that cathepsin G may stimulate antigen-specific T as well as B cell responses, at least in part, through concomitant production of both Th1 and Th2 lymphokines by activated T cells. Inactivation of cathepsin G enzymatic activity by PMSF or by boiling completely abolished its proliferative activity for murine spleen cells and blocked its in vivo adjuvant effects. These results indicate that enzymatic activity of cathepsin G is required for the stimulation of murine lymphocytes and also antigenspecific Ig production. This is in agreement with previous reports showing that proteolytically active cathepsin G is necessary for its stimulation of DNA synthesis in murine B cells (15) and human lymphocytes (16, 17). Cathepsin G is a neutrophil-derived antimicrobial mediator capable of inducing connective tissue damage (32, 33). However, the possibility that the antigen-specific Ig production by cathepsin G results from a toxic effect of this enzyme is unlikely since in this study 5 ␮g/ml of cathepsin G did not affect the viability of cultured spleen cells, but instead increased the number of cultured cells. Moreover, Okrent et al. reported that cathepsin G is not cytotoxic to endothelial cells at concentration as high as 50 ␮g/ml (34). Since cathepsin G binds to human cells and induces pertussis toxin-sensitive leukocyte migration (18), it is likely, specific cellular receptor(s) exists and studies are underway to elucidate the identity of the putative cathepsin G receptor. In summary, our study shows that cathepsin G upregulates antigen-specific Ig production in mice as a result of T cell activation. These results, together with our previous observations with ␣-defensins (29) indicate that neutrophils not only participate in the innate host defense against microbial invasion, but also are actively involved in mobilizing adaptive immune responses by secreting multiple granule proteins. ACKNOWLEDGMENTS The authors thank Ms. Kathleen Bengali, Dr. Akio Hirano, and Mr. Steve Stull for technical assistance and Ms. Cheryl Fogle for secretarial support. We are grateful for critical reading of this paper by Drs. Scott Durum, Michael Grimm, and Ken Wasserman.

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