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The Journal of Immunology, 2002, 168: 4127-4134.
Copyright © 2002 by The American Association of Immunologists

IL-16 Regulation of Human Mast Cells/Basophils and Their Susceptibility to HIV-11

Jian Cheng Qi*, Richard L. Stevens, Robert Wadley{dagger}, Andrew Collins{dagger}, Margaret Cooley{dagger}, Hassan M. Naif{ddagger}, Najla Nasr{ddagger}, Anthony Cunningham{ddagger}, Gregory Katsoulotos*, Yewlan Wanigasek§, Basil Roufogalis§ and Steven A. Krilis2,*

* Department of Medicine, University of New South Wales, and Departments of Immunology, Allergy, and Infectious Disease, St. George Hospital, Kogarah, New South Wales, Australia; {dagger} Department of Medicine, Microbiology, and Immunology, University of New South Wales, Kogarah, New South Wales, Australia; {ddagger} Center of Virus Research, Westmead Millennium Institute, and University of Sydney, Westmeade, New South Wales, Australia; and § Faculty of Pharmacy, University of Sydney, New South Wales, Australia; and Department of Medicine, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
AIDS patients often contain HIV-1-infected mast cells (MCs)/basophils in their peripheral blood, and in vivo-differentiated MCs/basophils have been isolated from the blood of asthma patients that are HIV-1 susceptible ex vivo due to their surface expression of CD4 and varied chemokine receptors. Because IL-16 is a ligand for CD4 and/or an undefined CD4-associated protein, the ability of this multifunctional cytokine to regulate the development of human MCs/basophils from nongranulated progenitors residing in cord or peripheral blood was evaluated. After 3 wk of culture in the presence of c-kit ligand, IL-16 induced the progenitors residing in the blood of normal individuals to increase their expression of chymase and tryptase about 20-fold. As assessed immunohistochemically, >80% of these tryptase+ and/or chymase+ cells expressed CD4. The resulting cells responded to IL-16 in an in vitro chemotaxis assay, and this biologic response could be blocked by anti-IL-16 and anti-CD4 Abs as well as by a competitive peptide inhibitor corresponding to a sequence in the C-terminal domain of IL-16. The additional finding that IL-16 induces calcium mobilization in the HMC-1 cell line indicates that IL-16 acts directly on MCs and their committed progenitors. IL-16-treated MCs/basophils also are less susceptible to infection by an M/R5-tropic strain of HIV-1. Thus, IL-16 regulates MCs/basophils at a number of levels, including their vulnerability to retroviral infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mast cells (MCs)3 and basophils are related cells that play central roles in allergic inflammation due to their release of varied preformed and newly generated mediators when these cells are activated via their high affinity IgE receptors (Fc{epsilon}RI). MCs are rarely found in mice that have defects in c-kit ligand (KL; also known as stem cell factor) or its receptor CD117 (1, 2) Nevertheless, large numbers of MCs are not generated when mouse or human progenitors are cultured in medium supplemented with KL alone (3). Thus, it is now clear that accessory factors (e.g., IL-3, IL-4, IL-9, IL-10, IL-15, nerve growth factor, TGF-{beta}, and glucocorticoids) are needed to complete the KL-mediated differentiation and maturation processes. In this regard, we previously showed that the HBM-M cell line derived from a child with a bullous mastocytosis (4) produces undefined factors that enhance the KL-mediated differentiation and maturation of hemopoietic progenitors into relatively mature human MCs (5, 6). Others have generated similar cells using KL with varied combinations of IL-4, IL-6, IL-10, and PGE2 (7, 8, 9).

CD4+ T cells often accumulate in tissues at sites of MC activation (10), and it is believed that the recruited T cells exacerbate asthma and other inflammatory disorders (11). IL-16 (also known as lymphocyte chemoattractant factor) (12) is a pleiotropic cytokine that induces its effect via CD4 (13, 14) and/or an undefined receptor (15) that may be physically associated with CD4 on the surface of T cells and other IL-16-responsive cells. The precursor and active forms of IL-16 consist of 631 and 121 aa, respectively, and the bioactive C-terminal portion of this cytokine is generated by a caspase 3-dependent proteolytic event that occurs during the exocytosis process (16). IL-16 induces chemotaxis of CD4+ T cells, monocytes, and eosinophils (12, 17). This cytokine also induces CD4+/CD14+ monocytes and maturing macrophages to secrete IL-1{beta}, IL- 6, IL-15, and TNF-{alpha} (18). IL-16 also inhibits HIV replication (19, 20, 21, 22). Although many cell types produce IL-16, certain populations of human MCs synthesize and release biologically active IL-16 (23). Thus, MCs can attract circulating CD4+ lymphocytes into tissues via an IL-16-dependent mechanism.

Whether IL-16 regulates the development of human MCs/basophils by an autocrine mechanism has not been evaluated, presumably because it was initially believed that these immune cells do not express CD4. We and others recently discovered that some populations of human MCs and/or basophils and their progenitors express CD4 and are susceptible to M-tropic strains of HIV-1 ex vivo (9, 24, 25). In addition, HIV-1-infected MCs/basophils often are present in the blood of AIDS patients (24). Thus, in the present study, we examined what effects, if any, IL-16 would have on the human MC line HMC-1 (26) as well as on in vitro-differentiated nontransformed human MCs/basophils. We now report that IL-16 can dramatically enhance the KL-mediated expression of tryptase and chymase in human MCs/basophils developed from progenitors residing in cord and peripheral blood. We also show that IL-16 is a chemotactic factor for these cells as well as the HMC-1 cell line. Finally, we show that IL-16-treated cells become less susceptible to HIV-1 infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Recombinant human rIL-16 and KL were obtained from (Serotec, Oxford, U.K.) and R&D Systems (Minneapolis, MN), respectively. Mouse anti-human tryptase and anti-human chymase Abs, alkaline phosphatase (AP)-conjugated and biotinylated mouse anti-human chymase Abs were obtained from Chemicon International (Temecula, CA). Rabbit anti-mouse IgG, AP anti-AP complex, and peroxidase-conjugated rabbit anti-mouse IgG were obtained from Dakopatts (Glostrup, Denmark). Mouse anti-human CD4 Ab was obtained from BD Biosciences (San Jose, CA). Rabbit anti-human IL-16 Ab was obtained from PeproTech (Rocky Hill, NJ). The peptides RRKSLQSK and ETTAAGDS corresponding to residues 616–623 and 624–631, respectively, in human IL-16 (GenBank locus ID 3603) were synthesized by Mimotopes (Clayton, Australia).

Cell culture

Peripheral blood was obtained from asthma patients; umbilical cord blood was obtained from normal donors after informed consent on the day of delivery of full-term newborn infants. Blood was collected in heparinized tubes to prevent coagulation and was centrifuged at 800 x g for 10 min. The leukocyte-enriched buffy coats were collected and resuspended in erythrocyte lysis buffer (Sigma-Aldrich, St. Louis, MO) for 10 min at room temperature to eliminate residual erythrocytes. The nonlysed cells remaining after this treatment were washed with Dulbecco’s PBS and resuspended in MEM-{alpha} (Life Technologies, Grand Island, NY) supplemented with 10% FCS, 2 mM L-glutamine, and 100 µg/ml penicillin-streptomycin. Each cell suspension was seeded at a density of 106 cell/ml and cultured in the presence of 50 ng/ml recombinant KL and rIL-16 (1–40 ng/ml). For controls, replicate cells were cultured at the same density in medium containing KL with or without HBM-M cell-conditioned medium. The latter conditioned medium was used in the control studies for comparison because of its synergistic effects with KL on MC development (5). Triplicate cultures of 2 x 106 cells for each cytokine-treated sample were maintained for up to 4 wk at 37°C in a humidified atmosphere of 5% CO2. The entire volume of cytokine-supplemented medium was replaced on a weekly basis. Cell number and viability were determined weekly by counting the cells in the culture that exclude trypan blue. The cultured cells were analyzed immunohistochemically as described below after being centrifuged onto glass slides.

Immunocytochemistry and RT-PCR analysis

Cytocentrifugation preparations of cells were air-dried and placed in Carnoy’s fixative for 15 min at room temperature. After the slides were washed with TBS (pH 7.6), they were incubated with normal rabbit serum (1/10 dilution) for 10 min, followed by mouse anti-human tryptase Ab (0.5 µg/ml), mouse anti-human chymase Ab (0.5 µg/ml), or mouse anti-human CD4 Ab (0.5 µg/ml) for 1 h at room temperature. After the treated slides were washed with TBS containing 1% BSA, they were incubated with rabbit anti-mouse IgG (1/100 dilution) and AP/anti-AP complex (1/100 dilution) for 1 h at room temperature. Finally, the slides were incubated for 20 min in a solution containing 0.2 mg/ml naphthol AS-MX phosphate, 0.1 mg/ml Fast Red Texas Red, and levamisole in 0.1 M Tris-HCl (pH 8.2).

A double-immunocytochemistry procedure was used to confirm that the tryptase+ and/or chymase+ cells in the peripheral blood and cord blood cultures also express CD4. For this analysis, slides were sequentially placed in Carnoy’s fixative, 0.3% hydrogen peroxide in methanol, and normal rabbit serum (diluted 1/10 in TBS) for 15, 10, and 10 min, respectively. The treated slides were incubated with mouse anti-human CD4 (0.5 µg/ml) Ab overnight at 4°C, followed by peroxidase-conjugated rabbit anti-mouse Ig (6 µg/ml) for 1 h at room temperature. Slides were developed with a freshly prepared diaminobenzidene substrate solution (10 ml Tris-HCl buffer (pH 7.6) containing 0.03% hydrogen peroxide and 1 mg/ml 3,3'-diaminobenzidene tetrahydrochloride). For double staining with tryptase, the treated slides were then incubated with AP-conjugated mouse anti-tryptase Ab (2 µg/ml) at 37°C for 2 h. For double staining with chymase, the treated slides were incubated with biotinylated mouse anti-human chymase Ab (0.3 µg/ml) for 1 h at 37°C, followed by AP-conjugated streptavidin (5 µg/ml) for 1 h at room temperature. The slides were further stained by the addition of a freshly prepared AP substrate (0.2 mg/ml naphthol AS-MX phosphate containing 0.1 mg/ml Fast Red Texas Red and levamisole in 0.1 M Tris-HCl, pH 8.2) for 20 min. In this immunohistochemical analysis, CD4+ cells appear brown, whereas tryptase+ and chymase+ cells appear pink. CD4+/tryptase+ and CD4+/chymase+ double-stained cells are both pink and brown.

As noted below, HMC-1 cells were poorly stained when incubated with anti-CD4 Ab. Thus, a standard RT-PCR approach was used to evaluate whether this cell line contains CD4 mRNA. In this transcript assay, total cellular RNA was extracted from 2 x 106 HMC-1 cells using an RNA extraction kit (Qiagen, Valencia, CA). Samples were treated with RNase-free DNase and then reverse transcribed for derivation of the starting cDNA preparation. The RT step was performed using the Advantage RT-PCR kit from Clontech Laboratories (Palo Alto, CA). As previously described (27), equal amounts of the amplified cDNAs were used in concurrent 30-cycle PCRs using CD4 (Clontech Laboratories) and GAPDH (27) primers to amplify the relevant cDNAs. For a negative control, duplicate samples were subjected to the PCR steps, but not the RT steps. The PCR products were analyzed on a 2% agarose gel.

IL-16-mediated cellular activation

The IL-16-dependent migration of cord blood-derived human MCs/basophils and the HMC-1 MC line were evaluated using transwells (Millipore, Bedford, MA) containing fibronectin (Sigma-Aldrich)-coated polycarbonate filters with 8-µm pores, essentially as described by Nilsson and coworkers (28), for evaluating the IL-8/CXCR2-mediated migration of the HMC-1 cell line. Varying amounts of IL-16 were placed in the lower compartment; 2 x 106 cells were placed in each upper compartment. After a 3-h incubation at 37°C and 5% CO2, the filters were removed, gently washed with PBS, fixed with 95% ethanol and 5% acetic acid, and stained with anti-tryptase Ab. Alternately, the washed filters were fixed with 3% glutaraldehyde, and the cells were stained with hematoxylin. Cell migration was measured by counting the number of tryptase+ or hematoxylin+ cells that firmly attached to the lower surface of the filter in three high power fields. Each concentration of IL-16 was tested in triplicate, and the results were expressed as the mean percentage (±SD) of that obtained with non-IL-16-treated control cells (normalized to 100%). A checkerboard analysis of cellular mobility was conducted according to the method of Zigmond and Hirsch (29).

Others (30) have reported that the IL-16-derived C-terminal peptide RRKSLQSK inhibits the IL-16-induced chemotaxis of T cells, but not the IL-16-derived C-terminal peptide ETTAAGDS. Thus, the IL-16-mediated chemotaxis of MCs was evaluated using both peptides. In these competitive assays, 1–40 ng/ml rIL-16 was placed in the lower compartment of each chemotaxis chamber along with 10 µg/ml peptide. HMC-1 cells and cord blood-derived human MCs/basophils were placed in the upper compartment, and the chemotactic response was measured as described above. Anti-IL-16 Ab was used as a control to ensure that the active factor in the cytokine preparation was rIL-16 rather than a contaminant. In this latter competitive assay, HMC-1 cells and cord blood-derived human MCs/basophils were incubated in the upper chamber after varied concentrations of anti-IL-16 Ab 30 min before the start of the chemotaxis assay were incubated with rIL-16 in the lower chamber.

Intracellular Ca2+ was measured because the levels of this cation are increased inside IL-16-treated T cells. The HMC-1 cell line was cultured in IMDM supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 50 µg/ml streptomycin, and 1.2 mM {alpha}-thioglycerol. HMC-1 cells (5 x 106/ml) were incubated in Grey’s buffer (138 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1 mM NaHPO4, 5 mM NaHCO3, 5.5 mM glucose, 1 mM CaCl2, 20 mM HEPES, and 0.1% (w/v) BSA (pH 7.4)) supplemented with 1% FCS and 2.5 µM fura 2 (Molecular Probes, Eugene, OR) for 30 min at 37°C. The cells were washed and suspended at 5 x 106 cells/ml in Grey’s buffer. Two milliliters of each cell suspension were placed in a cuvette. The cell suspensions were stirred constantly during the experiment. Fluorescence measurements were performed with a luminescence fluorescence spectrophotometer (LS50B; PerkinElmer, Palo Alto, CA) with the dual-wavelength optional function set at wavelengths of 340/380 (excitation) and 505 nm (emission). Twenty seconds after the start of the experiment, 40 ng/ml IL-16 or 10 µM ionomycin were added to the cuvette. Data were collected every second for 2 min and are presented as the relative ratio of fluorescence excited at 340 and 380 nm.

The ability of IL-16 to alter the proliferation of HMC-1 cells also was evaluated. In this assay, cells were cultured at 37°C and CO2 for 6 days in the presence of IL-16. [3H]TdR (NEN, Boston, MA; sp. act., 20 Ci/mM) was added (1 µCi/well) to comparable numbers of cells, and the cultures were incubated for an additional 16 h. After the treated cells were washed with PBS, they were analyzed using a PerkinElmer liquid scintillation counter.

FACS, cell sorting, and HIV-1 infection

Two- and three-color FACS analysis and cell sorting were performed on a MoFlo MLS flow cytometer (Cytomation, Fort Collins, CO) equipped with an argon ion laser at 488 nm. Data acquisition was performed with Cyclops Summit software (Cytomation). Fluorescence emission was collected at 510–555, 555–590, and 650–690 nm for FITC, PE, and PC5, respectively. Compensation parameters were determined using single-stained cells. For two-color FACS analyses, ~106 cord blood cells were suspended in 100 µl buffer containing anti-CD4-FITC (0.6 µg/ml) and anti-CD117-PE (2.5 µg/ml) IgG. Anti-CD3-PC5 IgG (5 µg/ml) was added to the reaction buffer when three-color FACS analyses were conducted. The resulting cell suspensions were incubated for 45 min in the dark, washed twice with ice-cold PBS containing 5% FCS, and suspended in 0.5 ml PBS. Isotype-matched, irrelevant mouse anti-human IgG-PE, anti-human IgG-FITC, and anti-human IgG-PC5-conjugated mAbs were used to assess the degree of nonspecific binding to each cell preparation. The net percentage of positive cells was calculated by subtracting the positive cells obtained with an isotype-matched, negative control mAb from the percentage of positive cells in each preparation. Cell sorting was performed by first selecting live single cells by forward and side scatter and pulse width and then gating on the regions in the compensated bivariate histograms that contained those cells that expressed both CD4 and CD117. Gates were set at 99%. The resulting cells were sorted into MEM supplemented with 10% FCS.

Cord blood-derived leukocytes from two donors were cultured in the presence of 50 ng/ml KL and 40 ng/ml IL-16 for 3 wk. A FACS approach was then used to isolate the CD3-/CD4+/CD117+ MCs/basophils from the culture. These cells were inoculated in duplicate with the M-tropic, laboratory-adapted bronchoalveolar lavage (BAL) strain of HIV-1 as previously described (27, 31). The virus stock was filtered through filters with 0.22-µm pores (Millex-GS; Millipore) and then treated with DNase (20 µg/ml) for 1 h at room temperature in the presence of 10 mM MgCl2 to eliminate residual HIV-1 DNA contamination from lysed viral particles. The multiplicity of infection was 0.025 infectious virus/cell; the inocula were allowed to interact with the cells for 4 h in the presence or the absence of IL-16 (1 µg/ml). In the latter cultures, fresh IL-16-enriched medium was added every 3 days. Culture supernatants were collected on day 6 postinfection and evaluated for their levels of HIV-1 p24 Ag using an ELISA kit (Coulter, Miami, FL). The limit of detection of HIV p24 Ag in this assay is about 8 pg/ml. The levels of HIV-1 DNA in the treated cells on days 6 and 12 postinfection were determined with a semiquantitative PCR assay. Primers specific for the human {beta}-globin gene were included in the assay to normalize the amount of DNA input, as previously described (31).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-16-dependent development of human MCs/basophils from progenitors residing in cord blood and peripheral blood

To determine whether IL-16 can enhance the KL-mediated development of nontransformed human MCs/basophils in vitro, the buffy coat cells isolated from the peripheral blood of three asthma patients and the cord blood of three normal individuals were cultured for up to 21 days in the presence of KL supplemented with varied amounts of rIL-16. Relative to cells exposed to KL alone, a slight increase in the total number of cells was obtained when KL-treated cells were concomitantly exposed to 20 or 40 ng/ml IL-16 for 14–21 days (Fig. 1Go). On day 21, only ~55% of the cells in the KL-treated cultures excluded trypan blue. In contrast, ~90% of cells in the KL/IL-16-treated cultures excluded trypan blue on day 21. Slightly more cells were obtained using cord blood progenitors from normal individuals rather than peripheral blood progenitors from asthmatic patients. Nevertheless, the KL/IL-16 combination was effective on both populations of progenitors.



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  		FIGURE 1. Effects of KL and IL-16 on the growth and/or viability of cells derived from the cord blood of normal individuals (A) and the peripheral blood of asthma patients (B). Isolated buffy coat cells (2 x 106) were cultured for 3 wk in enriched medium alone (pink, +) or in enriched medium supplemented with KL (50 ng/ml) alone (light blue, {diamond}), KL and HBM-M cell-conditioned medium (dark blue, {triangledown}), or KL (50 ng/ml) with IL-16 at 1 ng/ml (black, {blacksquare}), 20 ng/ml (red, {circ}), and 40 ng/ml (green, {triangleup}). The cell numbers and viability were calculated weekly. The results are the mean ± SD of three experiments.

 
To investigate whether IL-16 influences the granule protease content of in vitro-differentiated human MCs, the buffy coat cells isolated from cord and peripheral blood were cultured separately in the medium supplemented with 50 ng/ml KL and 0–40 ng/ml IL-16. At weekly intervals, aliquots of cells in the resulting cultures were analyzed immunohistochemically for their expression of chymase and tryptase. As noted in Fig. 2Go, IL-16 exhibited a dose-dependent effect on the expression of both families of MC-restricted granule proteases when this cytokine was used in combination with KL. At the start of the culture, only 0.9 ± 0.2 and 0.5 ± 0.2% (mean ± SD; n = 3) of the cells in the buffy coat of cord blood cells derived from normal individuals contained tryptase (Fig. 2GoA) or chymase (Fig. 2GoB). After 21 days of culture in the presence of KL alone, 9.2 ± 0.3 and 5.8 ± 0.1% (mean ± SD; n = 3) of the cells in the resulting cultures expressed tryptase and chymase, respectively. Although this represents a substantial relative increase in the percentage of tryptase+/chymase+ cells, there were so few cells in the 21-day KL-treated cultures (Fig. 1GoA) that the absolute number of tryptase+/chymase+ cells was only marginally significant. In contrast to that obtained with KL alone, the percentages of tryptase+ and/or chymase+ cells increased to 17 ± 2.1 and 14 ± 0.7% (mean ± SD; n = 3), respectively, when replicate progenitors from these normal individuals were cultured in medium supplemented with both KL and IL-16 (40 ng/ml). Taking into account the total number of cells in the latter cultures, there was an ~20-fold increase in the number of tryptase+/chymase+ cells. The number of tryptase+/chymase+ cells in the cultures also increased when peripheral blood cells from three asthma patients were exposed for 21 days to medium containing 50 ng/ml KL and 40 ng/ml IL-16 (Fig. 2Go, A and B). When used in combination with KL, IL-16 actually was more effective than HBM-M cell-conditioned medium in inducing the progenitors to differentiate into tryptase+/chymase+ MCs/basophils. Greater than 75% of the tryptase+/chymase+ cells in the IL-16-treated cultures possessed segmented or lobulated nuclei (Fig. 2GoE). Although the generated cells have the granule phenotype of normal cutaneous MCs, their nuclear profiles are more reminiscent of normal human basophils.



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  		FIGURE 2. Effect of IL-16 on the development of cells that express tryptase (A, C, and E) and chymase (B and D) from cord blood (A, B, and E) and peripheral blood (C and D) progenitors. Isolated buffy coat cells (2 x 106) were cultured for 3 wk in enriched medium alone (pink, +) or in enriched medium supplemented with KL (50 ng/ml) alone (light blue, {diamond}), KL and HBM-M cell-conditioned medium (dark blue, {triangledown}), or KL (50 ng/ml) with IL-16 at 1 ng/ml (black, {blacksquare}), 20 ng/ml (red, {circ}), and 40 ng/ml (green, {triangleup}). At weekly intervals, samples of the cells were stained with anti-tryptase Ab (A, C, and E) or anti-chymase Ab (B and D). The results depicted in A–D are the mean ± SD of three experiments. Shown in E are the cells in one of the cord blood cultures stained with anti-tryptase Ab. These cells were developed by culturing progenitors in medium supplemented with 50 ng/ml KL and 40 ng/ml IL-16. The inset in E shows a tryptase+ cell in the culture at a higher magnification.

 
As assessed immunohistochemically, >80% of the nontransformed tryptase+ and/or chymase+ cells developed from blood progenitors with the IL-16/KL cytokine combination expressed substantial amounts of CD4 on their surfaces. Although the transformed HMC-1 cell line was poorly stained when incubated with anti-CD4 Ab (data not shown), RT-PCR analysis revealed that this cell line contains low levels of the CD4 transcript (Fig. 3GoA). IL-16 could act directly on human MCs/basophils and their progenitors. Alternately, the cytokine could induce contaminating cells in the cultures to release MC regulatory factors. To address whether IL-16 can affect human MCs/basophils directly, additional studies were conducted on the HMC-1 cell line. The incorporation of [3H]TdR into DNA was used to determine whether IL-16 can enhance the rate of proliferation of this cell line. Compared with untreated cells, HMC-1 cells exposed to 40 ng/ml IL-16 more than doubled their rate of incorporation of [3H]TdR into DNA (Fig. 3GoB). However, in contrast to those cells that developed when nontransformed cord or peripheral blood progenitors were cultured in the presence of IL-16, the 3-wk IL-16-treated HMC-1 cells did not develop segmented nuclei (data not shown).



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  		FIGURE 3. CD4 mRNA levels in HMC-1 cells and IL-16-mediated proliferation of this cell line. HMC-1 cellular RNA was reverse transcribed (+) and then evaluated for the presence of the CD4 transcript as well as the control GAPDH transcript (A). The PCR step was conducted in the control reaction, but not in the RT step (-). As noted in this semiquantitative assay, HMC-1 cells contain very low amounts of CD4 mRNA. Molecular weight standards are shown on the right to document that the CD4-derived product in the middle lane is 438 bp as expected. In B, the incorporation of [3H]TdR was evaluated in HMC-1 cells that were cultured for 6 days in the presence of 1–40 ng/ml IL-16. Results (mean ± SD; n = 3) are expressed as the percent increase in the rate of incorporation of [3H]TdR into DNA relative to that obtained with replicate cells cultured in medium lacking IL-16.

 
Chemotactic and Ca2+ mobilization responses of human MCs to IL-16

Human cord blood-derived MCs/basophils and the HMC-1 cell line were used in an IL-16 chemotaxis assay. The former cells were obtained by culturing progenitors for 28 days in medium supplemented with KL and HBM-M cell-conditioned medium. Thirty to 40% of the cells at the start of the experiment contained immunoreactive tryptase. Before use, the two populations of cells were washed in PBS and resuspended in fresh medium. Cell migration in medium lacking IL-16 was set at 100%. As noted in Fig. 4Go, both populations of cells underwent a significant chemotactic response when exposed to 40 ng/ml IL-16 alone. These data support the data presented in Fig. 3Go that indicated that human MCs/basophils are IL-16-responsive cells. However, an optimal chemotactic response occurred when the cells were concomitantly exposed to KL and IL-16. The chemotactic response was inhibited by anti-IL-16 Ab. The IL-16-derived peptide RRKSLQSK inhibits the IL-16-induced chemotaxis of T cells, but not the IL-16-derived peptide ETTAAGDS. In agreement with the anti-IL-16 Ab data, only the RRKSLQSK peptide inhibited the IL-16-dependent chemotactic response of human MCs/basophils and HMC-1 cells. These data indicate that IL-16 directly induces human MCs/basophils to undergo chemotaxis.



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  		FIGURE 4. IL-16-dependent chemotaxis of cord blood-derived human MCs/basophils (A) and the HMC-1 cell line (B). Cells were exposed to IL-16 alone (black, {blacksquare}), KL and IL-16 (red, {circ}), or IL-16 alone (green, {triangleup}; light blue, {diamond}; dark blue, {triangledown}) in the presence of anti-IL-16 Ab (green, {triangleup}), the IL-16-derived peptide RRKSLQSK (dark blue, {triangledown}), or the IL-16-derived peptide ETTAAGDS (light blue, {diamond}). The results are the mean ± SD of three independent experiments.

 
In separate experiments, cord blood-derived MCs/basophils were preincubated in medium containing various concentrations of anti-CD4 Ab 30 min before the start of the chemotaxis assay. As shown in Fig. 5Go, the chemotactic response to IL-16 (40 ng/ml; but not the chemotactic response to KL) was inhibited by anti-CD4 Ab. Although these findings suggest that IL-16 is acting directly via CD4, the fact that the chemotactic response was not totally abolished by anti-CD4 Ab treatment raises the possibility that IL-16 also binds to an undefined receptor on the surface of cord blood MCs that is physically associated with CD4. Because changes in the intracellular levels of Ca2+ often occur during chemotactic responses, HMC-1 cells were loaded with the fluorescent Ca2+ indicator fura-2-AM before exposure to IL-16. As noted in Fig. 6Go, IL-16 induced a rapid increase in cytosolic free calcium that peaked 30 s after exposure to the cytokine. This pattern of Ca2+ influx also was seen with ionomycin. The ability of IL-16 to stimulate directional migration (chemotaxis) vs random migration (chemokinesis) was then evaluated using a checkerboard analysis. As shown in Table IGo, chemotaxis was observed only when the concentration of IL-16 in the lower chamber was greater than that in the upper chamber. Thus, IL-16 does not appear to be a chemokinetic factor for HMC-1 cells.



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  		FIGURE 5. Effect of anti-CD4 Ab on the IL-16-mediated chemotaxis of human MCs/basophils. Cord blood-derived human MCs/basophils were developed by culturing progenitors in the presence of KL and HBM cell-conditioned medium for 21 days. The resulting tryptase+ cells were washed, resuspended in PBS containing the indicated amounts of anti-CD4 Ab, and incubated for 30 min before the start of the chemotaxis assay. The subsequent chemotactic responses of the cells to IL-16 (40 ng/ml; {blacksquare}) and KL (50 ng/ml; {diamondsuit}) were determined. Results (mean ± SD; n = 3) are expressed as a percentage of those obtained for replicate cells that were cultured without IL-16.

 


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  		FIGURE 6. IL-16-mediated mobilization of calcium in HMC-1 cells. HMC-1 cells (5 x 106/ml) were loaded with fura-2. IL-16 or ionomycin in Grey’s buffer was added at the indicated time point, and the intracellular levels of Ca2+ were determined. Similar findings were obtained in two other experiments.

 

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  		Table I. Checkerboard analysis of the chemotactic response of HMC-1 cells to IL-161

 
IL-16-mediated inhibition of HIV-1 infection of human MCs/basophils

MCs/basophils were infected with the BAL strain of HIV-1 in the presence or the absence of 1 µg/ml IL-16. A 40–50% inhibition of HIV-1 replication was observed when MCs/basophils were exposed to the retrovirus in the presence of IL-16, as assessed by the levels of p24 Ag in the 6- and 12-day conditioned medium (Fig. 7Go). The specificity of the effect was evaluated with rabbit anti-human IL-16 (1 µg/ml) polyclonal Ab. This Ab completely counteracted the inhibitory effect of IL-16 on the HIV-1 infection of the cells (data not shown). Relative to non-IL-16-treated cells, the rate of HIV-1 DNA synthesis was modestly decreased in IL-16-treated cultures on day 6 during the early stage of reverse transcription (data not shown).



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  		FIGURE 7. Inhibition of HIV-1 infection of human MCs/basophils by IL-16. CD3-/CD4+/CD117+ MCs/basophils were exposed to the M-tropic BAL strain of HIV-1 in the presence ({blacksquare}) or the absence ({square}) of IL-16. Six and/or 12 days after infection, samples of the cultures were evaluated for the presence of the viral protein p24.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show for the first time that IL-16 synergizes with KL to induce the progenitors in the cord blood of normal individuals to preferentially differentiate into human MCs/basophils that express tryptase, chymase, and CD4. We demonstrate that IL-16 is a strong chemotactic factor for human cord blood-derived MCs/basophils and for the human MC line HMC-1, and that this chemotactic response is associated with increased calcium mobilization. Finally, we show that IL-16 inhibits the susceptibility of these cells to HIV-1 infection.

As occurs in the mouse, human MCs are highly dependent on KL and its receptor c-kit/CD117. KL by itself can induce human progenitors to slowly differentiate in vitro into relatively immature populations of cells that express at least one tryptase (32). Nevertheless, KL-derived human MCs do not express chymase and carboxypeptidase A and do not express high levels of Fc{epsilon}RI unless the developing cells come in contact with fibroblasts, IL-4, IL-6, or other MC regulatory factors. Thus, it has been apparent for some time that the human MC-committed progenitors residing in the bone marrow and peripheral blood must encounter accessory factors to complete their differentiation and maturation processes in tissues. While trying to understand what receptors the MCs/basophils (33) residing in the blood of asthma patients possess on their surfaces, we noted that these CD3- cells express the cytokine receptor c-kit and the chemokine receptors CCR3, CCR5, and CXCR4 (24). The major surprise of those studies was the finding that the MCs/basophils additionally expressed CD4. The potential role of IL-16 in MC/basophil development and/or function has not been examined as far as we are aware. Nevertheless, the identification of CD4 on these cells raised the possibility that some populations of human MCs/basophils might be responsive to IL-16. In the presence of KL, IL-16 induced the progenitors in the cord blood of normal individuals to increase their expression of tryptase and chymase ~20-fold (Figs. 1Go and 2Go). The numbers of tryptase+ and/or chymase+ cells in the 3-wk cultures were greater than those obtained with KL and HBM cell-conditioned medium. Although a head-to-head comparison was not conducted, the number of chymase+ cells obtained with the KL/IL-16 cytokine combination appears to equal or exceed that obtained by others using the KL/IL-6/IL-10 (9) or KL/IL-6/PGE2 (7) combination of factors. Thus, the KL/IL-16 cytokine combination is a technological advance for those interested in generating increased numbers of human MCs for in vitro study.

IL-16 induced the HMC-1 cell line to proliferate (Fig. 3GoB). Although the IL-16-treated HMC-1 cells did not undergo nuclear segmentation, most of the cord blood-derived MCs/basophils possess segmented nuclei after 3 wk of culture in the presence of IL-16. It is highly unlikely that a cell with such a nuclear profile can proliferate. Thus, IL-16 does not appear to be comparable to IL-3 in the mouse in terms of its ability to induce the proliferation of normal, nontransformed human MCs and their progenitors. Alternately, there is another factor produced by contaminating cells in the cord and peripheral blood cultures that inhibits the proliferation activity of IL-16 by promoting nuclear segmentation of the developing MCs.

Chemotactic movement of a cell toward a chemical gradient of a specific substance reflects a complex biological process involving numerous discrete cellular events. A chemotactic factor will engage cell surface receptors, resulting in a cascade of secondary signals. This, in turn, leads to cytoskeleton reorganization, which allows for unidirectional movement. IL-16 is a powerful chemotactic factor for CD4+ T cells. Arguably, the most impressive finding obtained in our study was the chemotactic response of HMC-1 cells and cord blood-derived MCs/basophils to IL-16 (Fig. 4Go). IL-16 (1 ng/ml) was sufficient to induce chemotaxis. Exposure to anti-CD4 Ab or an inhibitory peptide (30) that corresponds to a C-terminal region in IL-16 diminished the ability of cord blood-derived MCs to respond to the native cytokine (Fig. 5Go). Thus, IL-16 exerts its effects via CD4 or a CD4-associated protein. In this regard, many members of the tetraspan superfamily of surface proteins (e.g., CD9, CD37, CD53, CD63/ME491, CD81/TAPA-1, or CD82/kangai 1) physically associate with CD4 in other cell types. Normal human MCs and the transformed MCs in aggressive/malignant mastocytosis patients express CD9, CD63, CD81, and/or CD82 (34, 35, 36, 37, 38, 39). Because IL-16-responsive HMC-1 cells contain very low amounts of CD4 mRNA (Fig. 3GoA), the primary IL-16R on the surface of this transformed cell line may be one of these four tetraspans. In support of this possibility, IL-16-responsive T cells also express CD81.

Exposure of T cells to IL-16 results in rapid phosphorylation of the CD4-associated protein p56lck (40). This early signaling event results in a rise in the intracellular levels of inositol trisphosphate and calcium (41). A motility response then occurs in the IL-16-treated T cells along with an up-regulation of IL-2R and MHC class II expression. Although there is much to be learned about the IL-16-mediated signaling pathway in human MCs/basophils, the in vitro studies conducted with the HMC-1 cell line suggest that IL-16 activates a similar Ca2+-dependent pathway (Fig. 6Go).

HIV-1 infection requires CD4 and at least one chemokine coreceptor. CCR5 is a receptor for RANTES (42), and CCR5 is used by certain M/R5-tropic strains of HIV-1 to infect CD4+ cells (43). MCs undergo a chemotactic response to physiologic concentrations of RANTES in vitro (44, 45), and the number of MCs in muscle increase dramatically 4 h after 20 ng recombinant RANTES is injected into this tissue site (46). Thus, MCs use CCR5 in vivo. The finding that the MCs/basophils in the blood of asthma patients are susceptible to an M-tropic strain of HIV-1 ex vivo led to the subsequent discovery that the MCs/basophils in the blood of some AIDS patients are infected with HIV-1 (24). The finding that a nonpeptide inhibitor of CCR5 blocks viral entry is additional evidence that those M-tropic strains that infect human MCs/basophils preferentially use CD4 and CCR5 (25). The susceptibility of in vivo-differentiated human MCs/basophils and their progenitors is an explanation for why MCs are rarely found in the jejunum of advanced AIDS patients (47). IL-16 inhibits the susceptibility of T cells to T-tropic strains of HIV-1 (48). Because CD4 is a putative IL-16R, we evaluated whether IL-16 alters the susceptibility of human MCs/basophils to an M-tropic strain of HIV-1. As noted in Fig. 7Go, IL-16 inhibited the susceptibility of these cells to the BAL M-tropic strain of HIV-1. These findings now suggest that IL-16 therapy could be beneficial in hindering the infection of human MCs/basophils by M-tropic strains of HIV-1.


   Acknowledgments
 
We thank the Obstetrics Department of St. George’s Hospital for providing umbilical cord blood.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI23483, HL36110, and HL63284 and National Health and Medical Research Council (Australia) Grants. Back

2 Address correspondence and reprint requests to Dr. Steven A. Krilis, Department of Immunology, Allergy, and Infectious Disease, St. George Hospital, Kogarah, New South Wales 2217, Australia. E-mail address: s.krilis{at}unsw.edu.au Back

3 Abbreviations used in this paper: MC, mast cell; AP, alkaline phosphatase; BAL, bronchoalveolar lavage; Fc{epsilon}RI, high affinity IgE receptor; KL; c-kit ligand; RT, reverse transcription. Back

Received for publication December 17, 2001. Accepted for publication February 13, 2002.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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