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The Journal of Immunology, 1998, 160: 5874-5879.
Copyright © 1998 by The American Association of Immunologists

IL-16 Activates the SAPK Signaling Pathway in CD4+ Macrophages1

Stefan Krautwald2

Department of Immunobiology, Fraunhofer Institute for Toxicology and Molecular Biology, Hannover, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-16 has been reported as a modulator of T cell activation and was shown to function as chemoattractant factor. The chemotactic activity of IL-16 depends on the expression of CD4 on the surface of target cells, but the intracellular signaling pathways are only now being deciphered. This report describes IL-16 as an additional activator of the stress-activated protein kinase (SAPK) pathway in CD4+ macrophages. Treatment of these cells with recombinant expressed IL-16 leads to the phosphorylation of SEK-1, resulting in activation of the SAPKs p46 and p54. IL-16 stimulation also leads to the phosphorylation of c-Jun and p38 MAPK (mitogen-activated protein kinase), without inducing MAPK-family members ERK-1 and ERK-2. Interestingly, the IL-16-mediated activation of SAPKs and p38 MAPK in macrophages alone induces no detectable apoptotic cell death. These observations suggest specific regulatory functions of IL-16 distinct from the proinflammatory cytokines TNF-{alpha} and IL-1ß.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin 16 was originally described as a lymphocyte chemoattractant factor (LCF) derived from PBMCs (1, 2). IL-16 mRNA is constitutively expressed in CD4+ and CD8+ T cells. The biologically active protein is secreted from activated CD8+ cells and promotes migration of CD4+ lymphocytes, monocytes, and eosinophils (3). Interestingly, the deduced protein sequence of human IL-16 lacks a hydrophobic signal sequence for secretion of the peptide (3), a phenomenon that has also been reported for the secreted cytokines IL-1{alpha} and IL-1ß (4, 5). IL-16 shows no overt biologic function or sequence homology to other interleukins or chemokines. To gain biologic activity, the IL-16 monomer (14 kDa) must form a noncovalently linked tetrameric structure (3).

Interestingly, IL-16 has been reported to inhibit HIV replication in PBMCs (6, 7). Deletional analysis of IL-16 showed (3) that the inhibition of HIV replication is achieved by the C-terminal part of the molecule. This was further shown in HIV-1-susceptible CD4+ Jurkat cells stably transfected with the C-terminal portion (130 aa) of hIL-16 (8). These cells constitutively secreted IL-16, and HIV replication was 99% inhibited after infection.

A great deal of evidence suggests that IL-16 uses CD4 as its specific receptor independently of CD3/TCR (9, 3, 10). IL-16 stimulation of resting T cells induces a rise in intracellular [Ca2+], generation of phosphatidylinositol 1,4,5-trisphosphate, activation of p56lck, and an increased expression of the IL-2 receptor (11, 4, 3, 12). T lymphocyte activation is extremely complex; associates intracellular signaling pathways are not well understood. One of the major pathways in mammalian cells by which extracellular signals are converted into intracellular responses is mediated by the activation of members of the mitogen-activated protein kinase (MAPK)3 subfamilies (13, 14, 15), which are 1) the extracellular signal-regulated kinases ERK-1 and ERK-2 (16), 2) the c-Jun-N-terminal/stress-activated protein kinases (JNK/SAPK) (17, 18), and 3) the subgroups of p38 kinase (19, 20).

The MAPK pathway is extremely well conserved in higher eukaryotes (21) and involves serine/threonine kinases that are rapidly activated in cells induced by a variety of extracellular stimuli. These stimuli include mitogenic growth factors, cytokines, T cell Ags, tumor promoters, and hormones (22, 23), all leading to the formation of membrane-associated signaling complexes that deliver a Ras-mediated signal to a downstream kinase cascade involving Raf-1 kinase, MEK (mitogen-activated/extracellular signal-regulated kinase), and the MAPKs (24, 25, 26).

The SAPKs/JNKs are part of a signal transduction cascade that is related to the MAPK pathway (18). The SAPKs/JNKs are activated in response to a variety of physiologic and stressful stimuli, including growth factors, osmotic shock, gamma and ultraviolet irradiation, protein synthesis inhibitors, and the inflammatory cytokines TNF-{alpha} and IL-1ß (18, 17, 27, 28, 29). The stress-activated pathway involves the sequential phosphorylation and activation of the proteins MEKK, JNKK/SEK-1, and SAPK/JNK (18, 15, 30, 31, 32). The SAPKs/JNKs have been shown to phosphorylate and regulate the activity of several transcription factors including Elk-1, SAP-1, ATF-2, and c-Jun (33, 34, 27, 18). The transcriptional activity of c-Jun (35), a component of AP-1, is stimulated by phosphorylation through SAPKs at serines 73 and 63 (36, 37).

To investigate the signaling cascade induced by IL-16, the CSF-1-dependent, murine macrophage cell line BAC-1.2F5 (38) presents a well suited and studied model system for CD4+ cells. These cells are mature splenic macrophages immortalized by transfection with origin-defective SV40 DNA (39). The cell line possess all characteristics of macrophages (38), and signal transduction pathways have been well characterized in response to growth factor and LPS stimulation (40, 41, 42). CSF-1 stimulates macrophage survival, growth, and differentiation (43). It is known that treatment of macrophages with the mitogen CSF-1 or LPS leads to an activation of the MAPK pathway, including the proteins Raf-1, MEK, and MAPK (40, 41, 42, 44). Both signals result in the phosphorylation of two distinct ternary complex factors (TCFs), identified, respectively, as TCF/Elk and TCF/SAP (40, 45).

In the present study it is demonstrated that IL-16 is a specific activator of the SAPK pathway without inducing the MAPK-family members ERK-1 and ERK-2. Treatment of CD4+ macrophages with IL-16 led to the phosphorylation of SEK-1, which in turn activates the SAPKs (JNKs) p46 and p54, resulting in the phosphorylation of c-Jun at Ser63. The IL-16-mediated phosphorylation of p38 MAPK at Tyr182, resulting in the activation of the functionally distinct p38 MAPK pathway, too. In summary, these results indicate that IL-16 is a novel addition to the group of factors leading to the activation of the SAPK and p38 MAPK pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of recombinant proteins and bacterial expression

Construct encoding murine full-length c-Jun was cloned into a pET-15b His-Tag vector (Novagen, Madison, WI). Total RNA prepared from murine spleen (46) cells was used for RT-PCR.

The forward primer 5'-GGCATATGACTGCAAAGATGGAAACGACC-3' and the reverse primer 5'-CCCTCGAGTCAAAACGTTTGCAACTGCTG-3' were used for amplification. The relevant cDNA was recovered from the PCR mixture, digested with the restriction enzymes NdeI and XhoI, and cloned into the NdeI/XhoI-cut pET-15b vector. This construct was transformed into Escherichia coli BL21 (DE3) bacteria. An 800-ml LB culture was inoculated and grown to an optimal density of 0.8 measured at 600 nm. Bacteria were then induced with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) for 3 h and harvested by centrifugation (5,000 x g, 15 min, 4°C). After aspirating the supernatant, cells were resuspended in 50 mM Tris-HCl, pH 8.5, containing 10 mM EDTA and lysed by sonication. The c-Jun inclusion bodies were recovered by centrifugation (10,000 x g, 15 min, 4°C), and the pellet was solubilized in 8 M urea, 50 mM Tris-HCl, pH 8.5, containing 1 mM EDTA and 10 mM DTT. c-Jun was renatured at 4°C by dialysis against decreasing concentrations of urea. For purification of the recombinant protein, immobilized metal ion chromatography (IMAC) on chelating Sepharose loaded with Ni2+ was conducted according to the protocols provided by Qiagen (Hilden, Germany).

Construct encoding 390 nucleotides of the mature 130-aa human IL-16 protein (6, 3, 8) was cloned into a pET-15b His-Tag vector (Novagen). cDNA generated from total RNA prepared from PBMCs (46) was used as a template for PCR with forward 5'-GGCATATGCCCGACCTCAACTCCTCCACT-3' and reverse 5'-CCGGATCCTCAGGAGTCTCCAGCAGCTGTGGTTTCC-3' primers. The relevant cDNA was recovered from the PCR mixture, digested with the restriction enzymes NdeI and BamHI, and cloned into the NdeI/BamHI-cut pET-15b vector. This construct was transformed into E. coli BL21 (DE3) bacteria. Expression of the recombinant protein was as described above. Soluble IL-16 was purified according to the manufacturer’s instructions (Qiagen).

Preparation and isolation of recombinant baculoviruses

For expression of the recombinant human IL-16 in insect cells the plasmid (IL-16 cDNA in pET-15b) was digested with BglII/BamHI, isolated, and ligated into the BglII/BamHI-cut pVL1392 baculovirus transfer vector (Dianova, Hamburg, Germany). The resulting construct harboring IL-16 cDNA under the viral polyhedrin promoter was cotransfected with the wild-type AcNPV (Autographa californica nuclear polyhedrosis virus) DNA into Spodoptera frugiperda (Sf21) insect cells according to the manufacturer’s instructions (Dianova). Serial plaque purification was used to isolate recombinant viruses. Cells were cultured at 27°C in TNM-FH medium supplemented with 10% FCS and infected (multiplicity of infection of 5–10) by adding the virus directly to the culture at a density of 7 x 106 cells/10 ml according to standard methods (47). The cells were collected 72 h postinfection by centrifugation (1,000 x g, 10 min, 4°C). Isolation and purification of the intracellular polyhistidine tag containing recombinant protein was performed according to standard methods (48).

Flow cytometric analysis

To demonstrate that the macrophage cell line BAC-1.2F5 expresses the CD4 Ag on the cell surface, flow cytometric measurements were performed as described previously (49). Briefly, 5 x 105 BAC-1.2F5 cells were used for each analysis. After blocking Fc receptors with 40 µl goat serum (12 mg/ml, Dianova), 5 µl murine CD4 Ab (L3T4, Phycoerythrin labeled, MEDAC Hamburg, Germany) or an isotype control was added. The cells were incubated in a final volume of 50 µl for 30 min on ice. During this incubation, period cells were mixed twice and finally washed with PBS supplemented with 2% FCS and 0.05% NaN3. Cells were analyzed on a FACScan (Becton Dickinson, Heidelberg, Germany) by setting a gate on the BAC-1.2F5 cells. For each measurement, 6500 events were recorded. For data analysis, the shift in fluorescence intensity (FL-2) was measured by histogram overlays and determination of mean channel fluorescence.

Cell culture, stimulation, and cell lysis

BAC-1.2F5 cells were cultured in DMEM supplemented with 10% FCS and 20% L cell-conditioned medium as a source of CSF-1 (43). Confluent cultures (107 cells per 100-mm diameter dish) were incubated for different periods with either 1 µg of bacterial LPS (E. coli O111:B4; Sigma, Deisendorf, Germany) per ml, 0.63 nM mouse recombinant CSF-1 (50), or 0.1 µM human recombinant IL-16. For stimulation experiments with CSF-1 and LPS, confluent cells were cultured in medium without CSF-1 for 16 h to up-regulate CSF-1 receptors. Stimulations were terminated by aspirating the medium and washing five times with ice-cold PBS. Cells were lysed in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1% Triton X-100, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, 2 µM ZnCl2, and 1 mM PMSF. Insoluble material was removed by centrifugation (15,000 x g, 20 min, 4°C); protein concentration was determined (Bradford method) according to the manufacturer’s instructions (Bio-Rad, München, Germany). Equal protein amounts (22.5 µg/lane) were resolved on a 7.5% SDS-PAGE gel and transferred onto Hybond-C extra nitrocellulose membrane (Amersham, Braunschweig, Germany) for immunoblotting. Blots were probed with 1) phospho-specific SEK-1 (Thr223) Ab, 2) phospho-specific c-Jun (Ser63) Ab, 3) p38 MAPK Ab, or 4) phospho-specific p38 MAPK (Tyr182) Ab (all from New England Biolabs, Beverly, MA). Immune complexes were visualized by enhanced chemiluminescence (Amersham).

"In-gel" kinase assay

Samples containing 7.5 µg protein buffered in 62.5 mM Tris (pH 6.8), 2.3% SDS, 5 mM EDTA, 10% glycerol, and 100 mM DTT were incubated at 85°C for 5 min, then subjected to 12.5% SDS-PAGE. Separating gels were polymerized with 0.2 mg/ml myelin basic protein (MBP) (Sigma) or 40 µg/ml recombinat murine c-Jun to assay for ERK or SAPK activity, respectively. After electrophoresis, gels were denatured in 6 M guanidine HCl, renatured, and assayed for kinase activity as described previously (51, 52).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning and expression of recombinant IL-16

To investigate a possible involvement of SAPK and/or MAPK in IL-16-stimulated signal transduction pathways, this cytokine was cloned and expressed as a recombinant protein.

The 390-base pair coding region that has been shown to be sufficient for the biologic activity of IL-16 (6, 8) was cloned into an E. coli expression vector (data not shown). The modified construct (Materials and Methods) in the pVL1392 baculovirus transfer vector and wild-type AcNPV DNA were cotransfected into Sf21 insects cells to obtain the recombinant protein (Fig. 1Go). Expressed IL-16 (> 95% pure) was obtained via IMAC (using Ni2+-NTA agarose, Qiagen). Bacterial and eukaryotic expression systems produced identical biologically active proteins with a relative mass of ~17 kDa as estimated by SDS-PAGE (Fig. 1Go). However, to exclude the possibility of an LPS contamination interfering with the specific IL-16 signal, all results were obtained using insect cell-derived recombinant IL-16.



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  		FIGURE 1. Purification of recombinant IL-16. Coomassie blue-stained 12.5% SDS-PAGE gel of Sf21 insect cells (baculovirus system) expressing recombinant human IL-16. Cells were harvested 72 h after transfection. Lane 1, standard protein marker (New England Biolabs); lane 2, uninfected whole cell lysates (control); lane 3, infected whole cell lysate; lane 4, soluble protein fraction; lane 5, standard protein marker (New England Biolabs); lane 6, effluent of the IMAC; lane 7, wash (5 mM imidazole); lane 8, wash (60 mM imidazole); lane 9, eluate of the IMAC (250 mM imidazole). Equal volumes of protein solutions were loaded in each lane.

 
BAC-1.2F5 macrophages express the CD4 Ag on their cell surface

Earlier studies suggested that IL-16-mediated events might occur following interaction of IL-16 with CD4 or a CD4-associated molecule (3). A good candidate for study of an IL-16 effect is the macrophage cell line BAC-1.2F5. These cells have been shown to possess many properties of primary monocytes/macrophages. To ensure BAC-1.2F5 cells can process IL-16 stimuli properly, cell surface expression of CD4 Ag was investigated. The cells express ample CD4 on their surface (Fig. 2Go), providing a strong basis for further investigations of IL-16 signaling. Nevertheless, the following studies, also conducted with primary bone marrow monocytes/macrophages derived from C57BL/6J and C3H/HeN mice, delivered identical results (data not shown).



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  		FIGURE 2. Demonstration of CD4 Ag expression levels on BAC-1.2F5 macrophages. The histogram overlay shows the staining of the BAC-1.2F5 cells with the isotype control (dotted line) and with the murine phycoerythrin-labeled CD4 Ab L3T4 (solid line).

 
Phosphorylation of SEK-1 and p38 MAPK induced by IL-16

The stress-activated protein kinase activator SEK-1 is a dual specificity kinase that serves as one of the immediate upstream activators of the SAPKs/JNKs (15, 32, 53). Known substrates of SEK-1 are the {alpha}, ß, and {gamma} isoforms of p46/p54 SAPK subfamily of MAPKs and the p38 MAPK subfamily (30). SEK-1 has been shown to be phosphorylated in response to a variety of extracellular stimuli such as environmental stress or mitogenic factors (51, 14). The IL-16 concentration for the following experiments was calculated from the monomeric form (~17 kDa) shown in Figure 1Go. Confluent BAC-1.2F5 cells were stimulated for 15 min with various concentrations of recombinant IL-16, ranging from 10-10 to 10-6 M, and after lysis were subjected to Western analysis for phosphorylated SEK-1 (Fig. 3Go). This dose range of IL-16 was reported previously to possess biologic activity (11). Significant, dose-dependent effects were observed with concentrations from 10-6 to 10-8 M. Concentrations of IL-16 below 10-8 M are not sufficient to induce the described signaling pathway, yet the concentration of the actual biologically active tetrameric form (9, 3) may be much lower. Since no significant differences could be detected at IL-16 concentrations higher than 10-7 M, the following studies were performed at this dose.



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  		FIGURE 3. Dose-dependent phosphorylation of SEK-1 by IL-16. BAC-1.2F5 macrophages were cultured at 37°C and treated for 15 min with indicated concentrations of recombinant IL-16. Equal protein amounts (22.5 µg/lane) were resolved on a 7.5% SDS-PAGE gel and transferred onto Hybond-C extra nitrocellulose membrane for immunoblotting. Blots were probed with phospho-specific SEK-1 (Thr223) Ab.

 
To investigate an involvement of IL-16 in signal transduction pathways, BAC-1.2F5 macrophages were lysed after different stimulation periods with 10-7 M IL-16 and subjected to Western analysis for SEK-1 and p38 MAPK. Phospho-, residue-specific SEK-1 Ab revealed an IL-16-induced phosphorylation of SEK-1 the first time after 15 min (Fig. 4Go). The signal decreased after 30 min and was not observed after 60 min.



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  		FIGURE 4. Phosphorylation of SEK-1 by IL-16 stimulus. BAC-1.2F5 macrophages were treated with IL-16 (0.1 µM) at 37°C for the indicated times. Equal protein amounts (22.5 µg/lane) were resolved on a 7.5% SDS-PAGE gel and transferred onto Hybond-C extra nitrocellulose membrane for immunoblotting. Blots were probed with phospho-specific SEK-1 (Thr223) Ab.

 
Activated SEK-1 has been shown to function as an activator of p38 MAPK (15, 32), whose specific activators have been shown to be MKK-3 and MKK-6 (54). The mechanism of p38 MAPK activation is mediated by a dual phosphorylation on Thr180 and Tyr182 (15, 55). Like the SEK-1 phosphorylation, the tyrosine phosphorylation of p38 MAPK on residue 182 could be detected between 15 and 30 min of IL-16 stimulation via a phospho-specific p38 MAPK Ab (Fig. 5GoA).



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  		FIGURE 5. IL-16-induced phosphorylation of p38 MAPK in BAC-1.2F5 macrophages. Cells were treated with IL-16 (0.1 µM) at 37°C for the indicated times. Equal protein amounts of (22.5 µg/lane) were resolved on a 7.5% SDS-PAGE gel and transferred onto Hybond-C extra nitrocellulose membrane for immunoblotting. The blot was probed with phospho-specific p38 MAPK (Tyr182) Ab (A). As control for sample variations the blot was stripped, according to the manufacturer’s instructions (Amersham) and reprobed with p38 MAPK Ab, which detects total p38 MAPK status independent of its phosphorylation, showing equal levels of p38 protein (B).

 
An Ab directed against total p38 MAPK (phosphorylation-state independent) showed an equal abundance of p38 MAPK after different IL-16 stimulation periods (Fig. 5GoB). Further, between 15 and 30 min a shift of the phosphorylated p38 MAPK was detectable. Due to no detectable phosphorylation of SEK-1 and p38 MAPK before 15 min, the following kinetics do not include earlier time points.

BAC-1.2F5 cells were also pretreated for 2 h with 10 µM SB 203580 (Calbiochem, Bad Soden, Germany), followed by a 15-min incubation with 10-7 M IL-16. This specific p38 MAPK inhibitor (56, 57) was present throughout the incubation period of IL-16 and led only to the inhibition of p38 MAPK phosphorylation but not of SEK-1 (data not shown). Phosphorylation of neither SEK-1 nor p38 MAPK was detectable after stimulation of BAC-1.2F5 macrophages with 0.63 nM mouse recombinant CSF-1 (data not shown). Furthermore, no differences of SEK-1 and p38 MAPK phosphorylation after IL-16 stimulation were detectable in macrophages starved for 16 h, as is required for the CSF-1-induced pathways (data not shown).

IL-16 activates SAPKs/JNKs

Since phosphorylated SEK-1 has been shown to activate SAPKs by phosphorylating residues Thr183 and Tyr185 (17), the effect of IL-16 on SAPKs activation was investigated. SAPKs activity was visualized via in-gel kinase assay. This technique assesses SAPKs activity in whole cell extracts. Extracts were resolved by SDS-PAGE in a gel containing purified recombinant c-Jun (Fig. 6GoA) or MBP (Fig. 6GoB) as a substrate. After renaturation, the activities of p46 and p54 SAPKs were visualized by the transfer of radioactive phosphate to c-Jun. A strong activation of SAPKs after 15 min was detectable only in the gel containing c-Jun (Fig. 6GoA) as a substrate. A phosphorylation of MBP by activated SAPKs could not be observed after IL-16 stimulation (Fig. 6GoB). The SAPKs activation was nearly abolished after 30 min.



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  		FIGURE 6. Effect of IL-16 on SAPK activation. BAC-1.2F5 macrophages were treated with IL-16 (0.1 µM) at 37°C for indicated times. The activity of SAPKs (p46 and p54 are indicated) was measured in an in-gel kinase assay with recombinant murine c-Jun (A) or MBP (B) as a substrate. Whole cell lysates (7.5 µg per lane) were separated by 12.5% SDS-PAGE.

 
IL-16 could not activate the MAPKs ERK-2 and ERK-1

Another well-characterized pathway in mammalian cells leads to the activation of the extracellular signal regulated kinases ERK-1 and ERK-2 (16), members of the mitogen-activated protein kinases (MAPKs) subfamilies (13, 14, 15). In macrophages, proliferative stimuli, such as CSF-1, and inflammatory agents, such as LPS, lead to an activation of ERK-1 and ERK-2 (40, 41, 42, 44). The endotoxin LPS has also been shown to activate the SAPK pathway (58). To investigate whether IL-16 is capable of activating the SAPK as well as the MAPK pathway, an in-gel kinase assay was performed using MBP as a substrate. Figure 7Go shows an activation of ERK-1 and ERK-2 after LPS stimulation of macrophages but not after IL-16 stimulation. Although some extracellular stimuli such as LPS activate both ERKs and SAPKs (58), IL-16 activates only the SAPK subgroup.



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  		FIGURE 7. Effects of IL-16 or LPS on MAPK activation. BAC-1.2F5 macrophages were treated with IL-16 (0.1 µM) or LPS (1 µg/ml) at 37°C for indicated times. The activity of MAPKs (ERK-2 and ERK-1 are indicated) was measured in an in-gel kinase assay, with MBP as a substrate. Whole cell lysates (7.5 µg per lane) were separated by 12.5% SDS-PAGE.

 
IL-16 stimulation leads to phosphorylation of c-Jun

Activated SAPKs have been described to activate c-Jun (55, 59), a component of the transcription factor AP-1 (60), in response to proinflammatory cytokines. The transcriptional activity of c-Jun is stimulated by phosphorylation at Ser63 and Ser73 within its N-terminal activation domain (61). Figure 8Go shows serine phosphorylation of c-Jun on residue 63 15 min after IL-16 stimulation. The decreased phosphorylation after 30 min correlates with the activation of the stress kinases placed upstream of c-Jun.



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  		FIGURE 8. IL-16-mediated phosphorylation of c-Jun. BAC-1.2F5 macrophages were stimulated with 0.1 µM IL-16 at 37°C for the indicated times. Equal protein amounts (22.5 µg/lane) were resolved on a 7.5% SDS-PAGE gel. Phosphorylation of c-Jun at Ser63 was analyzed by Western blotting with phospho-specific c-Jun Ab.

 
IL-16 could not induce apoptosis

It has been shown that activation of both SAPK and p38 MAPK signaling pathways by TNF-{alpha} induced apoptotic cell death (18, 55). Therefore, it was examined whether treatment of macrophages with IL-16 resulted also in apoptosis. Cells were stimulated with various concentrations of IL-16, ranging from 10-9 to 10-6 M at 37°C for 4, 8, 24, 48, 72, and 96 h, followed by staining with a FITC conjugate of Annexin V and subsequent FACS analysis. Later stages of apoptosis were looked at using the genomic DNA fragmentation method. Experiments were conducted according to the material and protocols provided by Clontech (Heidelberg, Germany) and Boehringer (Mannheim, Germany), respectively. For up to 96 h, no differences of untreated control cells and IL-16-stimulated macrophages regarding apoptotic events or death rate were detectable in the performed assays (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The cellular responses of cells of the immune system such as macrophages require intracellular signaling pathways (62, 40). It has been shown that cascades of protein phosphorylation are involved in these processes (44). The present study demonstrates that the proinflammatory cytokine IL-16 activates the p38 MAPK and SAPK/JNK pathways in CD4+ macrophages in a dose-dependent manner (Fig. 3Go). Treatment of these cells with the recombinant IL-16 leads to phosphorylation of p38 MAPK (Fig. 5GoA) as well as to sequential phosphorylation and activation of SEK-1 (Fig. 4Go), p46/p54 SAPK (Fig. 6Go), and c-Jun (Fig. 8Go) and identifies IL-16 as a new upstream activator of the stress-activated protein kinases. In contrast to this, IL-16 is not able to activate the extracellular signal-regulated kinases ERK-1 and ERK-2 (Fig. 7Go). Although all MAPKs are >40% homologous, the results of this paper reflect once more (63) that the members of the MAPK family possess a distinct set of upstream activators. A structural basis for these specifications has not been currently demonstrated, but the SAPKs/JNKs and the p38 MAPK pathways have been shown to be preferentially activated in response to UV radiation, environmental stresses, and proinflammatory cytokines, which have little or no effect on ERK activity (17, 55). As for TNF-{alpha} and IL-1ß (64, 65), the signal transduction induced by IL-16, causing activation of SAPKs/JNKs as well as p38 MAPK, supports the existence of an additional member of proinflammatory cytokines that is able to activate these two groups of MAPKs, too. It might be possible that IL-16 activates SAPK/JNK and p38 MAPK by parallel signal transduction pathways, but it may be that SEK-1 is the common MAPK kinase that activates both pathways.

On the other side, it is noticeable that the signaling pathway utilized by IL-16 shows a significant difference to the proinflammatory cytokines TNF-{alpha} and IL-1ß. In contrast to IL-16 (Fig. 7Go), the pleiotropic cytokines TNF-{alpha} and IL-1ß have been shown to activate SAPKs, p38 MAPK, and the MAPK members ERK-1 and ERK-2 (64, 65). These findings emphasize the combinatorial nature of signal transduction and the resulting cellular responses, depending on stimulus and the cell type. Nevertheless, the data reported here show for the first time that a proinflammatory cyotokine does not necessarily activate all of the three MAP kinase subgroups. Further studies are required to characterize the physiologic significance of these observations.

The dynamic balance between the MAPK pathways may be important in determining whether a cell survives or undergoes apoptosis and is currently the subject of intense investigations. It has been shown that stress-induced apoptosis requires a functional SAPK/JNK signaling pathway (66) and that, under some circumstances, SAPK/JNK signaling alone might be sufficient to cause cell death (67). In the case of IL-16, the activation of SAPK/JNK and the related p38 MAPK in macrophages alone induces no detectable apoptotic cell death, measured by apoptosis-associated plasma membrane lipid changes with Annexin V or cleavage of cellular DNA into histone-associated DNA-fragments (data not shown).

In the last two years IL-16 achieved great interest through the reported activity on HIV replication in PBMCs (6, 7). Although the mechanism of HIV suppression by IL-16 is unknown, it was recently shown that human CD4+ cells that were stably transfected with IL-16 cDNA constitutively secrete IL-16 at nanomolar concentrations and that this amount of protein does not simply block virus entry by competing with the gp120 surface glycoprotein of HIV for CD4 binding sites (8). Nevertheless, these transfectants, which do not alter growth rate or CD4 expression, are resistant to HIV infection. Finally, the identification of IL-16, as an activator of the stress-activated protein kinases and a function of the described pathways in progression of HIV disease, might seem very interesting because CD4+ macrophages were previously identified as a highly productive source of HIV (68). But resolution of this question and the exact relationship remain to be established.


   Acknowledgments
 
I thank Anke Bialowons and Maren Steyer for technical assistance, and Andreas Emmendörffer for flow cytometric analysis. I am grateful to Dirk Büscher for valuable suggestions and for critically reading the manuscript.


   Footnotes
 
1 The nucleotide sequences for recombinant proteins reported in this paper were deposited in the GenBank database as follows: human IL-16, accession No. M90391; murine c-Jun, accession No. J04115. Back

2 Address correspondence and reprint requests to Dr. Stefan Krautwald, Fraunhofer Institute for Toxicology and Molecular Biology, Department of Immunobiology, Nikolai-Fuchs-Strasse 1, D-30625 Hannover, Germany. E-mail address: Back

3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated/extracellular single-regulated kinase; Ser, serine; Thr, threonine; Tyr, tyrosine; IMAC, immobilized metal ion chromatography; TCF, ternary complex factor; MBP, myelin basic protein; aa, amino acid. Back

Received for publication November 5, 1997. Accepted for publication February 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Center, D. M., W. W. Cruikshank. 1982. Modulation of lymphocyte migration by human lymphokines. I. Identification and characterization of chemoattractant activity for lymphocytes from mitogen-stimulated mononuclear cells. J. Immunol. 128:2563.[Medline]
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