AIDS Kaposi sarcoma-derived cells produce and respond to interleukin 6.

Cell lines derived from Kaposi sarcoma lesions of patients with AIDS (AIDS-KS cells) produce several cytokines, including an endothelial cell growth factor, interleukin 1 beta, and basic fibroblast growth factor. Since exposure to human immunodeficiency virus increases interleukin 6 (IL-6) production in monocytes and endothelial cells produce IL-6, we examined IL-6 expression and response in AIDS-KS cell lines and IL-6 expression in AIDS Kaposi sarcoma tissue. The AIDS-KS cell lines (N521J and EKS3) secreted large amounts of immunoreactive and biologically active IL-6. We found both IL-6 and IL-6 receptor (IL-6-R) RNA by slot blot hybridization analysis of AIDS-KS cells. The IL-6-R was functional, as [3H]thymidine incorporation by AIDS-KS cells increased significantly after exposure to human recombinant IL-6 (hrIL-6) at greater than 10 units/ml. When AIDS-KS cells (EKS3) were exposed to IL-6 antisense oligonucleotide, cellular proliferation decreased by nearly two-thirds, with a corresponding decrease in the production of IL-6. The decrease from IL-6 antisense in AIDS-KS cell proliferation was reversed by the addition of hrIL-6. We confirmed that AIDS-KS cells produced IL-6 in vivo by preparing RNA and tissue sections from involved and uninvolved skin from a patient with AIDS Kaposi sarcoma. We detected immunoreactive IL-6 in the involved tumor areas and to a lesser extent in the surrounding normal epidermis. Slot blot hybridization showed a great excess of IL-6 and IL-6-R RNA in involved skin compared to uninvolved skin. These results show that both IL-6 and IL-6-R are produced by AIDS-KS cells and that IL-6 is required for optimal AIDS-KS cell proliferation, and they suggest that IL-6 is an autocrine growth factor for AIDS-KS cells.     

Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor).

The control of vascular endothelial and smooth muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical vein endothelial cells, express HBGF-I mRNA. Smooth muscle cells also synthesize an HBGF-I-like polypeptide since (i) extract prepared from smooth muscle cells will compete with 125I-labeled HBGF-I for binding to the HBGF-I cell surface receptor, and (ii) the competing ligand is eluted from heparin-Sepharose affinity resin at a NaCl concentration similar to that required by purified bovine brain HBGF-I and stimulates endothelial cell proliferation in vitro. Furthermore, like endothelial cells, smooth muscle cells possess cell-surface-associated HBGF-I receptors and respond to HBGF-I as a mitogen. These results indicate the potential for an additional autocrine component of vascular smooth muscle cell growth control and establish a vessel wall source of HBGF-I for endothelial cell division in vivo.     

Sympathetic neurons can produce and respond to interleukin 6

Neuronal expression of cytokines is an area of active investigation in the contexts of development, disease, and normal neural function. Although cultured rat sympathetic neurons respond very weakly to exogenous interleukin 6 (IL-6), we find that addition of soluble IL-6 receptor (sIL-6R) and IL-6 enhances neuronal survival in the absence of nerve growth factor. Neutralizing monoclonal antibodies against IL-6 block these effects. Addition of IL-6 and sIL-6R also induces a subset of neuropeptide and transmitter synthetic enzyme mRNAs identical to that demonstrated for leukemia inhibitory factor, ciliary neurotrophic factor, and oncostatin M. Both of these effects are duplicated by addition of a highly active fusion protein of sIL-6R and IL-6, covalently linked by a flexible peptide chain, which is designated H-IL-6. In addition, we show that sympathetic neurons produce IL-6. In situ hybridization indicates a neuronal localization of IL-6 mRNA in superior cervical ganglia, and bioactive IL-6 protein is detected in ganglion culture supernatants. Interestingly, the IL-6 produced by sympathetic neurons does not lead to survival of these cells in culture unless sIL-6R is added. Thus, sympathetic neurons can produce IL-6 and may respond to it in an autocrine/paracrine manner if sIL-6R is present. Moreover, the prior findings of sIL-6R in serum and inflammatory fluids now have added interest in the context of neuro–immune interactions.     

Intestinal epithelial cells secrete exosome-like vesicles

BACKGROUND & AIMS: Given the observations that intestinal epithelial cells (IECs) can present antigens to CD4(+) T lymphocytes and that professional antigen-presenting cells secrete exosomes (antigen-presenting vesicles), we hypothesized that IECs may secrete exosomes carrying molecules implicated in antigen presentation, which may be able to cross the basement membrane and convey immune information to noncontiguous immune cells. METHODS: Human IEC lines HT29-19A and T84-DRB1*0401/CIITA were grown on microporous filters. Release of exosomes under basal or inflammatory conditions was evaluated in conditioned apical and basolateral media after differential ultracentrifugations. Morphologic and biochemical characterization of exosomes was performed using immunoelectron microscopy, Western blotting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. RESULTS: The intestinal cell lines released 30-90-nm-diameter vesicles from the apical and basolateral sides, and this release was significantly increased in the presence of interferon gamma. MHC class I, MHC class II, CD63, CD26/dipeptidyl-peptidase IV, and A33 antigen were present in epithelial-derived exosomes. CONCLUSIONS; Human IEC lines secrete exosomes bearing accessory molecules that may be involved in antigen presentation. These data are consistent with a model in which IECs may influence antigen presentation in the mucosal or systemic immune system independent of direct cellular contact with effector cells.     

Intestinal epithelial cells in inflammatory bowel diseases

The pathogenesis of inflammatory bowel diseases (IBDs) seems to involve a primary defect in one or more of the elements responsible for the maintenance of intestinal homeostasis and oral tolerance. The most important element is represented by the intestinal barrier, a complex system formed mostly by intestinal epithelial cells (IECs). IECs have an active role in producing mucus and regulating its composition; they provide a physical barrier capable of controlling antigen traff ic through the intestinal muco...     

Primary differentiated respiratory epithelial cells respond to apical measles virus infection by shedding multinucleated giant cells

Measles virus (MeV) is highly infectious by the respiratory route and remains an important cause of childhood mortality. However, the process by which MeV infection is efficiently established in the respiratory tract is controversial with suggestions that respiratory epithelial cells are not susceptible to infection from the apical mucosal surface. Therefore, it has been hypothesized that infection is initiated in lung macrophages or dendritic cells and that epithelial infection is subsequently established through the basolateral surface by infected lymphocytes. To better understand the process of respiratory tract initiation of MeV infection, primary differentiated respiratory epithelial cell cultures were established from rhesus macaque tracheal and nasal tissues. Infection of these cultures with MeV from the apical surface was more efficient than from the basolateral surface with shedding of viable MeV-producing multinucleated giant cell (MGC) syncytia from the surface. Despite presence of MGCs and infectious virus in supernatant fluids after apical infection, infected cells were not detected in the adherent epithelial sheet and transepithelial electrical resistance was maintained. After infection from the basolateral surface, epithelial damage and large clusters of MeV-positive cells were observed. Treatment with fusion inhibitory peptides showed that MeV production after apical infection was not dependent on infection of the basolateral surface. These results are consistent with the hypothesis that MeV infection is initiated by apical infection of respiratory epithelial cells with subsequent infection of lymphoid tissue and systemic spread.

Measles virus (MeV) is a highly contagious enveloped, nonsegmented negative-strand RNA virus that is transmitted by the respiratory route to cause a systemic rash disease that includes pulmonary infection in both humans and nonhuman primates. Despite availability of an effective live attenuated MeV vaccine (LAMV), the worldwide incidence of measles cases and deaths has recently tripled (1, 2). Because of the efficient respiratory spread of the virus, a major hurdle for control of measles is the requirement for high vaccine-induced population immunity (92 to 95%) to interrupt transmission (3, 4).Although the respiratory tract is the site of wild-type (WT) MeV initiation of infection and aerosol delivery of LAMV induces protective immunity (5), the biology of how MeV so efficiently initiates infection after aerosol or respiratory droplet exposure is controversial. Two fundamentally different models of MeV pathogenesis have been proposed: 1) Initial replication of MeV occurs in respiratory epithelial cells through apical infection with subsequent systemic spread to other cells and tissues; 2) Initial replication of MeV occurs in myeloid cells of the respiratory tract (alveolar macrophages/dendritic cells) with spread to other tissues and subsequent delivery by infected lymphocytes to the basolateral surface of respiratory epithelial cells for pulmonary infection and virus transmission (6, 7). The first model is supported by the presence of giant cells in broncho-alveolar lavage fluid from measles patients and identification of epithelial cell infection by histology or immunohistochemistry in the airway and lung during measles in humans and macaques (8–10). In addition, LAMV can efficiently establish pulmonary infection without producing a viremia (11), suggesting that prior infection of lymphocytes is not necessary for respiratory epithelial cell infection. The alternative model is suggested by the observation that, after pulmonary infection of macaques with recombinant MeV-expressing eGFP, the predominant eGFP⁺ cells in the respiratory epithelium were CD150⁺ cells and dendritic cells, not epithelial cells (12, 13). That epithelial cells are not the site of initial infection is further supported by the apparent resistance of polarized epithelial cells derived from airway tissue to infection with WT MeV via the apical surface (14, 15). Questions remain for both models.Entry of MeV into susceptible cells is determined mainly by interaction of the hemagglutinin (H) and fusion (F) virion surface glycoproteins with receptor molecules on the host cell. The three identified cellular receptors for MeV are CD46 expressed ubiquitously in all nucleated cells (16, 17), CD150 (signaling lymphocyte activation molecule/SLAM) expressed on activated immune cells (18), and nectin-4 expressed by epithelial cells (19, 20). WT MeV can use CD150 and nectin-4 as receptors while LAMVs can utilize all three receptors. Nevertheless, in vitro and in vivo spread of LAMV is more restricted than that of WT MeV (21).The identification of nectin-4 as a functional receptor for MeV on epithelial cells (19, 20) has shed light on the pathogenesis of MeV in the respiratory tract. As an adherens junction protein, nectin-4 is located at the basolateral surfaces of epithelial cells and thus can be used by MeV to spread back into the airways after replicating systemically (22). The importance of nectin-4 in MeV transmission is highlighted by infection of macaques with a recombinant virus unable to recognize nectin-4. This nectin-4-blind virus is defective in shedding virus into the airways, although most other aspects of the infection are indistinguishable from infection caused by WT MeV (7). The observed competency of the nectin-4-blind MeV to enter and establish infection in macaques has been viewed as supporting evidence for the alternative model of MeV pathogenesis in the respiratory tract (20, 23–25). However, the mode of MeV entry into epithelial cells in vivo remains in question and the fact that nectin-4 is located on the basolateral surface of epithelial cells could also indicate that MeV enters respiratory epithelial cells from the apical surface in a nectin-4-independent manner. In particular, other modes of entry, such as endocytosis and macropinocytosis used by several paramyxoviruses, may be important for MeV entry into primary cells (26–28).To better understand MeV infection of the respiratory tract, we have developed and characterized a primary differentiated respiratory epithelial cell culture system using tracheal and nasal epithelial cells derived from rhesus macaques (rmTEC and rmNEC), a highly relevant animal model for measles (29, 30), and compared the outcome of apical and basolateral exposure of these cells to WT MeV and LAMV. Infection occurred through both the apical surface and the basolateral surface. Apical infection was efficient and induced rapid shedding of viable MeV-infected multinucleated giant cells (MGCs) from the epithelial surface into the lumen while maintaining epithelial barrier integrity. Basal infection was less efficient with infected cells retained in the epithelial monolayer and with barrier disruption, as well as with shedding of MGCs from the apical surface.     

Human cholangiocarcinomas express somatostatin receptors and respond to somatostatin with growth inhibition

Cholangiocarcinoma, a malignancy of biliary epithelia, is usually fatal because of absence of tests for early detection and lack of effective therapy. Somatostatin (SS) receptors are expressed in several malignancies and in rodent biliary epithelia. We tested the hypothesis that SS receptors are present in cholangiocarcinomas. We examined tissue from seven surgically resected human cholangiocarcinomas and a human bile duct cancer cell line for the messenger RNA for one subtype of SS receptors (SSTR2) and studied binding and growth-active properties of SS and its analogues. SSTR2 messenger RNA was expressed in all seven human cholangiocarcinoma specimens. Experiments with the human cholangiocarcinoma cell line showed specific, saturable binding of an SS analogue (MK-678) with high affinity for SSTR2 on cholangiocarcinoma membranes; inhibition in vitro of tumor cell proliferation by SS-14 and its analogue, octreotide; and inhibition in vivo of tumor growth in athymic mice implanted with human cholangiocarcinoma cells and treated with lanreotide, another SS analogue. Experiments to elucidate a possible mechanism of growth inhibition by SS showed it was not through changes in cellular cyclic adenosine monophosphate or calcium levels. Using gamma camera imaging with an ¹¹¹In-SS analogue, we localized a histologically proven cholangiocarcinoma in a patient. These results suggest that SS analogues may be useful for diagnostic localization and treatment of biliary tract malignancies.     

Common and pre-B acute lymphoblastic leukemia cells express interleukin 2 receptors, and interleukin 2 stimulates in vitro colony formation

The role of interleukin 2 (IL 2) as a possible regulator of in vitro proliferation and differentiation of non-T acute lymphoblastic leukemia (ALL) cells was investigated. For this purpose, leukemic cells from the blood or bone marrow of eight untreated patients with common or pre-B ALL were analyzed using the anti-Tac monoclonal antibody (reactive with the IL 2 receptor) in indirect immunofluorescence. The receptors for IL 2, which were initially absent from the cell surface, were induced on high percentages of the ALL cells after the in vitro exposure to the lectin phytohemagglutinin or the phorbol ester 12-O- tetradecanoylphorbol-13-acetate in six patients, suggesting that the cells had become sensitive to IL 2. In colony cultures to which feeder leukocytes and IL 2 had been added, colony growth was obtained in five of eight cases. Whereas the cells from one patient formed colonies in the absence of exogenous stimuli, the cells from others were dependent on the addition of feeder leukocytes plus IL 2. In the latter cases, feeder leukocytes alone, releasing some IL 2, stimulated growth suboptimally at different cell concentrations. Their stimulative effect was significantly enhanced when leukocyte-derived IL 2 or pure recombinant IL 2 was supplemented. Alone, IL 2 (up to 500 U/mL) did not support colony formation. Apparently, IL 2 and feeder leukocytes are both required for the induction of colonies in these cases of ALL. From cell sorting of fluorescent anti-common ALL antigen (CALLA) stained cells it appeared that colonies descended from cells with high as well as low or negative CALLA expression. Immunophenotyping demonstrated the presence of the original leukemia markers on colony cells, but was not indicative of maturation of ALL toward more differentiated B cells. We suggest that IL 2 can stimulate the in vitro proliferation of certain neoplastic B lymphocyte progenitors.     

Interleukin 2 and interleukin 15 differentially predispose natural killer cells to apoptosis mediated by endothelial and tumour cells

Human natural killer (NK) cells constitutively express the beta- and gamma-chains of the interleukin 2 (IL-2)/IL-15 receptor, and both IL-2 and IL-15 are able to activate NK cell proliferation and cytotoxicity. When IL-2-primed human NK cells are exposed to sensitive targets (i.e. K562) they undergo apoptosis mediated by the beta(2)-integrin CD18. Here, we demonstrate that: (i) endothelial cells, similar to K562 tumour target cells, induce apoptosis of IL-2-primed NK cells; (ii) endothelial- and K562 cell-induced apoptosis is significantly lower in IL-15 than in IL-2-stimulated NK cells; (iii) a critical role in the apoptosis of IL-2-primed NK cells is played by the alpha-chain of the IL-2 receptor. Our data show for the first time that IL-2-activated NK cells can die by apoptosis upon contact with the vascular endothelium, which is a necessary step for their extravasation, with a direct pathophysiological relevance on the strategy of adoptive immunotherapy of cancer. On the other hand, IL-15, although generating a similar level of activation of NK cells, largely prevents their apoptotic fate. Therefore, IL-15 produced early in the immune response, when T cells are not yet activated, generates lymphokine-activated killer cells that are efficient killers relatively protected from apoptosis. Once activated, T cells produce IL-2 that overcomes the effect of IL-15 on NK cells, paving the way for their death by apoptosis.     

Intestinal dendritic cells and epithelial barrier dysfunction in Crohn's disease

Crohn's disease (CD) is a chronic gastrointestinal inflammatory disorder considered to be the result of an inappropriate and exaggerated mucosal immune reaction to yet undefined triggers from the gut flora in genetically predisposed individuals. This inflammatory phenomenon has been characterized by an adaptive T-cell response in addition to an abnormal function of the innate immune system. Dendritic cells (DCs) are constituents of this innate system, inducing T-cell activation via antigen presentation. In the gut, mucosal DCs are separated from the luminal milieu by a monolayer of cylindrical epithelial cells that forms an anatomical and physiological barrier that controls the normal traffic of antigens between both compartments. An imbalance of colonic and ileal DC distribution in tissues from CD patients as well as functional differences between DCs isolated from normal and diseased intestinal samples have been demonstrated. Moreover, a gut barrier defect in the para- and transepithelial routes in addition to a significant reduction in the intestinal secretion of epithelial products involved in barrier function has been well documented in CD. Therefore, this may expose the diseased mucosa to overwhelming amounts of antigens, resulting in abnormal DC activation and a subsequent imbalance in their distribution. In conclusion, this review provides a summary of relevant progress in CD, intestinal epithelial permeability, and DCs highlighting a potential relationship between increased epithelial permeability and abnormal DC distribution during the pathogenesis of intestinal inflammation.     

Fractionated populations of normal human marrow cells respond to both human colony-stimulating factors with granulocyte-macrophage activity

Populations of normal human colony-forming cells (blast cells) and cluster-forming cells (promyelocytes-myelocytes) were obtained from bone marrow by using the monoclonal antibody WEM G11 and the fluorescence-activated cell sorter (FACS). Both populations were shown to be responsive to both human colony-stimulating factors (CSFs) with granulocyte-macrophage activity (CSF alpha and CSF beta), with the cluster-forming cell population being more responsive to CSF beta than the colony-forming cell population. The clonal proliferation of promyelocytes-myelocytes was transient, and the clones generated were of subcolony size (less than 40 cells) regardless of the CSF used. Clone transfer experiments demonstrated that progeny of promyelocytes-myelocytes initiated using one stimulus (CSF alpha or CSF beta) were also responsive to the other stimulus.     

Role of interleukin 15 and interleukin 18 in inflammatory response

Interleukin 15 (IL15) and interleukin 18 (IL18) are cytokines produced principally by macrophages during innate immune response and subsequently profoundly influence adaptive immunity. Recent studies have shown that IL15 and IL18 play an influential part in inflammatory response. Here we present recent data mainly from our own laboratories illustrating the importance of IL15 and IL18 in the induction and perpetuation of chronic inflammation during experimental and clinical rheumatoid synovitis.     

Human endothelial cells express CCR2 and respond to MCP-1: direct role of MCP-1 in angiogenesis and tumor progression

Although several CXC chemokines have been shown to induce angiogenesis and play roles in tumor growth, to date, no member of the CC chemokine family has been reported to play a direct role in angiogenesis. Here we report that the CC chemokine, monocyte chemotactic protein 1 (MCP-1), induced chemotaxis of human endothelial cells at nanomolar concentrations. This chemotactic response was inhibited by a monoclonal antibody to MCP-1. MCP-1 also induced the formation of blood vessels in vivo as assessed by the chick chorioallantoic membrane and the matrigel plug assays. As expected, the angiogenic response induced by MCP-1 was accompanied by an inflammatory response. With the use of a rat aortic sprouting assay in the absence of leukocytic infiltrates, we ruled out the possibility that the angiogenic effect of MCP-1 depended on leukocyte products. Moreover, the direct effect of MCP-1 on angiogenesis was consistent with the expression of CCR2, the receptor for MCP-1, on endothelial cells. Assessment of supernatant from a human breast carcinoma cell line demonstrated the production of MCP-1. Treatment of immunodeficient mice bearing human breast carcinoma cells with a neutralizing antibody to MCP-1 resulted in significant increases in survival and inhibition of the growth of lung micrometastases. Taken together, our data indicate that MCP-1 can act as a direct mediator of angiogenesis. As a chemokine that is abundantly produced by some tumors, it can also directly contribute to tumor progression. Therefore, therapy employing antagonists of MCP-1 in combination with other inhibitors of angiogenesis may achieve more comprehensive inhibition of tumor growth.     

Intestinal epithelial cells as a source of inflammatory cytokines and chemokines.

The intestinal epithelium has long been known to provide nonspecific defences such as mucus, lysozyme and transport of secretory immunoglobulin via the polyimmunoglobulin receptor. In the past decade, the realization emerged that enterocytes secrete molecules (cytokines) that regulate inflammation. As the focus tightened on this new role as sentinel, so has the interest in enterocyte production of cytokines with chemoattractant properties for leukocytes - the chemokines. Neutrophils are a prominent feature of the cellular infiltrate in various inflammatory diseases, and early reports indicated that epithelial cells secrete neutrophil chemoattractants. More recently, it has been shown that the cells also secrete chemokines for monocytes and lymphocytes. Some of these chemokines appear to be important in the uninflamed intestine but become increased during disease. While a great deal of knowledge has been gained regarding the circumstances leading to chemokine production by epithelial cells, the application of this understanding to the treatment of human intestinal diseases is lacking. Closing this gap is necessary to take advantage of emerging therapies aimed at blocking chemokine function.     

Chondrogenic progenitor cells respond to cartilage injury

Objective

Hypocellularity resulting from chondrocyte death in the aftermath of mechanical injury is thought to contribute to posttraumatic osteoarthritis. However, we observed that nonviable areas in cartilage injured by blunt impact were repopulated within 7–14 days by cells that appeared to migrate from the surrounding matrix. The aim of this study was to assess our hypothesis that the migrating cell population included chondrogenic progenitor cells that were drawn to injured cartilage by alarmins.

Methods

Osteochondral explants obtained from mature cattle were injured by blunt impact or scratching, resulting in localized chondrocyte death. Injured sites were serially imaged by confocal microscopy, and migrating cells were evaluated for chondrogenic progenitor characteristics. Chemotaxis assays were used to measure the responses to chemokines, injury‐conditioned medium, dead cell debris, and high mobility group box chromosomal protein 1 (HMGB‐1).

Results

Migrating cells were highly clonogenic and multipotent and expressed markers associated with chondrogenic progenitor cells. Compared with chondrocytes, these cells overexpressed genes involved in proliferation and migration and underexpressed cartilage matrix genes. They were more active than chondrocytes in chemotaxis assays and responded to cell lysates, conditioned medium, and HMGB‐1. Glycyrrhizin, a chelator of HMGB‐1 and a blocking antibody to receptor for advanced glycation end products (RAGE), inhibited responses to cell debris and conditioned medium and reduced the numbers of migrating cells on injured explants.

Conclusion

Injuries that caused chondrocyte death stimulated the emergence and homing of chondrogenic progenitor cells, in part via HMGB‐1 release and RAGE‐mediated chemotaxis. Their repopulation of the matrix could promote the repair of chondral damage that might otherwise contribute to progressive cartilage loss.

     

Glucocorticoid up-regulation of high-affinity interleukin 6 receptors on human epithelial cells.

Interleukin 6 (IL-6) is a potent pleiotropic cytokine, known, among others, to stimulate immunoglobulin production by B cells and to trigger acute-phase protein synthesis by hepatocytes. Similar to IL-1, it is produced by monocytes and macrophages following an inflammatory challenge. Analysis of IL-6 receptor (IL-6R) expression on different human cell lines indicates that dexamethasone could up-regulate the number of IL-6R on one epithelial cell line (UAC) and on two hepatoma cell lines (HepG2 and Hep3B). This effect was confirmed by Scatchard analysis of binding experiments, using [35S]methionine and [35S]cysteine metabolically labeled IL-6. It was confirmed at the level of mRNA expression by Northern blot analysis. These results provide evidence for a link between IL-6 and glucocorticoids. They could represent an example of a system in which one role of glucocorticoids is to define more accurately the target of cytokines, and they could explain, at least partly, the frequently observed synergy between IL-6 and glucocorticoids, notably in the case of hepatocytes.