Il-9 stimulates release of chemotactic factors from human bronchial epithelial cells.

Interleukin (IL)-9 is a T helper 2 cytokine implicated as a candidate gene and contributor to human asthma. We hypothesized that the inflammatory potential of bronchial epithelium is affected by its local environment and explored this hypothesis with respect to the effect of IL-9 on bronchial epithelium. We investigated the response of primary and immortalized human bronchial epithelial cells to IL-9 stimulation with respect to the release of T-cell chemoattractant factors. In response to IL-9, the HBE4-E6/E7 cell line, but not BEAS-2B cells, released the T-cell chemoattractants IL-16 and regulated on activation, normal T cells expressed and secreted (RANTES) in a dose-dependent fashion. We found a similar dose response to IL-9 in primary cells from bronchial brushings of healthy subjects and that nearly all of the T-cell chemoattraction was attributable to IL-16 and RANTES. Reverse transcriptase/polymerase chain reaction of BEAS-2B, HBE4-E6/E7, and primary cells from two subjects revealed messenger RNA for IL-9 receptor (IL-9R) alpha but not in BEAS-2B cells. Fluorescence-activated cell sorter analysis of HBE4-E6/E7 and primary cells confirmed surface expression of the IL-9 receptor. Costimulation of both cell types with IL-9 and antibody to either gamma-common chain or IL-9Ralpha completely blocked the release of T-cell chemoattractant activity, confirming the primary role of a functioning IL-9 receptor for IL-9 signaling in HBE4-E6/E7 and primary bronchial epithelial cells. We conclude that IL-9 is a stimulus for airway epithelial cell release of T-cell chemoattractant factors, which in turn may modulate the immune response in allergic airway inflammation.

Lipopolysaccharide binding protein potentiates airway reactivity in a murine model of allergic asthma.

The development of allergic asthma is influenced by both genetic and environmental factors. Epidemiologic data often show no clear relationship between the levels of allergen and clinical symptoms. Recent data suggest that bacterial LPS may be a risk factor related to asthma severity. Airborne LPS is typically present at levels that are insufficient to activate alveolar macrophages in the absence of the accessory molecule LPS binding protein (LBP). LBP levels are markedly elevated in bronchoalveolar lavage fluids obtained from asthmatic subjects compared with those in normal controls. We hypothesized that LBP present in the lung could augment the pulmonary inflammation and airway reactivity associated with allergic asthma by sensitizing alveolar macrophages to LPS or other bacterial products and triggering them to release proinflammatory mediators. We compared wild-type (WT) and LBP-deficient mice using a defined Ag immunization and aerosol challenge model of allergic asthma. Immunized LBP-deficient mice did not develop substantial Ag-induced airway reactivity, whereas WT mice developed marked bronchoconstriction following aerosol Ag sensitization and challenge with methacholine. Similarly, production of NO synthase 2 protein and the NO catabolite peroxynitrite was dramatically higher in the lungs of WT mice following challenge compared with that in LBP-deficient mice. Thus, NO production appears to correlate with airway reactivity. In contrast, both mice developed similar pulmonary inflammatory cell infiltrates and elevated mucin production. Thus, LBP appears to participate in the development of Ag-induced airway reactivity and peroxynitrite production, but does not seem to be required for the development of pulmonary inflammation.

Nuclear translocation of the N-terminal prodomain of interleukin-16.

Interleukin-16 (IL-16) is a pleiotropic cytokine that functions as a chemoattractant factor, a modulator of T cell activation, and an inhibitor of human immunodeficiency virus (HIV) replication. These diverse functions are exclusively attributed to the secreted C-terminal peptide of 121 amino acids (mature IL-16), which is cleaved from the precursor protein (pro-IL-16) by caspase-3. Human pro-IL-16 is comprised of 631 amino acids with three PDZ domains, one of which is present in secreted mature IL-16. No cellular localization or biologic functions have been ascribed to the unusually large and highly conserved N-terminal prodomain formed as a result of proteolytic release of the third PDZ domain of pro-IL-16. Here we show that the N-terminal prodomain of pro-IL-16 translocates into the nucleus following cleavage of the C-terminal segment. The nuclear localization signal of pro-IL-16 consists of a classical bipartite nuclear targeting motif. We also show that the nuclear targeting of the IL-16 prodomain induces a G(0)/G(1) arrest in the cell cycle. Taken together, the high degree of conservation of the prodomain among species, the presence of two PDZ motifs, and the nuclear localization and subsequent inhibitory effect on cell cycle progression suggest that pro-IL-16 is cleaved into two functional proteins, a C-terminal-secreted cytokine and an N-terminal product, which affects the cell cycle.

Desensitization of CXC chemokine receptor 4, mediated by IL-16/CD4, is independent of p56lck enzymatic activity.

CCR5 and CXC chemokine receptor 4 (CXCR4) are coreceptors for CD4 as defined by HIV-1 glycoprotein (gp) 120 binding. Pretreatment of T cells with gp120 results in modulation of both CCR5 and CXCR4 responsiveness, which is dependent upon p56(lck) enzymatic activity. The recent findings that pretreatment of T cells with a natural CD4 ligand, IL-16, could alter cellular responsiveness to macrophage-inflammatory protein-1ss (MIP-1ss) stimulation, prompted us to investigate whether IL-16 could also alter CXCR4 signaling. These studies demonstrate that IL-16/CD4 signaling in T lymphocytes also results in loss of stromal derived factor-1alpha (SDF-1alpha)/CXCR4-induced chemotaxis; however, unlike MIP-1ss/CCR5, the effects were not reciprocal. There was no effect on eotaxin/CCR3-induced chemotaxis. Desensitization of CXCR4 by IL-16 required at least 10-15 min pretreatment; no modulation of CXCR4 expression was observed, nor was SDF-1alpha binding altered. Using murine T cell hybridomas transfected to express native or mutated forms of CD4, it was determined that IL-16/CD4 induces a p56(lck)-dependent inhibitory signal for CXCR4, which is independent of its tyrosine catalytic activity. By contrast, IL-16/CD4 desensitization of MIP-1ss/CCR5 responses requires p56(lck) enzymatic activity. IL-16/CD4 inhibition of SDF-1alpha/CXCR4 signals requires the presence of the Src homology 3 domain of p56(lck) and most likely involves activation of phosphatidylinositol-3 kinase. These studies indicate the mechanism of CXCR4 receptor desensitization induced by a natural ligand for CD4, IL-16, is distinct from the inhibitory effects induced by either gp120 or IL-16 on CCR5.

Increased expression of IL-16 immunoreactivity in bronchial mucosa after segmental allergen challenge in patients with asthma.

BACKGROUND: We have previously shown increased expression of the CD4(+) cell chemoattractant IL-16 in bronchial mucosa of patients with asthma. We investigated the effects of allergen challenge on airway IL-16 expression. METHODS: We investigated the expression of IL-16 immunoreactivity in bronchial biopsy samples obtained from atopic asthmatic subjects (n = 19) and normal subjects (n = 6) 24 hours after segmental allergen challenge. Control biopsy samples were obtained either at baseline or after diluent challenge. IL-16 expression was correlated to numbers of CD4(+) cells, CD25(+) cells, and activated eosinophils. IL-16 bioactivity was assessed in bronchoalveolar fluid obtained from patients with asthma. RESULTS: IL-16 expression was higher in control biopsy specimens obtained from subjects with asthma compared with normal subjects (P<.05). In patients with asthma, numbers of IL-16 immunoreactive cells were significantly higher in biopsy specimens obtained after allergen challenge compared with control biopsy specimens (P<.001). Allergen provocation was associated with release of IL-16 in bronchoalveolar fluid in patients with asthma. In normal subjects, there was no difference in the number of IL-16-immunoreactive cells in biopsy specimens obtained after allergen challenge compared with biopsy specimens obtained after diluent challenge. Allergen challenge was associated with an increase in the numbers of EG2(+) eosinophils in patients with asthma but not in normal subjects. IL-16 expression correlated with the numbers of CD4(+) cells and CD25(+) cells after allergen challenge in asthmatic subjects with a provocative concentration required to decrease the FEV(1) by 20% of its baseline value (PC(20)FEV(1)) < 4 mg/mL. IL-16-immunoreactive cells were identified mainly as T cells and eosinophils in asthmatic subjects after allergen challenge. CONCLUSION: Endobronchial allergen provocation in atopic asthmatic patients resulted in increased airway expression of IL-16 and release of bioactive IL-16 in airways. IL-16 may contribute to the immunoregulation of the inflammatory infiltrate in the airways in response to antigen.

Interleukin 16 and T-cell chemoattractant activity in bronchoalveolar lavage 24 hours after allergen challenge in asthma.

IL-16 has been shown to be one of the earliest CD4(+) cell chemoattractants present in BAL 4-6 h after antigen challenge but little is known about its persistence and biological activity after 6 h. We determined the concentration of IL-16 using ELISA and the T-cell chemoattractant activity using a modified Boyden chamber assay in unconcentrated BAL fluid from 13 patients with mild asthma and 9 nonatopic control subjects at baseline and 24 h after segmental allergen or saline challenge. Furthermore, the percentage of IL-16-producing T cells was determined in the different samples of BAL fluid using a flow cytometric intracellular cytokine assay. Although no substantial levels of IL-16 protein were detectable in BAL fluid from control subjects and patients with asthma at baseline and after saline challenge, IL-16 concentrations were significantly elevated in patients with asthma after allergen challenge (median, 97 pg/ml; range, 38-362 pg/ml; p < 0.01). Furthermore, there was an increased T-cell chemoattractant activity after allergen challenge in patients with asthma (p < 0.01), which could be blocked by preincubation with anti-IL-16 antibodies and which correlated significantly with the IL-16 protein levels (R = 0.90, p < 0.01) and with the level of Fas ligand expression on BAL CD4(+) cells (R = 0. 80, p < 0.05). A high percentage (mean 70-90%) of CD4(+) and CD8(+) cells stained positively for IL-16 in both patients with asthma and control subjects without differences after allergen or saline challenge. These data demonstrate that the increased chemotactic activity for T cells in patients with asthma is mainly attributable to IL-16. Although T cells by themselves are able to produce IL-16, other cells, such as epithelial cells, have to be considered as further sources for this cytokine in patients with asthma.

Interleukin-16.

Interleukin 16 (IL-16) was initially described in 1982 as the first T cell chemoattractant. Through interaction with CD4, IL-16 has now been characterized as a chemoattractant for a variety of CD4+ immune cells. Recent in vivo studies have more fully characterized IL-16 as an immunomodulatory cytokine that contributes to the regulatory process of CD4+ cell recruitment and activation at sites of inflammation in association with asthma and several autoimmune diseases. Since its cloning in 1994, IL-16 structure and function have been studied extensively. This review addresses the current data regarding IL-16 protein and gene structure; the expanding list of cells capable of generating IL-16; the direct interaction of IL-16 with its receptor, CD4; and the functional bioactivities of IL-16 as they relate to inflammation and HIV-1 infection. In addition, potential therapeutic modalities for IL-16 relating to inflammation and immune reconstitution in HIV-1 infection are also discussed.

Interleukin 16: implications for CD4 functions and HIV-1 progression.

In this article, David Center and colleagues clarify the controversies that have emerged over the unique structure of interleukin 16 and its anti-HIV-1 activity. Interleukin 16 is a ligand for CD4, and this implies CD4 acts as a sentinel receptor that can switch CD4+ T cells between immune and inflammatory functions.

Cultured human fibroblasts express constitutive IL-16 mRNA: cytokine induction of active IL-16 protein synthesis through a caspase-3-dependent mechanism.

Human fibroblasts can express numerous regulatory molecules that influence immune function. IL-16, a ligand for CD4, is a chemoattractant molecule expressed by lymphocytes, eosinophils, mast cells, and lung epithelium. It appears that the sole target for IL-16 is the CD4-bearing cell. Here we demonstrate that fibroblasts from several tissues can express IL-16 mRNA and protein as well as IL-16-dependent chemoattractant activity. The transcript is expressed abundantly under basal culture conditions as a 2.5-kb band on Northern analysis, similar to that observed in lymphocytes. IL-16 protein and activity are undetectable in fibroblast cultures under these same control conditions. However, when treated with proinflammatory cytokines such as IL-1beta, they express very high levels of IL-16 protein and chemoattractant activity, a substantial component of which can be blocked with IL-16-neutralizing Abs. The amount of IL-16 protein released into the medium is 3- to 4-fold greater, on a per cell basis, than that observed in lymphocytes. The induction of IL-16 protein by IL-1beta can be attenuated with specific inhibition of caspase-3, which could be detected in IL-1beta-treated fibroblasts. IL-1beta also induces RANTES mRNA, protein, and activity, and most of the chemoattractant activity released from fibroblasts not derived from IL-16 can be attributed to RANTES. Human fibroblasts appear to be an important source of IL-16 and through expression of this molecule may have key roles in the recruitment of CD4+ cells to sites of inflammation. IL-16 expression and the mechanism involved in its regulation appear to be cell type specific.

Role of B7-CD28/CTLA-4 costimulation and NF-kappa B in allergen-induced T cell chemotaxis by IL-16 and RANTES.

The mechanisms that cause T cell recruitment into inflamed airways of asthmatic individuals are poorly understood. It has been shown previously that both natural exposure to allergen and challenge in the laboratory induce T cell accumulation in the bronchial mucosa of sensitized asthmatics. To study the mechanisms involved in this process, we have used an explant model in which bronchial biopsies taken from mild atopic asthmatic volunteers during fiberoptic bronchoscopy were stimulated in culture for 24 h by the common aeroallergen house dust mite (Dermatophagoides pteronyssinus (Der p)). Analysis of culture supernatants showed that stimulation with Der p significantly enhanced both the generation of T cell chemotactic activity by the mucosal tissue, as assayed in microchemotaxis chambers, and the production of IL-16 and RANTES. Neutralization experiments showed that IL-16 contributed more to the chemotactic activity than RANTES. The fusion protein CTLA-4-Ig, blocking B7:CD28 costimulation, and dexamethasone both significantly reduced the ex vivo production of chemotactic activity and release of IL-16 and RANTES. The proteasome inhibitor Cbz-Ile-Glu(OtBu)-Ala-leucinal also had a significant inhibitory effect on T cell chemotactic activity and IL-16 but not RANTES generation, indicating a role for nuclear factor NF kappa B activation. These results indicate that allergen stimulates cells within the bronchial mucosa to increase IL-16 and RANTES release, both of which contribute to T cell accumulation in asthmatic airways. The allergen-induced chemotactic activity is dependent on cell activation via CD28 and involves, at least partly, NF-kappa B.

Reciprocal desensitization of CCR5 and CD4 is mediated by IL-16 and macrophage-inflammatory protein-1 beta, respectively.

The ability of HIV-1 gp120 to inhibit chemokine signaling prompted us to determine whether signaling through CD4 by a natural ligand, IL-16, could alter cellular responsiveness to chemokine stimulation. These studies demonstrate that IL-16/CD4 signaling in T lymphocytes results in a selective loss of macrophage-inflammatory protein (MIP)-1 beta/CCR5-induced chemotaxis. There was no effect on monocyte chemoattractant protein-2/CCR1, -2, or -3-induced chemotaxis. Desensitization of CCR5 by IL-16 required at least 10 min of pretreatment; no modulation of CCR5 expression was observed, nor was MIP-1 beta binding to CCR5 altered. Using murine T cell hybridomas transfected to express native or mutated forms of CD4, it was determined that IL-16/CD4 induces a p56lck-dependent signal that results in desensitization of CCR5. The desensitization process is reciprocal and again selective, as prior CCR5 stimulation, but not CCR1, -2, or -3 stimulation, completely inhibits IL-16/CD4-induced T cell migration. Of interest, while p56lck enzymatic activity is not required for IL-16-induced migration, it was required for desensitization of CCR5. These studies indicate the existence of reciprocal receptor cross-desensitization between CD4 and CCR5 induced by two proinflammatory cytokines and suggest a selective relationship between the two receptors.

Polarized type 1 cytokine profile in bronchoalveolar lavage T cells of patients with hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is characterized by an inflammatory lymphocytic alveolitis comprised of both CD8+ and CD4+ T cells. Animal models suggest that HP is facilitated by overproduction of IFN-gamma, and that IL-10 ameliorates severity of the disease, indicating a Th1-type response. To determine whether a Th1 phenotype in HP also exists clinically, bronchoalveolar lavage (BAL) and peripheral blood (PB) T cells were obtained from HP individuals and analyzed for Th1 vs Th2 cytokine profiles. It was determined that soluble OKT3-stimulated BAL T cells cocultured with alveolar macrophages produced more IFN-gamma and less IL-10 than PB T cells cocultured with monocytes, but no difference was observed in IL-4 production. The monocytic cells did not account for this difference, as CD80 and CD86 expressions were similar, and coculturing PB T cells with alveolar macrophages resulted in no difference in IFN-gamma production. Similarly, there was no difference in IL-12 production between stimulated BAL or PB T cells; however, addition of rIL-12 significantly increased production of IFN-gamma by BAL T cells, but not by PB T cells. This effect was due to a difference in IL-12R expression. High affinity IL-12R were only present in association with BAL T cells. These studies indicate that clinical HP is characterized by a predominance of IFN-gamma-producing T cells, perhaps resulting from a reduction in IL-10 production and an increase in high affinity IL-12R compared with blood T cells.

Identification of domains in IL-16 critical for biological activity.

IL-16 is a proinflammatory cytokine implicated in the pathogenesis of asthma and other conditions characterized by recruitment of CD4+ T cells to sites of disease. It is postulated that CD4 is an IL-16 receptor, although other receptors or coreceptors may exist. Among several known functions, IL-16 is a chemoattractant factor for CD4+ T cells and it inhibits MLR. We previously reported that an oligopeptide corresponding to the 16 C-terminal residues of human IL-16 inhibits chemoattractant activity. To identify functional domains with greater precision, shorter oligonucleotides containing native or mutated C-terminal IL-16 sequences were tested for IL-16 inhibition. Within the 16 C-terminal residues, the minimal peptide RRKS (corresponding to Arg106 to Ser109) was shown to mediate inhibition of IL-16 chemoattractant activity. Inhibition was lost when either arginine was substituted with alanine. Point mutations in IL-16 revealed that Arg107 is critical for chemoattractant activity, but MLR inhibition was unaffected by mutation of Arg107 or even deletion of the C-terminal tail through Arg106. Deletion of 12 or 22 N-terminal residues of IL-16 had no impact on chemoattractant activity, but MLR inhibition was reduced. Deletion of 16 C-terminal plus 12 N-terminal residues abolished both chemoattractant and MLR-inhibitory activity of IL-16. These data indicate that receptor interactions with IL-16 that activate T cell migration are not identical with those required for MLR inhibition, and suggest that both N-terminal and C-terminal domains in IL-16 participate in receptor binding or activation.

Identification of a CD4 domain required for interleukin-16 binding and lymphocyte activation.

Interleukin-16 (IL-16) activates CD4(+) cells, possibly by direct interaction with CD4. IL-16 structure and function are highly conserved across species, suggesting similar conservation of a putative IL-16 binding site on CD4. Comparison of the human CD4 amino acid sequence with that of several different species revealed that immunoglobulin-like domain 4 is the most conserved extracellular region. Potential interaction of this domain with IL-16 was studied by testing murine D4 sequence-based oligopeptides for inhibition of IL-16 chemoattractant activity and inhibition of IL-16 binding to CD4 in vitro. Three contiguous 12-residue D4 region peptides (designated A, B, and C) blocked IL-16 chemoattractant activity, with peptide B the most potent. Peptides A and B were synergistic for inhibition, but peptide C was not. Peptides A and B also blocked IL-16 binding to CD4 in vitro, whereas peptide C did not. CD4, in addition to its known function as a receptor for major histocompatibility complex class II, contains a binding site for IL-16 in the D4 domain. The D4 residues required for IL-16 binding overlap those previously shown to participate in CD4-CD4 dimerization following class II major histocompatibility complex binding, providing a mechanistic explanation for the known function of IL-16 to inhibit the mixed lymphocyte reaction.

Phenotype of IL-16-producing cells in bronchial mucosa: evidence for the human eosinophil and mast cell as cellular sources of IL-16 in asthma.

BACKGROUND: We have previously shown increased expression of the CD4+ cell chemoattractant interleukin (IL)-16 in bronchial biopsies of atopic asthmatic subjects compared to normal controls. IL-16 immunoreactive cells were identified as both epithelial cells and non-epithelial inflammatory cells. The aim of this study was to characterize and compare the phenotype of non-epithelial inflammatory cells that express IL-16 immunoreactivity in bronchial biopsies from non-atopic normal controls and atopic asthmatic subjects. METHODS: Sections from endobronchial biopsies obtained from non-atopic normal controls and atopic asthmatics were processed for double immunocytochemistry. IL-16 immunoreactivity was assessed using a polyclonal anti-IL-16 antibody and the avidin-biotin complex-diaminobenzidine method. The phenotype of IL-16 immunoreactive cells was assessed using anti-CD3, anti-MBP, anti-tryptase and anti-CD68 mAbs and the alkaline phosphatase complex-Fast Red method. RESULTS: In normal subjects, the majority of IL-16 immunoreactive cells were CD3+ T cells (71.1+/-10.3%) and CD68+ macrophages (22.4+/-8.1%). IL-16 immunoreactivity coexpressed with tryptase+ mast cells in 4 of 7 normal subjects whereas IL-16 immunoreactivity coexpressed with MBP+ eosinophils in only 1 normal subject. In atopic asthmatic subjects, IL-16 immunoreactive cells were mainly CD3+ T cells (60.8+/-8.7%) and MPB+ eosinophils (16.8+/-8.2%). IL-16 immunoreactivity also coexpressed with tryptase+ mast cells (10.6+/-4.0%) in all asthmatic subjects. The number of IL-16 immunoreactive cells that coexpressed MBP was higher in asthmatic subjects compared to normal controls (p = 0.003). CONCLUSION: Our data show that T cells are the major non-epithelial cellular source of IL-16 in normal and asthmatic airways. Eosinophils and mast cells comprised other potential cellular sources of IL-16 in asthmatic airways.

Processing and release of IL-16 from CD4+ but not CD8+ T cells is activation dependent.

IL-16 is synthesized as a precursor molecule of 68 kDa (pro-IL-16) that is processed by caspase-3, a member of the IL-1 converting enzyme (ICE) family. This cleavage results in a 13-kDa carboxy terminal peptide, which constitutes the bioactive secreted form of IL-16. We have previously reported constitutive IL-16 mRNA expression and pro-IL-16 protein in CD4+ and CD8+ T cells. Although bioactive IL-16 protein is present in unstimulated CD8+ T cells, there is no bioactive IL-16 present in CD4+ T cells. Along these lines, unstimulated CD8+ T cells contain active caspase-3. In the current studies we investigated the regulation of IL-16 protein and mRNA expression in CD4+ T cells and determined the kinetics of secretion following stimulation of the TCR. CD4+ T cells release IL-16 protein following antigenic stimulation, and this release is accelerated in time by costimulation via CD28. However, CD3/CD28 costimulation did not alter IL-16 mRNA appearance or stability in either CD4+ or CD8+ T cells. The secretion of bioactive IL-16 from CD4+ T cells correlated with the appearance of cleavage of pro-caspase-3 into its 20-kDa active form. Thus, resting CD8+ T cells contain active caspase-3 that is capable of cleaving pro-IL-16, whereas CD4+ T cells require activation for the appearance of active caspase-3. The mechanism of release or secretion of bioactive IL-16 is currently unknown, but does not correlate with cellular apoptosis.

Association of increased CD4+ T-cell infiltration with increased IL-16 gene expression in atopic dermatitis.

BACKGROUND: The mechanisms involved in the initiation and the maintenance of skin inflammation in atopic dermatitis (AD) are poorly understood. Previous studies have demonstrated increased numbers of infiltrating CD4+ T cells in acute lesions compared with normal control skin. IL-16 is a cytokine that has selective chemotactic activity for CD4+ cells. OBJECTIVE: We sought to examine whether IL-16 expression might be upregulated in acute versus chronic AD. METHODS: We investigated the expression of IL-16 mRNA in skin biopsy specimens from acute and chronic skin lesions, as well as from the uninvolved skin of patients with AD and normal skin. Cryostat sections from 4% paraformaldehyde-fixed skin biopsy specimens were processed for in situ hybridization by using cRNA coding for IL-16 mRNA. Numbers of infiltrating CD4+ and CD8+ cells were also determined by immunocytochemistry. RESULTS: There were positive signals for IL-16 mRNA both in the basal layer of the epidermis and in the dermis of AD skin biopsy specimens from all subjects studied. The numbers of epidermal and dermal IL-16 mRNA+ cells were significantly increased in acute skin lesions compared with chronic (P <.01) and uninvolved (P <.001) skin lesions and compared with normal skin (P <.001). The number of CD4+ cells was significantly increased in acute skin lesions compared with chronic (P <.01) skin lesions and uninvolved skin (P <.01) and compared with normal skin (P <.01). Significant correlations were found between the numbers of CD4+ cells and the numbers of epidermal (r = 0.82, P <.001) and dermal (r = 0.71, P <.001) IL-16 mRNA+ cells. CONCLUSION: The results demonstrate that upregulation of IL-16 mRNA expression in acute AD is associated with increased numbers of CD4+ cells, suggesting that IL-16 may play a role in the initiation of skin inflammation, presumably through recruitment of CD4+ cells.

Tissue and T cell distribution of precursor and mature IL-16.

IL-16 is a novel cytokine, which is chemoattractant for CD4+ T cells, macrophages, and eosinophils. Recently, it was reported that IL-16 is synthesized as an approximately 80-kDa precursor molecule, pro-IL-16. Since little is known about the processing and tissue distribution of IL-16 and pro-IL-16, we investigated the distribution of IL-16 mRNA and protein in human lymphoid tissue. Northern blotting identified IL-16 mRNA predominantly in normal lymphoid organs, including PBMC, spleen, and thymus. Immunohistochemistry of human lymph node localized IL-16 protein to lymphocyte cytoplasm within T cell zones and occasionally in lymphocytes in B cell zones. Flow cytometric detection of intracellular IL-16 showed that >70% of CD4+ and CD8+ T cells constitutively expressed IL-16 protein. Western blot analysis of PBMC revealed nearly all of this protein to be approximately 80-kDa pro-IL-16 in unstimulated PBMC, and upon cell activation, the amino terminus of pro-IL-16 is processed into multiple fragments. These results show that pro-IL-16 is widely and constitutively expressed and suggest that the amino terminus of the protein can be processed upon cell activation.

Identification of IL-16 as the lymphocyte chemotactic activity in the bronchoalveolar lavage fluid of histamine-challenged asthmatic patients.

OBJECTIVE: We have previously demonstrated that the earliest lymphocyte chemotactic factors present in bronchoalveolar lavage fluid (BALF) of subjects with atopic asthma after subsegmental antigen challenge are IL-16 and MIP-1alpha, of which IL-16 appears to contribute a majority of the chemotactic activity. Because IL-16 is released in vitro after histamine stimulation of CD8+ T cells and epithelial cells, we evaluated the potential role of histamine in the release of IL-16 into the airways of allergic asthmatics in vivo. METHODS: Eight allergic asthmatic subjects, six normal subjects, and six atopic nonasthmatic subjects were challenged with saline in the lingula and with serial concentrations of histamine (1 x 10(-7) to 5 x 10(-5) mol/L) in the right middle lobe followed by bronchoalveolar lavage (BAL) 15 minutes and 6 hours later. RESULTS: The BALF from saline- and histamine-challenged lobes of normal subjects and atopic nonasthmatic subjects contained no significant lymphocyte chemoattractant activity. In six of the eight atopic asthmatic subjects, the histamine-challenged but not saline-challenged segment contained IL-16 chemotactic activity but no other identifiable lymphocyte chemoattractant activities at 6 hours. CONCLUSIONS: IL-16 appears in the airways after histamine challenge and therefore could contribute to the earliest infiltration of CD4+ T cells and eosinophils observed after antigen challenge due to histamine release from mast cells.