Identification of interleukin-16 production on tumor aggravation in hepatocellular carcinoma by a proteomics approach.

BACKGROUND: Cytokines play an important role in the immune response, angiogenesis, cell growth, and differentiation in hepatocellular carcinoma (HCC). OBJECTIVE: We performed a comprehensive study to identify tumor-related cytokines and pathways involved in HCC pathogenesis. METHODS: Cytokine production was evaluated in human HCC tissues and adjacent non-tumor tissues using an antibody-based protein array technique. We compared cytokine expression in HCC tissues with that of hepatic hemangioma (HH), liver metastasis of colorectal cancer, and noncancerous liver tissues from transplantation donors. The protein levels and localization of the candidate cytokines were analyzed by western blotting and immunohistochemistry. RESULTS: Increased expression of interleukin (IL)-1 receptor antagonist, macrophage migration inhibitory factor, and IL-16 was observed in HCC and paired adjacent non-tumor tissues compared with noncancerous livers. In addition, there were increased IL-16 levels in HCC tissues compared with HH. IL-16 treatment significantly increased cell proliferation in vitro. The expression of extracellular signal-regulated kinase (ERK)1/2 and cyclin D1 was markedly increased in cells from two HCC cell lines, Huh7 and HepG2, in a dose- and time-dependent manner. Phosphorylated to total ERK1/2 ratio was increased in Huh7 cells following IL-16 50 ng/ml, but not HepG2 cells. ERK phosphorylation have occurred earlier than protein accumulation at 48 h. Pretreatment with the ERK inhibitor, FR18024, or an anti-IL-16 antibody reduced the increase in IL-16 production in HCC cells. CONCLUSIONS: These results suggest that cell proliferation induced by IL-16 is mediated through the ERK pathway, thus, we identified a new factor associated with HCC tumor growth.

Interleukin-16 inhibits sodium channel function and GluA1 phosphorylation via CD4- and CD9-independent mechanisms to reduce hippocampal neuronal excitability and synaptic activity.

Interleukin 16 (IL-16) is a cytokine that is primarily associated with CD4+ T cell function, but also exists as a multi-domain PDZ protein expressed within cerebellar and hippocampal neurons. We have previously shown that lymphocyte-derived IL-16 is neuroprotective against excitotoxicity, but evidence of how it affects neuronal function is limited. Here, we have investigated whether IL-16 modulates neuronal excitability and synaptic activity in mouse primary hippocampal cultures. Application of recombinant IL-16 impairs both glutamate-induced increases in intracellular Ca2+ and sEPSC frequency and amplitude in a CD4- and CD9-independent manner. We examined the mechanisms underlying these effects, with rIL-16 reducing GluA1 S831 phosphorylation and inhibiting Na+ channel function. Taken together, these data suggest that IL-16 reduces neuronal excitability and synaptic activity via multiple mechanisms and adds further evidence that alternative receptors may exist for IL-16.

CCR4+T cell recruitment to the skin in mycosis fungoides: potential contributions by thymic stromal lymphopoietin and interleukin-16.

Mycosis fungoides (MF) is characterized by skin accumulation of CCR4+CCR7- effector memory T cells; however the mechanism for their recruitment is not clearly identified. Thymic Stromal Lymphopoietin (TSLP) is a keratinocyte-derived cytokine that triggers Th2 immunity and is associated with T cell recruitment to the skin in atopic dermatitis. Interleukin-16 (IL-16) is a chemoattractant and growth factor for CD4+T cells. We hypothesized that TSLP and IL-16 could contribute to recruitment of malignant T cells in MF. We found elevated TSLP and IL-16 in very early stage patients' plasma and skin biopsies, prior to elevation in CCL22. Both TSLP and IL-16 induced migratory responses of CCR4+TSLPR+CD4+CCR7-CD31+cells, characteristic of malignant T cells in the skin. Co-stimulation also resulted in significant proliferative responses. We conclude that TSLP and IL-16, expressed at early stages of disease, function to recruit malignant T cells to the skin and contribute to their enhanced proliferation.

[Recombinant human IL-16 inhibits proliferation and induces apoptosis in MT-4 cells].

OBJECTIVE: To investigate the effects of recombinant human IL-16 (rhIL-16) on the proliferation and apoptosis of MT-4 cells. METHODS: MT-4 cells were infected with adenovirus expressing rhIL-16 at different time points. Then the proliferation of MT-4 cells was measured by the MTT assay. The apoptosis of the MT-4 cells was evaluated by flow cytometry. The expression of two molecular markers for apoptosis, Bax and Bcl-2, were analyzed by Western blotting. RESULTS: The MTT assay results showed that pAd-IL-16 transfectants had inhibitory effect on the proliferation of MT-4 cells, compared with untreated cells at various time points. Flow cytometry revealed that apoptotic rate of MT-4 cells was higher than that of control group. Furthermore, Western blotting indicated that the Bax protein expression increased while Bcl-2 protein expression decreased in a time-dependent manner in MT-4 cells infected with the rhIL-17-expressing adenovirus. CONCLUSION: These results demonstrated that IL-16 plays an important role in the proliferation and apoptosis of MT-4 cells, and the molecular mechanism underlying this phenomenon may involve the modulation of Bax and Bcl-2 expression.

IL-16 induces intestinal inflammation via PepT1 upregulation in a pufferfish model: new insights into the molecular mechanism of inflammatory bowel disease.

Inflammatory bowel disease (IBD) has long been a worldwide health care problem with a persistently increasing incidence. Although its clinical features have been well described, its etiology and pathogenesis remain unclear. IL-16 is a chemoattractant cytokine with various effects on cellular activities and diseases. However, the involvement of IL-16 in IBD remains poorly understood. In this study, to our knowledge we report for the first time the mechanism by which IL-16 induces intestinal inflammation by upregulating the expression of oligopeptide transporter member 1 (PepT1) in a Tetraodon nigroviridis fish model. The dextran sodium sulfate-induced colitis model in this species revealed that IL-16 levels significantly increase accompanied by elevations in PepT1 in the colon. Moreover, the signs of colitis were dramatically attenuated by IL-16 depletion using anti-IL-16 Abs. In vivo IL-16 administration induced remarkable intestinal inflammation with typical ulcerative colitis-like features, including histologic damage, inflammatory cell infiltration, increased myeloperoxidase activity, and proinflammatory cytokines expression, which corresponded with significant PepT1 upregulation in the colon. The IL-16-induced PepT1 expression and its upregulated fMLF transport were also demonstrated in vitro. To our knowledge, our study provides the first evidence of the connection between IL-16 and PepT1, which provides new insights into the molecular mechanism underlying IBD development. Additionally, this study suggests that fish species are an attractive model for studying IBD. By providing a better understanding of IL-16 biology from fish to mammals, this study should aid the development of IL-16-based therapies for IBD.

Myeloid differentiation primary response protein 88 blockade upregulates indoleamine 2,3-dioxygenase expression in rheumatoid synovial fibroblasts.

Indoleamine 2,3-dioxygenase (IDO) is a key negative regulator of immune responses and has been implicated in tumor tolerance, autoimmune disease and asthma. IDO was detected in the joint synovial tissue in the inflammatory microenvironment of rheumatoid arthritis (RA), but IDO expression in joint synovial tissue is not sufficient to overcome the inflamed synovial environment. This study aimed to unravel the mechanisms involving the failure to activate tolerogenic IDO in the inflamed joint. We demonstrate that both poly (I:C) and lipopolysaccharide (LPS) induce expression of IDO in synovial fibroblasts. However, inflammatory cytokines such as IL-17, TNF-alpha, IL-12, IL-23 and IL-16 did not induce IDO expression. Poly (I:C) appeared to induce higher IDO expression than did LPS. Surprisingly, toll-like receptor (TLR)4-mediated IDO expression was upregulated after depletion of myeloid differentiation primary response protein 88 (MyD88) in synovial fibroblasts using small interfering RNA (siRNA). IDO, TLR3 and TLR4 were highly expressed in synovial tissue of RA patients compared with that of osteoarthritis patients. In addition, RA patients with severe disease activity had higher levels of expression of IDO, TLR3 and TLR4 in the synovium than patients with mild disease activity. These data suggest that upregulation of IDO expression in synovial fibroblasts involves TLR3 and TLR4 activation by microbial constituents. We showed that the mechanisms responsible for IDO regulation primarily involve MyD88 signaling in synovial fibroblasts, as demonstrated by siRNAmediated knockdown of MyD88.

The role of CD4-dependent signaling in interleukin-16 induced c-Fos expression and facilitation of neurite outgrowth in cerebellar granule neurons.

Neuronal interleukin 16 (NIL-16) is the larger neural-specific splice variant of the interleukin-16 (IL16) gene and shows restricted expression to post-mitotic neurons of the mammalian hippocampus and cerebellum. Although the N-terminus of NIL-16 is unique to the neuronal variant, the C-terminus is identical to pro-IL-16, the IL-16 precursor expressed primarily in T-cells. IL-16 was originally described as a proinflammatory cytokine and has diverse immunoregulatory effects which involve signaling through CD4. NIL-16-expressing neurons can secrete IL-16 and may express CD4; moreover, treatment of cultured cerebellar granule neurons (CGCs) with IL-16 increases the expression of c-Fos, an immediate-early gene which transcriptionally regulates genes directing survival, proliferation, and growth. Taken together, we hypothesize that IL-16 functions as a neuroregulatory cytokine which signals through neuronal CD4 receptors. In this study, we investigated the role of CD4 in IL-16-induced c-Fos expression in CGCs, as well as the effects of IL-16 on neuronal survival and growth. We detected components involved in IL-16-signaling in lymphocytes, including CD4 and the associated tyrosine kinase p56(lck), in CGCs using qRT-PCR and immunoblotting. We also show that IL-16 induces c-Fos expression in wild-type CGCs, but not CD4-deficient CGCs or following inhibition of p56(lck). Finally, treatment of CGCs with IL-16 enhanced neurite outgrowth, an effect also observed in CD4-deficient CGCs. Taken together, our results indicate that IL-16-signaling affects neuronal gene expression and growth through CD4-dependent and independent pathways.

IL-16 effects on A549 lung epithelial cells: dependence on CD9 as an IL-16 receptor?

Interleukin-16 (IL-16) is a pro-inflammatory cytokine released by many types of cells found in the lungs, including normal airway and alveolar epithelial cells. Though a chemotactin for CD4(+) cells and eosinophils, IL-16 also modulates their production of factors that influence inflammatory lung diseases, e.g., asthma and allergic rhinitis. To date, little is known about any potential autocrine-like regulatory effects of IL-16. Using a model human alveolar basal epithelial A549 cell line, the present study sought to assess lung epithelial cell responses to IL-16. Potential induced effects on cell growth/function were assessed using MTT reduction, lactate dehydrogenase release, and 5-bromo-2-deoxyuridine incorporation assays. As IL-16 (at locally high levels) can induce CD4(+) cell death via apoptosis, this potential outcome among the A549 cells was also evaluated using TUNEL and changes in expression of caspase-3 and the pro-apoptotic and anti-apoptotic proteins of Bcl-2 family. The data here indicated that IL-16 inhibited A549 cell growth/function and this was associated with a marked increase in apoptosis characterized by DNA fragmentation, activation of caspase-3, and altered pro-apoptotic protein expression. Since lung epithelial cells lack the CD4 that may bind IL-16, it has been suggested that CD9 may act as an alternate receptor for this cytokine (i.e., an IL-16R). Thus, these studies also sought to determine the extent of CD9 expression on A549 cells and if any/all observed IL-16-induced changes were mediated by CD9. Flow cytometric analyses revealed the cells to be CD9(+)CD4(-). However, neutralization of the purported IL-16R with anti-CD9 antibody could not block the cytotoxic/growth inhibiting effects of IL-16. The only exception appeared to be a mitigation of a chemotactic effect of IL-16; however, studies with an equal amount of non-specific antibody (of same isotype as the anti-CD9) revealed this effect to be artefactual. The neutralization study results thus suggest to us that as-yet undefined pathway(s) exist through which IL-16 may act to exert growth inhibiting/apoptosis-inducing effects on A549 cells, a cell line routinely used as a model for lung epithelial cells.

Expression of CD4 on Epstein-Barr virus-immortalized B cells.

Human antigen presenting cells commonly express CD4 but the significance of this phenomenon has not been clarified. We analyzed a panel of Epstein-Barr virus-immortalized B cells (so called lymphoblastoid cell lines, LCL) by using flow cytometry, DNA-microarray analysis, and reverse transcriptase-polymerase chain reaction (RT-PCR). The number of CD4(+) cells varied from cell line to cell line but expression of CD4 was detected by flow cytometry and RT-PCR in all investigated cell lines. To characterize CD4 expressing LCL in more detail, we separated CD4(+) and CD4(-) cells from single cell lines by using immunomagnetic beads. When we cultured sorted CD4(+) and CD4(-) cells, we observed that CD4 expression was stable for several passages. However, the number of CD4(+) cells decreased with time in culture. We never observed that CD4(-) cell lines returned back to a CD4(+) phenotype. DNA-microarray analysis of isolated CD4(+) and CD4(-) cells indicated that the overall gene expression profile of both cell populations was highly similar. In addition, CD4(+) and CD4(-) cells showed the same allostimulatory capacity. CD4(+) LCL showed a slightly increased interleukin-16 induced chemotaxis. Differences in the gene expression profile of CD4(+) and CD4(-) cell lines suggested that loss of CD4 expression occurred during a differentiation step involving achaete-scute complex homolog-like 1.

IL-16 can synergize with early acting cytokines to expand ex vivo CD34+ isolated from cord blood.

We previously reported that interleukin (IL)-16 can induce CD34(+) hematopoietic cells to proliferate and differentiate in vitro into phenotypically and functionally mature dendritic cells. In this study, we investigated the effects of IL-16 on the expansion of CD34(+) cells from human cord blood (CB). CD34(+) CB cells were cultured for 14 days in medium containing a basal cocktail (BC) containing stem cell factor, Flt-3 ligand, thrombopoietin, IL-6, and IL-3 with and without IL-16 as a control. Interleukin-16 added to BC significantly enhanced the expansion of CD34(+) cells (66.47 +/- 1.46-fold vs. 36.23 +/- 1.67-fold), as well as CD34(+)CD38(-) early stem cells (106.67 +/- 2.34-fold vs. 63.42 +/- 1.89-fold) and progenitor cells [colony-forming unit (CFU) -mixed -(GEMM)] and multilineage-committed progenitors [burst-forming unit (BFU-E), CFU-granulocyte, macrophage (-GM), CFU-megakaryocyte (-MK)]. Interleukin-16 also significantly increased long-term culture-initiating cells (160.8 +/- 3.45-fold vs. 83 +/- 2.89-fold with BC alone). Moreover, CD34(+) cells expanded with IL-16 maintained the capacity to differentiate into the lymphoid-B and -NK lineage. The addition of IL-16 to BC increased the migratory capacity of expanded CD34(+) cells compared to BC alone, leaving the expression of CXCR4 unaffected, and decreased the percentage of CD34(+)CD4(+) cells. We showed that IL-16 released endogenously affected the ex vivo expansion of CD34(+) cells. Overall, this study suggests that IL-16 may have a new role in promoting the expansion of hematopoietic stem cells and may represent a new tool for the expansion of CD34(+) cells for clinical applications.

Preferential migration of T regulatory cells induced by IL-16.

As a natural ligand for CD4, IL-16 has been shown to preferentially induce migration in Th1 cells, and, in long-term cultures with IL-2, IL-16 facilitates the expansion of CD4(+)CD25(+) cells. In addition, IL-16 has an immunomodulatory role in asthmatic inflammation, as exogenous administration significantly reduces inflammation and airway hyperreactivity. The mechanism for this, however, is not clear. Based on its functional characteristics and potential immunomodulatory role, we investigated the ability of IL-16 to recruit and influence the development of T regulatory (Treg) cells. We now demonstrate that IL-16 preferentially induces migration in a CD25(+)CTLA-4(+) human T cell subset and that responding cells produce IFNgamma and TGFbeta but not IL-10. These cells are relatively unresponsive to antigenic stimulation and can suppress proliferation and IL-5, but not IFNgamma, production by autologous T cells. We further demonstrate that IL-16-recruited cells are enriched for Forkhead box P3 (Foxp3). In addition, we find that IL-16 stimulation may facilitate de novo induction of Foxp3(+) Treg cells, because the stimulation of FoxP3-negative T cells for 48 h results in the expression of FoxP3 mRNA and protein. These data indicate that at sites of inflammation IL-16 may contribute to selective Treg cell expansion through the preferential induction of a migratory response from existing Treg cells, as well as by the induction of de novo generation of FoxP3(+) cells. These findings offer a potential mechanism for the immunosuppressive effects of IL-16 seen in Th2-mediated inflammation.

Chemokine receptor CXCR3 desensitization by IL-16/CD4 signaling is dependent on CCR5 and intact membrane cholesterol.

Previous work has shown that IL-16/CD4 induces desensitization of both CCR5- and CXCR4-induced migration, with no apparent effect on CCR2b or CCR3. To investigate the functional relationship between CD4 and other chemokine receptors, we determined the effects of IL-16 interaction with CD4 on CXCR3-induced migration. In this study we demonstrate that IL-16/CD4 induced receptor desensitization of CXCR3 on primary human T cells. IL-16/CD4 stimulation does not result in surface modulation of CXCR3 or changes in CXCL10 binding affinity. This effect does require p56(lck) enzymatic activity and the presence of CCR5, because desensitization is not transmitted in the absence of CCR5. Treatment of human T cells with methyl-beta-cyclodextrin, a cholesterol chelator, prevented the desensitization of CXCR3 via IL-16/CD4, which was restored after reloading of cholesterol, indicating a requirement for intact cholesterol. These studies demonstrate an intimate functional relationship among CD4, CCR5, and CXCR3, in which CCR5 can act as an adaptor molecule for CD4 signaling. This process of regulating Th1 cell chemoattraction may represent a mechanism for orchestrating cell recruitment in Th1-mediated diseases.

HIV-1 infection is facilitated in T cells by decreasing p56lck protein tyrosine kinase activity.

Several studies have suggested an important role for the protein tyrosine kinase p56lck (Lck) in HIV infection; however, the exact nature of this role remains unclear. Using a series of well characterized Jurkat-derived cell lines having a wide range of Lck kinase activity, our results showed that, while the entry of HIV-1 into these cell lines was similar, the kinetics of virus production by these cells were very different. Cells expressing a kinase-inactive Lck showed accelerated viral replication, whereas, cells expressing Lck with normal or elevated enzymatic activity showed a delay in virus replication that was proportional to the initial level of endogenous Lck activity. The cell line having the highest initial Lck kinase activity showed the slowest rate of productive HIV-1 infection. Analysis of 2-LTR circles revealed that this inhibitory effect of Lck was not due to inhibition of reverse transcription of HIV-1 genome or migration of the proviral DNA into the nuclei. This affect of Lck was confirmed in additional studies that used either the S1T cell line lacking completely Lck or where the Lck activity was altered in Jurkat cells prior to infection. S1T cells showed a 3- to 12-fold increase in the level of infection compared to Jurkat cells despite similar CD4 and chemokine coreceptor expression and cell doubling times. Pretreatment of Jurkat with an antisense lck oligodeoxynucleotide inhibited the synthesis of functional Lck and facilitated the viral replication by the cells as did expressing a dominant-negative mutant Lck which increased the productive infection>3-fold. Conversely, whereas IL-16 had no affect on productive infection in S1T cells that lack Lck, IL-16 pretreatment of Jurkat cells resulted in an immediate (within 5 min) and sustained and gradual (over 5 h) increase in Lck activity that resulted in a reduction of HIV-1 replication that paralleled the increasing Lck kinase activity. These results show that the enzymatic activity of Lck kinase can affect viral replication, that a lack of, or decreased Lck activity facilitates viral replication. Conversely, Lck can mediate a delay in HIV-1 infection that is proportional to the initial endogenous Lck enzyme activity.

IL-16 promotes leukotriene C(4) and IL-4 release from human eosinophils via CD4- and autocrine CCR3-chemokine-mediated signaling.

Human eosinophils are potential sources of inflammatory and immunomodulatory mediators, including cysteinyl leukotrienes, chemokines, and cytokines, which are pertinent to allergic inflammation. We evaluated the means by which IL-16, a recognized eosinophil chemoattractant, might act on eosinophils to affect their capacity to release leukotriene C(4) (LTC(4)) or their preformed stores of chemokines (eotaxin, RANTES) or Th1 (IL-12) or Th2 (IL-4) cytokines. IL-16 dose dependently (0.01-100 nM) elicited new lipid body formation, intracellular LTC(4) formation at lipid bodies, and priming for enhanced calcium ionophore-activated LTC(4) release. IL-16 also elicited brefeldin A-inhibitable, vesicular transport-mediated release of preformed IL-4, but not IL-12, from eosinophils. CD4 is a recognized IL-16R, and accordingly anti-CD4 Fab, soluble CD4, and a CD4 domain 4-based IL-16 blocking peptide inhibited the actions of IL-16 on eosinophils. Although CD4 is not G-protein coupled, pertussis toxin inhibited IL-16-induced eosinophil activation. IL-16 actions were found to be mediated by the autocrine activity, not of platelet-activating factor, but rather of endogenous CCR3-acting chemokines. IL-16 induced the rapid vesicular transport-mediated release of RANTES. The effects of IL-16 were blocked by CCR3 inhibitors (met-RANTES, anti-CCR3 mAb) and by neutralizing anti-eotaxin and anti-RANTES mAbs, but not by platelet-activating factor receptor antagonists (CV6209, BN52021). RANTES and eotaxin each enhanced LTC(4) and IL-4 (but not IL-12) release. Therefore, IL-16 activation of eosinophils is CD4-mediated to elicit the extracellular release of preformed RANTES and eotaxin, which then in an autocrine fashion act on plasma membrane CCR3 receptors to stimulate both enhanced LTC(4) production and the preferential release of IL-4, but not IL-12, from within eosinophils.

The associated regulators and signal pathway in rIL-16/CD4 mediated growth regulation in Jurkat cells.

IL-16 is a ligand and chemotactic factor for CD4+ T cells. IL-16 inhibits the CD3 mediated lymphocyte activation and proliferation. The effects of IL-16 on the target cells are dependent on the cell type, the presence of co-activators etc. To understand the regulation function and mechanism of IL-16 on target cells, we used a 130 a.a. recombinant IL-16 to study its effects on the growth of Jurkat T leukemia cells in vitro. We found that the rIL-16 stimulated the proliferation of Jurkat cells at low dose (10(-9)M), but inhibited the growth of the cells at higher concentration (10(-5)M). Results showed that 10(-5) M of rIL-16 treatment induced an enhanced apoptosis in Jurkat cells. The treatment blocked the expression of FasL, but up-regulated the c-myc and Bid expression in the cells. Pre-treatment of PKC inhibitor or MEK1 inhibitor markedly increased or decreased the rIL-16 induced growth-inhibiting effects on Jurkat cells, respectively. The results suggested that the rIL-16 might be a regulator for the growth or apoptosis of Jurkat cells at a dose-dependent manner. The growth-inhibiting effects of rIL-16 might be Fas/FasL independent, but, associated with the activation of PKC, up-regulated expression of c-Myc and Bid, and the participation of the ERK signal pathway in Jurkat cells.

Theophylline inhibits TNF-alpha-induced CD4 expression on human eosinophils and CD4+ eosinophil migration.

BACKGROUND: Increasing evidence regarding asthma suggests that CD4+ cells are preferentially recruited to sites of bronchial inflammation. Interleukin (IL)-16 has been reported as playing an important role in the accumulation of CD4+ cells. We have shown that the CD4 molecule is expressed on normal human eosinophils by tumor necrosis factor (TNF)-alpha stimulation. METHODS: We evaluated the effects of theophylline, KF19514 [a selective phosphodiesterase (PDE) IV inhibitor] and dexamethasone on CD4 expression on eosinophils and eosinophil migration in response to IL-16, a natural soluble ligand of the CD4 molecule. RESULTS: The maximum eosinophil migration was observed when eosinophils were cultured with TNF-alpha at 10 ng/ml for 18 h and the concentration of IL-16 was 10 pg/ml. CD4+ eosinophil migration in response to IL-16 was mostly, if not fully, chemokinetic and this migration was significantly inhibited by Fab of anti-CD4 monoclonal antibody. Theophylline (10(-4)-10(-3) M), KF19514 (10(-7)-10(-6) M) and dexamethasone (10(-8)- 10(-6) M) significantly inhibited CD4 expression on eosinophils induced by TNF-alpha. Theophylline (10(-3) M) and KF19514 (10(-6) M) inhibited CD4+ eosinophil migratory responses induced by IL-16, but 10(-6) M dexamethasone did not. Theophylline and KF19514 augmented the intracellular adenosine-3',5'-cyclic monophosphate (cAMP) concentration in eosinophils, suggesting modulation by cAMP of CD4 expression and eosinophil migration. CONCLUSIONS: These data suggest that TNF-alpha-induced CD4+ eosinophils may contribute to eosinophil migratory responses induced by IL-16. Theophylline and selective PDE IV inhibitor may prevent airway inflammation by downregulating CD4 expression on eosinophils and inhibiting eosinophil migration through CD4 and IL-16 interaction.

Interleukin-16 supports the migration of Langerhans cells, partly in a CD4-independent way.

Migration of cutaneous dendritic cells is essential for the induction of primary immune responses. Chemotaxis plays an important part in guiding migrating cells through the skin. Therefore, we investigated the influence of interleukin-16, a potent chemoattractant, on the migratory properties of cutaneous dendritic cells. Interleukin-16 added to murine and human skin explant cultures, enhanced emigration of Langerhans cells as well as dermal dendritic cells out of the skin. In contrast to tumor necrosis factor-alpha, intradermally injected interleukin-16 did not reduce the density of Langerhans cells suggesting a chemotactic rather than a mechanistic migration-inducing effect of interleukin-16. In support of these findings, the known migration-promoting effect of tumor necrosis factor-alpha in skin explant cultures could be neutralized by anti-interleukin-16 antibody and vice versa, indicating different but cooperative ways of action for both cytokines. In whole skin explant cultures blocking of the interleukin-16 effect was also achieved with a monoclonal antibody against CD4, the receptor for interleukin-16. In contrast, in cultures of murine epidermis alone no blocking by anti-CD4 became obvious and in CD4-deficient mice Langerhans cell migration in response to interleukin-16 was maintained. This suggests that another receptor for interleukin-16 might be operative for Langerhans cells in the mouse epidermis. Finally, we detected interleukin-16-positive cells in the dermis of skin explants, tumor necrosis factor-alpha-treated and contact allergen-treated skin. Taken together, it seems likely that locally secreted interleukin-16 might serve to enhance the migration of cutaneous dendritic cells and optimize the response to foreign antigen encountering the skin.

Interleukin (IL)-4/IL-9 and exogenous IL-16 induce IL-16 production by BEAS-2B cells, a bronchial epithelial cell line.

Previous studies have suggested that bronchial epithelial cells may perpetuate airway inflammation. We have reported that the bronchial epithelial cell line BEAS-2B can produce interleukin (IL)-16, a potent chemoattractant for CD4+ T cells. IL-16 is thought to regulate airway inflammation in asthmatics. Recent studies showed that IL-4 induces inflammatory cytokines in bronchial epithelial cells and that IL-9 is a candidate gene for development of asthma. The present study demonstrated that BEAS-2B cells produced specifically IL-16 by synergistic effects of IL-4 + IL-16, or IL-9 + IL-16, and that the synthesized IL-16 induced migration of CD4+ T cells. This study is a first report indicating that IL-16 production may be maintained by an autocrine machinery by epithelial cell-derived IL-16 with IL-4 and IL-9 in asthma.

Immunosuppressive effect on T cell activation by interleukin- 16-cDNA-transfected human squamous cell line.

It is well known that it is difficult to induce an immunotolerance with allogeneic skin transplantation. We attempted to find the immunosuppressive protocol for prolonging skin allograft rejection by using interleukin-16 because IL-16 is considered one of the natural ligands to CD4 molecules. First we examined whether synergistic immunosuppressive effects of recombinant IL-16 plus anti-CD4 mAbs are induced in mixed lymphocyte reaction (MLR). Next we used IL-16-cDNA-transfected OSC-20 (human oral squamous cell carcinoma cell line) as an in vitro model of the epidermal keratinocyte equivalent and examined whether this transfectant could inhibit the activation of allogeneic T cells. Our data indicated that IL-16 clearly inhibited human MLR and that IL-16 increased synergistically the immunosuppressive effect of anti-CD4 mAb. We also used IL-16 transfectant and this produced more than 50 ng/ml of IL-16 in the supernatant by which human MLR was significantly inhibited. Furthermore, this transfectant also inhibited the activation of allogeneic lymphocytes stimulated directly with transfectant cells. These results indicated that the IL-16-producing allogeneic skin graft might have a local immunosuppressive action that would prolong graft survival.

Recombinant human CD40 ligand inhibits simian immunodeficiency virus replication: a role for interleukin- 16.

CD40 ligand (CD40L), expressed on activated T cells, binds its receptor, CD40, on dendritic cells, B cells, and monocytes/ macrophages. Human immunodeficiency virus (HIV)-infected individuals exhibit normal B-cell CD40 expression but diminished expression of CD40L on CD4 + T cells. Thus, we studied recombinant human CD40L (huCD40L) in an in vitro rhesus macaque model of acquired immunodeficiency syndrome (AIDS). huCD40L induced peripheral blood mononuclear cell (PBMC) proliferation independent of mitogenic cytokines and led to a 70% reduction in p27 production by simian immunodeficiency virus (SIV) mac239 infected PBMCs (P < 0.05). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed reduced expression of SIV gag and increased expression of interleukin (IL)-16 mRNA. Supernatants from huCD40L-stimulated PBMC and control cultures contained similar amounts of IL-16, suggesting an intracellular antiviral effect by IL-16. Phytohemagglutinin (PHA)-stimulated PBMCs similarly cultured with huCD40L showed only slight increases in chemokine production (P > 0.05). These results suggest that huCD40L inhibits replication (antigen and mRNA production) of SIVmac239. This response involves huCD40L induction of IL16 mRNA expression and appears to be independent of beta-chemokines.