Anti-Interleukin-16-Neutralizing Antibody Attenuates Cardiac Inflammation and Protects against Cardiac Injury in Doxorubicin-Treated Mice.

BACKGROUND: Interleukin-16 (IL-16) is an important inflammatory regulator and has been shown to have a powerful effect on the regulation of the inflammatory response. Cardiac inflammation has been reported to be closely related to doxorubicin- (DOX-) induced cardiac injury. In this study, the role of IL-16 in DOX-induced cardiac injury and the possible mechanisms were examined. METHODS: Cardiac IL-16 levels were first measured in DOX- or saline-treated mice. Additionally, mice were pretreated with the anti-IL-16-neutralizing antibody (nAb) or isotype IgG for 1 day and further administered DOX or saline for 5 days. Then, cardiac injury, cardiac M1 macrophage levels, and cardiomyocyte apoptosis were analyzed. The effects of the anti-IL-16 nAb on macrophage differentiation and cardiomyocyte apoptosis were also investigated in vitro. RESULTS: DOX administration increased IL-16 expression in cardiac macrophages compared with that of saline treatment. The anti-IL-16 nAb significantly decreased serum levels of lactate dehydrogenase (LDH), myocardial-bound creatine kinase (CK-MB), and cardiac troponin T (cTnT) and elevated cardiac function in DOX-induced mice. Treatment with the anti-IL-16 nAb also reduced p65 pathway activation, decreased M1 macrophage-related marker and cytokine expression, and protected against cardiomyocyte apoptosis in DOX-induced mice. In cell studies, the anti-IL-16 nAb also reduced DOX-induced M1 macrophage differentiation and alleviated apoptosis in cardiomyocytes cocultured with macrophages. CONCLUSIONS: The anti-IL-16 nAb protects against DOX-induced cardiac injury by reducing cardiac inflammation, and IL-16 may be a promising target to prevent DOX-related cardiac injury.

High-Dose Dexamethasone Alters the Increase in Interleukin-16 Level in Adult Immune Thrombocytopenia.

Adult primary immune thrombocytopenia (ITP) is an autoimmune-mediated haemorrhagic disorder. Interleukin-16 (IL-16) can directly affect cellular or humoural immunity by mediating the cellular cross-talk among T cells, B cells and dendritic cells. Several studies have focused on IL-16 as an immunomodulatory cytokine that takes part in Th1 polarization in autoimmune diseases. In this study, we investigated IL-16 expression in the bone marrow supernatant and plasma of ITP patients and healthy controls. What's more, we detected IL-16 expression in ITP patients with the single-agent 4-day high-dose dexamethasone (HD-DXM) therapy. In patients with active ITP, bone marrow supernatant and plasma IL-16 levels increased (P < 0.05) compared with those of healthy controls. In the meantime, the mRNA expression in BMMCs (pro-IL-16, caspase-3) and PBMCs (pro-IL-16, caspase-3 and T-bet) of ITP patients was increased (P < 0.05) relative to those of healthy controls. In patients who responded to HD-DXM therapy, both plasma IL-16 levels and gene expression in PBMCs (pro-IL-16, caspase-3, and T-bet) were decreased (P < 0.05). In summary, the abnormal level of IL-16 plays important roles in the pathogenesis of ITP. Regulating Th1 polarization associated with IL-16 by HD-DXM therapy may provide a novel insight for immune modulation in ITP.

Colitogenic effector T cells: roles of gut-homing integrin, gut antigen specificity and γδ T cells.

Disturbance of T-cell homeostasis could lead to intestinal inflammation. Naive CD4 T cells undergoing spontaneous proliferation, a robust proliferative response that occurs under severe lymphopenic conditions, differentiate into effector cells producing Th1- and/or Th17-type cytokines and induce a chronic inflammation in the intestine that resembles human inflammatory bowel disease. In this study, we investigated the key properties of CD4 T cells necessary to induce experimental colitis. α4ß7 upregulation was primarily induced by mesenteric lymph node (mLN) resident CD11b(+) dendritic cell subsets via transforming growth factor beta (TGFß)/retinoic acid-dependent mechanism. Interestingly, α4ß7 expression was essential but not sufficient to induce inflammation. In addition to gut-homing specificity, expression of gut Ag specificity was also crucial. T-cell acquisition of the specificity was dramatically enhanced by the presence of γδ T cells, a population previously shown to exacerbate T-cell-mediated colitis. Importantly, interleukin (IL)-23-mediated γδ T cell stimulation was necessary to enhance colitogenicity but not gut antigen reactivity of proliferating CD4 T cells. These findings demonstrate that T-cell colitogenicity is achieved through multiple processes, offering a therapeutic rationale by intervening these pathways.

IL-16 promotes T. whipplei replication by inhibiting phagosome conversion and modulating macrophage activation.

The replication of Tropheryma whipplei (the agent of Whipple's disease) within human macrophages is associated with the expression of IL-16, a cytokine known for its chemotactic and inflammatory properties. In this study, we asked whether IL-16 acts on T. whipplei replication by interfering with the endocytic pathway. We observed that in macrophages, T. whipplei was located within late phagosomes that were unable to fuse with lysosomes; in monocytes, T. whipplei was eliminated in phagolysosomes. Moreover, adding IL-16 to monocytes induced bacterial replication and inhibited phagolysosome formation. On the other hand, blocking IL-16 activity, either with anti-IL-16 antibodies in human macrophages or by using murine IL-16(-/-) bone marrow-derived macrophages, inhibited T. whipplei replication and rescued phagolysosome biogenesis. Furthermore, we propose that IL-16-mediated interference with the endocytic pathway is likely related to macrophage activation. First, IFNγ induced T. whipplei elimination and phagolysosome formation and inhibited IL-16 production by macrophages. Second, the full transcriptional response of murine macrophages to T. whipplei showed that T. whipplei specifically modulated the expression of 231 probes in IL-16(-/-) macrophages. Gene Ontology analysis revealed that 10 of 13 over-represented terms were linked to immune responses, including proinflammatory transcriptional factors of the NF-κB family. Our results demonstrated a previously unreported function for IL-16 in promoting bacterial replication through inhibited phagolysosome biogenesis and modulated macrophage activation program.

Neutralization of interleukin-16 protects nonobese diabetic mice from autoimmune type 1 diabetes by a CCL4-dependent mechanism.

OBJECTIVE: The progressive infiltration of pancreatic islets by lymphocytes is mandatory for development of autoimmune type 1 diabetes. This inflammatory process is mediated by several mediators that are potential therapeutic targets to arrest development of type 1 diabetes. In this study, we investigate the role of one of these mediators, interleukin-16 (IL-16), in the pathogenesis of type 1 diabetes in NOD mice. RESEARCH DESIGN AND METHODS: At different stages of progression of type 1 diabetes, we characterized IL-16 in islets using GEArray technology and immunoblot analysis and also quantitated IL-16 activity in cell migration assays. IL-16 expression was localized in islets by immunofluorescence and confocal imaging. In vivo neutralization studies were performed to assess the role of IL-16 in the pathogenesis of type 1 diabetes. RESULTS: The increased expression of IL-16 in islets correlated with the development of invasive insulitis. IL-16 immunoreactivity was found in islet infiltrating T-cells, B-cells, NK-cells, and dendritic cells, and within an insulitic lesion, IL-16 was derived from infiltrating cells. CD4(+) and CD8(+) T-cells as well as B220(+) B-cells were identified as sources of secreted IL-16. Blockade of IL-16 in vivo protected against type 1 diabetes by interfering with recruitment of CD4(+) T-cells to the pancreas, and this protection required the activity of the chemokine CCL4. CONCLUSIONS: IL-16 production by leukocytes in islets augments the severity of insulitis during the onset of type 1 diabetes. IL-16 and CCL4 appear to function as counterregulatory proteins during disease development. Neutralization of IL-16 may represent a novel therapy for the prevention of type 1 diabetes.

A homologous form of human interleukin 16 is implicated in microglia recruitment following nervous system injury in leech Hirudo medicinalis.

In contrast to mammals, the medicinal leech Hirudo medicinalis can completely repair its central nervous system (CNS) after injury. This invertebrate model offers unique opportunities to study the molecular and cellular basis of the CNS repair processes. When the leech CNS is injured, microglial cells migrate and accumulate at the site of lesion, a phenomenon known to be essential for the usual sprouting of injured axons. In the present study, we demonstrate that a new molecule, designated HmIL-16, having functional homologies with human interleukin-16 (IL-16), has chemotactic activity on leech microglial cells as observed using a gradient of human IL-16. Preincubation of microglial cells either with an anti-human IL-16 antibody or with anti-HmIL-16 antibody significantly reduced microglia migration induced by leech-conditioned medium. Functional homology was demonstrated further by the ability of HmIL-16 to promote human CD4+ T cell migration which was inhibited by antibody against human IL-16, an IL-16 antagonist peptide or soluble CD4. Immunohistochemistry of leech CNS indicates that HmIL-16 protein present in the neurons is rapidly transported and stored along the axonal processes to promote the recruitment of microglial cells to the injured axons. To our knowledge, this is the first identification of a functional interleukin-16 homologue in invertebrate CNS. The ability of HmIL-16 to recruit microglial cells to sites of CNS injury suggests a role for HmIL-16 in the crosstalk between neurons and microglia in the leech CNS repair.

Lymphocytes in the development of lung inflammation: a role for regulatory CD4+ T cells in indirect pulmonary lung injury.

Although roles for myelocytes have been suggested in the pathophysiology of indirect acute lung injury (ALI not due to a direct insult to the lung), the contribution of various regulatory lymphoid subsets is unknown. We hypothesized a role for lymphocytes in this process. Using a sequential model of indirect ALI induced in mice by hemorrhagic shock followed 24 h later by polymicrobial sepsis; we observed a specific and nonredundant role for each lymphocyte subpopulation in indirect ALI pathophysiology. In particular, we showed that CD4(+) T cells are specifically recruited to the lung in a dendritic cell-independent but IL-16-dependent process and diminish neutrophil recruitment through increased IL-10 production. Most importantly, this appears to be mediated by the specific subpopulation of CD4(+)CD25(+)Foxp3(+) regulatory T cells. Although indirect ALI has constantly been described as a proinflammatory pathology mediated by cells of the innate immune system, we now demonstrate that cells of the adaptive immune response play a major role in its pathophysiology as well. Most importantly, we also describe for the first time the nature of the regulatory mechanisms activated in the lung during indirect ALI, with CD4(+) regulatory T cells being central to the control of neutrophil recruitment via increased IL-10 production.

Generation of bleomycin-induced lung fibrosis is independent of IL-16.

Given that CD4+ cells are found in the lungs of patients with fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF) we hypothesized that IL-16, a potent chemoattractant for CD4+ cells, may be involved in the pathogenesis of this disease. We found that baseline IL-16 gene expression is greater in fibroblasts isolated from IPF patients compared to non-fibrotic fibroblasts. Furthermore, IL-16 gene expression increased in IPF fibroblasts following stimulation with either of the pro-fibrotic growth factors TGFb1 or PDGF. In contrast, PDGF had no effect on IL-16 gene expression in non-fibrotic lung fibroblasts, whereas TGFb1 down-regulated IL-16 gene expression in non-fibrotic fibroblasts. To gain a better understanding of an association of IL-16 with fibrosis, we used the bleomycin-induced mouse model of fibrosis to examine IL-16 gene expression. Our current study demonstrates that IL-16, and its activator caspase 3, are highly expressed at the mRNA level in the lungs of mice prior to the deposition of collagen following intratracheal bleomycin administration. We then sought to determine the role of IL-16 in the generation of fibrosis in the mouse by using IL-16KO mice. There were no differences observed between IL-16WT and IL-16KO mice (cellular infiltrate, collagen deposition, total lung collagen generation and cytokine expression) following bleomycin instillation. These results indicate that IL-16 is prominently expressed in both murine and human fibrosis however as complete loss of this cytokine did not modulate pulmonary fibrosis, IL-16 is a candidate biomarker for IPF.

CD4 expression on activated NK cells: ligation of CD4 induces cytokine expression and cell migration.

NK cells play an important role in the innate immune response. We have isolated NK cells from human lymphoid tissues and found that these cells express the CD4 molecule on their surface at levels higher than those found on peripheral blood NK cells. To study the functional role of the CD4 molecule on NK cells, we developed an in vitro system by which we are able to obtain robust CD4 expression on NK cells derived from blood. CD4+ NK cells efficiently mediate NK cell cytotoxicity, and CD4 expression does not appear to alter lytic function. CD4+ NK cells are more likely to produce the cytokines gamma-IFN and TNF-alpha than are CD4- NK cells. Ligation of CD4 further increases the number of NK cells producing these cytokines. NK cells expressing CD4 are also capable of migrating toward the CD4-specific chemotactic factor IL-16, providing another function for the CD4 molecule on NK cells. Thus, the CD4 molecule is present and functional on NK cells and plays a role in innate immune responses as a chemotactic receptor and by increasing cytokine production, in addition to its well-described function on T cells as a coreceptor for Ag responsive cell activation.

Not-so-sweet sixteen: the role of IL-16 in infectious and immune-mediated inflammatory diseases.

Over the past two decades, our understanding of interleukin-16 (IL-16) has increased substantially. Initial studies characterizing IL-16 as a chemotactic cytokine (but not a chemokine) just scratched the surface of the unique properties of this cytokine. Since then, scientists have determined that IL-16 has a wide range of effects on cells, including upregulation of CD25, induction of cells to progress to the G(1) phase, inhibition of antigen- specific proliferation yet with retained antigen nonspecific proliferative properties, and discovery of a novel neuronal form with unique properties. Recently, a plethora of studies have implicated IL-16 in exacerbation of infectious, immune-mediated, and autoimmune inflammatory disorders, including atopic dermatitis, irritable bowel syndrome, systemic lupus erythematosus, neurodegenerative disorders, and viral infections. Herein, we review the body of evidence supporting a role for IL-16 in infectious and immune-mediated inflammatory disorders and explore the known and possible mechanism of actions in the numerous diseases.

Leukotriene A4 hydrolase expression in PEL cells is regulated at the transcriptional level and leads to increased leukotriene B4 production.

Primary effusion lymphoma (PEL) is a herpesvirus-8-associated lymphoproliferative disease characterized by migration of tumor cells to serous body cavities. PEL cells originate from postgerminal center B cells and share a remarkable alteration in B cell transcription factor expression and/or activation with classical Hodgkin's disease cells. Comparative analysis of gene expression by cDNA microarray of BCBL-1 cells (PEL), L-428 (classical Hodgkin's disease), and BJAB cells revealed a subset of genes that were differentially expressed in BCBL-1 cells. Among these, four genes involved in cell migration and chemotaxis were strongly up-regulated in PEL cells: leukotriene A4 (LTA4) hydrolase (LTA4H), IL-16, thrombospondin-1 (TSP-1), and selectin-P ligand (PSGL-1). Up-regulation of LTA4H was investigated at the transcriptional level. Full-length LTA4H promoter exhibited 50% higher activity in BCBL-1 cells than in BJAB or L-428 cells. Deletion analysis of the LTA4H promoter revealed a positive cis-regulatory element active only in BCBL-1 cells in the promoter proximal region located between -76 and -40 bp. Formation of a specific DNA-protein complex in this region was confirmed by EMSA. Coculture of ionophore-stimulated primary neutrophils with BCBL-1 cells leads to an increased production of LTB4 compared with coculture with BJAB and L-428 cells as measured by enzyme immunoassay, demonstrating the functional significance of LTA4H up-regulation.

Human and mouse mast cells use the tetraspanin CD9 as an alternate interleukin-16 receptor.

Interleukin-16 (IL-16) induces the chemotaxis and activation of mast cells (MCs) and other cell types. While it has been concluded that CD4 is the primary IL-16 receptor on T cells, at least one other IL-16 receptor exists. We now show that the IL-16-responsive human MC line HMC-1 lacks CD4, and that the IL-16-mediated chemotactic and Ca2+ mobilization responses of this cell can be blocked by anti-CD9 monoclonal antibodies (mAbs) but not by mAbs directed against CD4 or other tetraspanins. Anti-CD9 mAbs also inhibited the IL-16-mediated activation of nontransformed human cord blood-derived MCs and mouse bone marrow-derived MCs by 50% to 60%. The chemotactic response of HMC-1 cells to IL-16, as well as the binding of the cytokine to the cell's plasma membrane, was inhibited by CD9-specific antisense oligonucleotides. CD9 is therefore essential for the IL-16-mediated chemotaxis and activation of the HMC-1 cell line. In support of this conclusion, IL-16 bound to CD9-expressing CHO cell transfectants. The ability of wortmannin and xestopongin C to inhibit the IL-16-mediated chemotactic response of these cells suggests that the cytokine activates a phosphatidylinositol 3-kinase (PI3K)/inositol trisphosphate-dependent signaling pathway in MCs. This is the first report of a tetraspanin that plays a prominent role in a cytokine-mediated chemotactic response of human MCs.

CD8+ T lymphocytes regulate the arteriogenic response to ischemia by infiltrating the site of collateral vessel development and recruiting CD4+ mononuclear cells through the expression of interleukin-16.

BACKGROUND: Previous studies have demonstrated that macrophages and CD4+ T lymphocytes play pivotal roles in collateral development. Indirect evidence suggests that CD8+ T cells also play a role. Thus, after acute cerebral ischemia, CD8+ T cells infiltrate the perivascular space and secrete interleukin-16 (IL-16), a potent chemoattractant for monocytes and CD4+ T cells. We tested whether CD8+ T lymphocytes contribute to collateral vessel development and whether the lack of circulating CD8+ T cells prevents IL-16 expression, impairs CD4+ mononuclear cell recruitment, and reduces collateral vessel growth after femoral artery ligation in CD8(-/-) mice. METHODS AND RESULTS: After surgical excision of the femoral artery, laser Doppler perfusion imaging demonstrated reduced blood flow recovery in CD8(-/-) mice compared with C57/BL6 mice (ischemic/nonischemic limb at day 28, 0.66+/-0.04 versus 0.87+/-0.04, respectively; P<0.01). This resulted in greater calf muscle atrophy (mean fiber area, 785+/-68 versus 1067+/-69 microm2, respectively; P<0.01) and increased fibrotic tissue content (10.8+/-1.2% versus 7+/-1%, respectively; P<0.01). Moreover, CD8(-/-) mice displayed reduced IL-16 expression and decreased CD4+ T-cell recruitment at the site of collateral vessel development. Exogenous CD8+ T cells, infused into CD8(-/-) mice immediately after femoral artery ligation, selectively homed to the ischemic hind limb and expressed IL-16. The restoration of IL-16 expression resulted in significant CD4+ mononuclear cell infiltration of the ischemic limb, faster blood flow recovery, and reduced hindlimb muscle atrophy/fibrosis. When exogenous CD8+ T cells deficient in IL-16 (IL-16(-/-)) were infused into CD8(-/-) mice immediately after femoral artery ligation, they selectively homed to the ischemic hind limb but were unable to recruit CD4+ mononuclear cells and did not improve blood flow recovery. CONCLUSIONS: These results demonstrate that CD8+ T cells importantly contribute to the early phase of collateral development. After femoral artery ligation, CD8+ T cells infiltrate the site of collateral vessel growth and recruit CD4+ mononuclear cells through the expression of IL-16. Our study provides further evidence of the significant role of the immune system in modulating collateral development in response to peripheral ischemia.

Increased levels of bioactive IL-16 correlate with disease activity during relapsing experimental autoimmune encephalomyelitis (EAE).

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T-cell mediated disease, which resembles immunopathology of multiple sclerosis (MS). Interleukin (IL)-16 is a CD4+ cell-specific chemoattractant cytokine. In CD4+ T cells, production of bioactive IL-16 from constitutive pro-IL-16 requires cleavage by active caspase-3. We reported reversal of established relapsing disease by IL-16 neutralization. To better understand role(s) of IL-16 in regulation of relapsing EAE, we comparatively analyzed levels of IL-16, active caspase-3 and CD4 in mice with severe relapsing-remitting [(B6xSJL) F1], and low-relapsing (B6), disease. Elevated levels of IL-16 along with an increase in active-caspase-3 and CD4 levels correlated with stages of clinically active disease in both strains. CNS levels of bioactive IL-16 were notably higher in F1 compared to B6 mice at all stages, being most prominent during relapse. Similar patterns of regulation for IL-16 and active caspase-3 were observed in peripheral lymphoid organs, and in T cells isolated from lymph nodes following T-cell activation in vitro. IL-16 was co-immunoprecipitated with CD4 from CNS of relapsing mice. Our data suggest that caspase-3 mediated production of IL-16 by infiltrating CD4+ T cells, contributes to ongoing neuroinflammation by chemoattraction of additional waves of CD4+ T cells.

IL-16 is critical for Tropheryma whipplei replication in Whipple's disease.

Whipple's disease (WD) is a rare systemic disease caused by Tropheryma whipplei. We showed that T. whipplei was eliminated by human monocytes but replicated in monocyte-derived macrophages (Mphi) by inducing an original activation program. Two different host molecules were found to be key elements for this specific pattern. Thioredoxin, through its overexpression in infected monocytes, was involved in bacterial killing because adding thioredoxin to infected Mphi inhibited bacterial replication. IL-16, which was up-regulated in Mphi, enabled T. whipplei to replicate in monocytes and increased bacterial replication in Mphi. In addition, anti-IL-16 Abs abolished T. whipplei replication in Mphi. IL-16 down-modulated the expression of thioredoxin and up-regulated that of IL-16 and proapoptotic genes. In patients with WD, T. whipplei replication was higher than in healthy subjects and was related to high levels of circulating IL-16. Both events were corrected in patients who successfully responded to antibiotics treatment. This role of IL-16 was not reported previously and gives an insight into the understanding of WD pathophysiology.

Pro-IL-16 regulation in activated murine CD4+ lymphocytes.

Prior DNA microarray studies suggested that IL-16 mRNA levels decrease following T cell activation, a property unique among cytokines. We examined pro-IL-16 mRNA and protein expression in resting and anti-CD3 mAb-activated primary murine CD4(+) T cells. Consistent with the microarray reports, pro-IL-16 mRNA levels fell within 4 h of activation, and this response is inhibited by cyclosporin A. Total cellular pro-IL-16 protein also fell, reaching a nadir at 48 h. Pro-IL-16 comprises a C-terminal cytokine domain and an N-terminal prodomain that are cleaved by caspase-3. Pro-IL-16 expressed in transfected tumor cells was previously shown to translocate to the nucleus and to promote G(0)/G(1) arrest by stabilizing the cyclin-dependent kinase inhibitor p27(Kip1). In the present study, we observed increased S-phase kinase-associated protein 2 mRNA expression in IL-16 null mice, but basal expression and activation-dependent regulation of p27(Kip1) were no different from wild-type mice. Stimulation with anti-CD3 mAb induced transiently greater thymidine incorporation in IL-16-deficient CD4(+) T cells than wild-type controls, but there was no difference in cell survival or in the CFSE dilution profiles. Analysis of CD4(+) T cell proliferation in vivo using BrdU labeling similarly failed to identify a hyperproliferative phenotype in T cells lacking IL-16. These data demonstrate that pro-IL-16 mRNA and protein expression are dynamically regulated during CD4(+) T cell activation by a calcineurin-dependent mechanism, and that pro-IL-16 might influence T cell cycle regulation, although not in a dominant manner.