Role of interleukin-16 in human diseases: a novel potential therapeutic target.

All major types of immune cells express IL-16. Overview of data is presented in Table 1 . Expression of IL-16 in different immune cell types. Ref., references; ND, no data. Human T cells are the most studied source and target of IL-16. T cell activation by antigens, mitogens or vasoactive amines (like histamine or serotonin) may result in activation of casp-3 and cleavage of mature IL-16, which can be secreted. This process was described in both CD4+ and CD8+ T cells. While casp-3 is constantly active in CD8+ cells, continuously produced and cleaved IL-16 is stored in secretory vesicles, activation of casp-3 in CD4+ T cells must be induced by a stimuli, and thereafter IL-16 can be cleaved ( 34 ). Interestingly, it may take different amount of time from cell stimulation to IL-16 secretion depending on what stimuli the CD8+ cell receives: for histamine and serotonin it takes several hours, while for antigens and mitogens it requires 12–24 hours. Importantly, addition of transcription and translation inhibitors may block IL-16 secretion from CD4+, but not from CD8+ cells (reviewed in ( 4 )). In vitro , IL-16 treatment of human peripheral CD4+T lymphocytes induces upregulation of IL-2Ra (CD25) and IL-2Rb within 24 h, enhancing IL-2 mediated T cell proliferation ( 9 , 47 ). Binding of bioactive IL-16 to CD4 preferentially induces chemotaxis of CD4+Th1 type T cells, rather than Th2 type ( 24 ). In the presence of CCR5 the response to IL-16 is enhanced, while IL-16 could not bind to CCR5 without CD4. CD4+CCR5+ cells are the typical Th1cells that are prevalent at the sites of inflammation and this mechanism likely plays a pivotal role for recruitment and activation of T cells ( 24 ). After the cells attraction the CCR5 receptor is desensitized, possibly in order to terminate the process. Interestingly, the pre-treatment of cells with MIP-1β, SDF-1α/CXCR4 resulted in loss of responsiveness to IL-16 stimulation ( 23 ). In a diabetes mellitus (DM) model, T cells are attracted to the place of insulitis via CCL4, and inhibition of CCL4 and IL-16 were protective from DM ( 48 ). Peripheral B cells express IL-16 at different proportions, ranging widely from 1.5%-94.4% ( 38 ). B cells express IL-16 mRNA and synthesize bioactive IL-16 protein, as observed in lymph node follicles ( 39 ). B cell supernatants induce migration of CD4+ Th cells, monocyte-derived DCs, and circulating DCs, which can be blocked via neutralization of IL-16. IL-16 seems important B-cell derived chemotactic factor involved in cellular cross-talk among T lymphocytes and DCs and their recruitment ( 39 ). Neutrophils express IL-16 and are active secretors of IL-16, while IL-16 is a potent attractor of neutrophils to the site of inflammatory damage ( 40 ). In addition, secondary necrotic neutrophils may passively release interleukin-16 ( 41 ). Human CD14+ monocytes constitutively express IL-16. If unstimulated within 6–8 h in vitro monocytes undergo apoptosis due to activation of casp-3, and release IL-16. It is not clear what is the stimulus and mechanism of this spontaneous release ( 42 ). Monocyte cell line THP-1 infected with methicillin-resistant staphylococcus aureus (MRSA) secrete IL-16 ( 43 ), which can be blocked with anti-epidermal growth factor receptor (EGFR), anti-FasL antibodies (Ab), while pan-casp inhibitor (Z-VAD-FMK) or casp-3 inhibitors ( 43 ). In MRSA infected mouse lungs infiltrating immune cells express IL-16 and neutralizing anti-IL-16 Ab improved clearance of MRSA pneumonia in mice ( 43 ). In vitro , stimulation with IFN-γ+LPS induce IL-16 mRNA upregulation in THP-1 monocyte cell line. Incubation of M0 macrophages with rIL-16 (150ng/ml) increase phagocytosis by 150%, and skew cytokine expression with upregulated IL-1α, IL-6 and IL-12, but downregulate IL-10; without affecting THP-1 cell proliferation ( 49 ). Interleukin-16 has an important role in DC differentiation. In vitro IL-16 can induce human cord blood cells and CD34(+) hematopoietic cells to proliferate and differentiate into phenotypically and functionally mature DCs ( 50 ). Monocyte exposure to IL-16 and thrombopoetin may lead to monocyte maturation to DCs ( 51 ). In atopic dermatitis IgE-bound allergens are presented to epidermal LCs via the high affinity IgE receptor, FcϵRI and induces expression of IL-16, what results in chemoattraction of DCs, CD4+ T cells and eosinophils ( 44 ). Importantly casp-1, but not casp-3, further process IL-16 in epidermal DCs. Mast cells express CD9 and lack CD4 receptor. Ligation of CD9 by IL-16 on MCs induces their chemotaxis and activation ( 25 ). Cell treatment with anti-CD9 mAbs inhibit the IL-16–mediated chemotactic response of human (H) MC-1 line and its activation, same effects were observed on non-transformed human cord blood–derived MCs and mouse bone marrow–derived MCs by 50% to 60% ( 25 ). NK cells can also express and produce IL-16, and smokers display decreased levels of IL-16 in circulating NK cells. What biological function IL-16 mediates in NK cells requires further studies ( 38 ). Interleukin-16 (IL-16) is known as a highly potent chemotactic and chemoattractant molecule for eosinophils ( 45 ). Tissue infiltrating eosinophils in eosinophilic conditions such as chronic eosinophilic rhinitis (ECRS) express IL-16 protein in parallel to other infiltrating cells such as MCs, lymphocytes and tissue epithelial cells. Data suggest that IL-16 may stimulate the migration and persistence of activated eosinophils in ECRS ( 46 ). Keratinocytes and intestinal and respiratory epithelial and endothelial cells express IL-16 ( 52 ). The IL-16 ligand on these cells is CD9 receptor ( 15 , 34 , 35 ). What is the IL-16 function in or on these cells is not known. Data indicate that IL-16 can be spontaneously released by unstimulated PBMCs in samples drawn and kept in rooms temperature. Importantly, this significant increase is observed in EDTA plasma, slighter increase in citrate plasma, but not at all in serum ( 53 ). Investigators suggested that abundant release of IL-16 in EDTA plasma could be due to presence of granulocytes as they are abundant in EDTA but not in citrate, and absent in serum. Investigators suggested that measuring IL-16 (and IL-8) in plasma could be an indicator of quality control if the samples were handled correctly prior to analysis. From the biological point of view these findings raise a relevant question: what environmental factor triggers release of IL-16 and if this process serves any physiologic function.

As reviewed above, circulating levels of IL-16 are increased in multiple autoimmune diseases, which have also been associated with impaired expression and regulation of both casp-3 and IL-16 ( 98 ). Thus, there could be alternative mechanisms of IL-16 cleavage and release. A recent study in endometriosis demonstrated that IL-16 can be cleaved and released during iron overload induced pyroptotic cell death via non-classical GASDME mediated pathway ( 70 ). Interestingly, pyroptosis is implicated in multiple autoimmune diseases and common triggers of autoimmunity such as immune-complexes, secondary necrotic cell debris, extracellular damaged DNA could possibly trigger pyroptosis, and hypothetical IL-16 release ( Figure 2 ) ( 100 ). In cancer, the expression of GASDME is lower in most tumor cells due to the epigenetic inactivation caused by methylation, while chemotherapy may activate casp-3, GADSME expression and facilitate tumor cell pyroptosis ( 101 , 102 ). It is not clear if cancer pyroptotic cell death results in release of cleaved IL-16, as was proven in the case in endometriosis ( Figure 2 ) ( 70 ).

The human IL-16 gene is encoded on chromosome 15q26.3, is 153 kilobase pair (kbp) long and consists of seven exons and six introns ( 1 ). IL-16 protein is highly conserved among species. Two translated IL-16 proteins have been found in humans: one is restricted to the central nervous system, entitled neuronal NIL-16 (130 kilo daltons (kDa) or 141kD according different sources) ( 2 ). The 80 kDa IL-16 is found in many other organ tissues, particularly lymphoid, and is generated as a 631-amino-acid precursor IL-16 molecule. After cell stimulation caspase-3 (casp-3) may cleave IL-16 at residue asparagine 253 into 66kDa N terminal also called pro-IL-16 and 14kDa C terminal mature IL-16 ( Figure 1A ) ( 3 – 5 ). IL-16 is highly conserved, up to 96-98%, across the species close to humans such as Rhesus, Macaques and monkeys ( 3 , 6 ).It is reported that functional activity of the secreted mature IL-16 is localized at the hydrophylic region of the carboxyterminal, and before secretion 14kDA chains form 56kDa homotetramers ( Figure 1B ) ( 6 ). The N terminus of IL-16 can be located in either the cytosol or the nucleus of the cells and may regulate cell growth ( 7 , 8 ). Cleaved or mature IL-16 is supposed to be secreted; however, certain cell types (i.e. cluster differentiation (CD) 8+ T cells) may contain cytoplasmic stores of mature, bioactive IL-16 which can be released upon stimulation, saving time from new synthesis ( 9 , 10 ). Secretion of bioactive IL-16 is regulated by mitogen-activated protein (MAP) kinase and has been postulated to be likened to secretion of IL-1β following cleavage by caspase-1 ( 11 ). IL-16 mRNA is regulated in calcineurin dependent manner ( 12 ). Casp-3 activation and IL-16 cleavage is not necessarily associated with the induction of apoptosis, but recently release of IL-16 has been described in endometriosis via Gasdermin (GASDM) E pyroptotic pathway ( Figure 2 ) ( 9 , 13 ). (A) Structure of IL-16 protein. (B) Processing of IL-16 within a cell. The cytoplasmic IL-16 is cleaved by active caspase-3 and mature C terminal IL-16 forms homo-tetramers than can be secreted and ligate its receptors CD4, CD9 or CCR5. N terminal pro-IL-16 enters nucleus and regulate cell cycle. Schematic illustration of classical pyroptosis pathway (left) and a relatively novel gasdermin-E (GSDME) pyroptosis pathway (right) resulting in IL-16 cleavage and release. The classical pyroptosis pathway is induced by recognition of danger associated molecular patterns (DAMPS), activation of inflammasome, cleavage of procaspase-1 to caspase -1, subsequent cleavage of GASDM-E and pro-IL-1b/18, multimerization of GASDME-N terminal products and a cell pore formation and release of proinflammatory mediators including IL-1b/18. The GASDME pathway can be activated by several stimuli including granzyme A and B, iron overload, extrinsic and intrinsic death receptors, and mitochondrial damage. Physiologically IL-16 is expressed by cells of lymphoid organs: bone marrow, spleen, lymph nodes, thymus, tonsils and appendix, while tissues of the lower gastrointestinal tract express IL-16, but at a lower level. The majority of other tissues express mRNA for IL-16, and presumably can translate and secrete IL-16 under certain conditions. Among other tissue cells IL-16 has been found in cutaneous, cardial, renal fibroblasts and bronchial epithelial cells ( 14 – 16 ). The majority of the immune cell types can express IL-16 protein. The CD4+ and CD8 positive T cells are the best studied expressors of IL-16, but also neutrophils, B-cells, eosinophils, dendritic cells, mast cells, macrophages and monocytes, natural killer cells all are found to express IL-16 ( 9 , 17 – 20 ). Microglial cells and astrocytes are also found to express IL-16, while neurons did not show IL-16 expression ( 21 ). It seems that IL-16 is a rapid response cytokine in certain cell types, for example in CD8+ T cells, eosinophils and also mast cells, where circulating cells carry an already pre-synthesized protein and are ready to release it immediately after stimulation with vasoactive amines, such as serotonin or histamine ( 22 ). Reports indicate that same types of cells express and secrete IL-16 in various categories of human diseases including infectious, inflammatory diseases and cancers. The overall data point that levels of circulating IL-16 are increased in the investigated diseases. Knowledge on what cell types are the producers and responders in respective conditions are limited and is covered in more details below. Data indicates that mechanisms which tune IL-16 are of importance and modulation might have therapeutic effect. Several receptors for IL-16 have been identified: CD4, CD9 and CCR5 ( 3 , 23 , 24 ). CD4 is regarded as the major receptor as it is expressed by a range of hematopoietic and immune cells, as well on neuronal cells ( 3 ). The main ligand of IL-16 is D4 domain of CD4 ( 3 ). Many types of lymphoid cells including T cells, eosinophils, dendritic cells, B cells, as well as mast cells, macrophages and monocytes respond to IL-16, but it is not completely clear what receptors IL-16 binds ( 15 , 19 , 24 – 28 ). In the spinal cord CD4 is expressed in microglia, located close to astrocytes and neurons ( 29 ). The best described cellular function of IL-16 is mediated by CD4 binding, which induces cell migration via chemoattraction. Upon binding to the D4 domain of CD4, IL-16 activates the CD4-associated src-related tyrosine kinase p56lck via autocatalysis ( 30 ). Protein kinase C subsequently translocate to the membrane, inducing downstream signaling pathways in the target cell, including a rise in intracellular calcium, inositol trisphosphate (IP) 3 and phosphoinositide 3-kinase (PI3K) activation ( 30 , 31 ). Additionally, IL-16 has also been shown to activate signal transducer and activator of transcription (STAT) 6 following interaction with CD4, though downstream signaling pathways and subsequent cellular responses have not yet been identified ( 31 ). Co-ligation of C-C chemokine receptor type 5 (CCR5) enhances cell response to the IL-16 signaling, and importantly CCR5 is preferentially expressed on T helper (Th) 1 cells, what seems to enhance migratory IL-16 effects on these cells ( 24 ). Findings indicate that before secretion, IL-16 preferentially forms multimers (homodimers or homo-tetramers) to mediate its effects more efficiently ( 4 ). Multimerization of IL-16 facilitates crosslinking of CD4 and CCR5 on the target cells and enhances cell signaling ( 4 ). Several studies have shown that IL-16/CD4 ligation may result in desensitization for other chemokine receptors, such as CCR5-, C-X-C chemokine receptor type (CXCR) 3 and CXCR4-induced migration ( 32 ), impairing macrophage inflammatory protein-1 beta (MIP-1b) and stromal cell-derived factor 1 (SDF-1a) mediated T cell chemoattraction ( 23 , 24 , 32 , 33 ). In the presence of IL-16, the chemokine ligands for receptors including C-C motif chemokine ligand 5 (CCL5 or RANTES), MIP-1a, MIP-1, and SDF-1a are unable to recruit T cells, which indicates that IL-16 may direct what cells should be recruited ( 9 ). The cells of monocyte and epithelial origin may respond to IL-16 via CD9 receptor as demonstrated in vitro on primary cells, cell lines and also in mouse models ( 15 , 25 , 34 , 35 ). Both the N-terminal pro-IL-16 and the secreted mature IL-16 may impact cell growth. Cleaved N-terminal or pro-IL-16 fragment translocate into the nucleus and has been suggested to serve as cell cycle regulator ( 10 ). Repeated studies confirmed that pro-IL-16 has a cell cycle regulator function in T cells: high nuclear expression of pro-IL-16 induces cell cycle arrest G0/G1, inhibit cell proliferation and is associated with increased levels of cyclin-dependent kinase inhibitor p27Kip1 ( 36 ). During T cell activation, mRNA of IL-16 is downregulated and less IL-16 is observed in cell nuclei, which may result in expression of S-phase kinase-associated protein 2 (Skp2), degradation of p27Kip1 and enable the cell to enter cell cycle ( 36 ). It could render stimulated and pre-exposed T cells refractory to antigenic stimulation, promoting anergy ( 37 ).

Several polymorphisms of IL-16 gene have been associated with cancer and autoimmune diseases. Two meta-analysis confirmed association of cancers with rs11556218T>G, and one meta-analysis confirmed association of cancers with rs4778889T>C polymorphisms. The rs11556218T>G polymorphism was also associated with the risk of cardiovascular disease in Chinese population ( 103 , 104 ). Genetic studies in autoimmune diseases found associations with polymorphisms in the same loci, but did not reach genome-wide association study power and will not be reviewed in this paper.

Neutralization of cytokines with in vitro produced anti-cytokine Abs has been used in daily practice in rheumatology and cancer care over the last several decades. It could be one of the modalities chosen for therapeutic inhibition of IL-16 function, as in experimental animal models described in section 6 anti-IL16 neutralizing Abs showed positive results ( 48 , 74 , 84 , 99 , 105 ). An alternative approach could be to block IL-16 receptor with anti-receptor Abs. The inhibition of CD4 receptor is not possible, as this receptor is vital for its role in protection against infections. However inhibition of CD9 or CD4 co-receptor CCR5 could be novel potential target. Hypothetically inhibition of CCR5 could reduce the potency of IL-16 stimulus to CD4 and could still guarantee the vital IL-16 signal. Data on hypothesis of possible therapeutic interventions is summarized in graphical abstract ( Figure 3 ). Graphical abstract. Panel (A) illustrates what immune cells express IL-16 and its receptors. ? – indicates that it is not clear if the cell type expresses any receptor for IL-16. NK - natural killer cell, Th1 – T helper 1 cell. Panel (B) illustrates overview of IL-16 roles in inflammation, infection and cancer (left) and possible outcomes of therapeutic IL-16 inhibition (right): neutralization of extracellular IL-16 could attenuate inflammation and cell recruitment to the inflammatory sites (upper panel); there is no clear information whether IL-16 targeting could benefit infections (middle panel); in cancers IL-16 inhibition could allow anti-tumour immune response and possibly block cell cycle progression (lower panel). In conclusion, the IL-16 protein has multiple functions in the human immune system, and impaired protein regulation or function or genetic polymorphisms are associated with a variety of life-threatening acute and chronic conditions. Overall data in inflammatory diseases indicate that inhibition of IL-16 could attenuate inflammatory responses and reduce recruitment of target cells to the inflammatory sites. In cancers, loss of nuclear IL-16 leads to hyperproliferative states, which are more difficult to target, but neutralization of secreted IL-16 could improve outcomes of chemotherapy and modulate tumor escape mechanism.

So far inhibition of IL-16 has been addressed only in animal models. In a model of spontaneous diabetes in NOD female mice, blockade of IL-16 with neutralizing Abs prevented insulitis, destruction of insulin-producing pancreatic islets and inhibited development of type 1 diabetes ( 48 ). Experimental autoimmune encephalomyelitis (EAE) is the typical model to study multiple sclerosis. In this model the majority of the immune cells infiltrating central nervous system express IL-16. Blockade of IL-16 with neutralizing Ab led to successful outcomes - reversed paralysis, ameliorated relapsing disease, reduced infiltration by CD4+ T cells and demyelination ( 74 ). In models of sepsis or cardiac injury, blockade of IL-16 reduced mortality and improved survival and reduced damage via activation of antioxidant pathways ( 84 , 105 ). Importantly neutralization of IL-16 proved adjuvant effect on novel cytostatic drugs and could improve outcomes in oncologic diseases ( 99 ).