The regulation and functional consequence of proinflammatory cytokine binding on human intestinal epithelial cells

Products of an activated immune system may affect cells within the immune system as well as nonlymphoid cells in the local environment. Given the immunologically activated state of the intestinal tract, it is conceivable that locally produced cytokines could regulate epithelial cell function. To assess whether epithelial cells are targets for particular cytokines, we initiated studies on the binding of a panel of proinflammatory cytokines in freshly isolated epithelial cells from normal and inflammatory bowel disease (IBD) patients as well as in cell lines. Isolated intestinal epithelial cells (IEC) were stained with phycoerythrin-conjugated or biotinylated cytokines to determine the expression and density of receptors for IL-1beta, IL-6, granulocyte-macrophage CSF (GM-CSF), and TNF-alpha. Receptors for IL-1beta, IL-6, and GM-CSF were readily detectable in all epithelial cell preparations at levels equal to (GM-CSFR) or lower than those seen on monocytes. However TNFalpha-R were not detectable on freshly isolated IECs. Receptor density was greater in surface vs crypt epithelial cells, but no significant differences were seen between normal and IBD epithelial cells. Expression of IL-1R and IL-6R was enhanced by LPS and IFN-gamma. Functionally, IL-1beta enhanced proliferation of the IEC cell line, DLD1, whereas GM-CSF treatment of de-differentiated crypt-like DLD1 and HT29 cells resulted in enhanced expression of ICAM-1. Furthermore, TNF-alpha treatment enhanced the secretion of IL-8 and GRO-alpha in HT29 cells, but not in freshly isolated IEC cultures. The differential binding and function of proinflammatory cytokines on IEC support the hypothesis that these cytokines may be involved in normal physiological processes as well as in regulating mucosal immune responses.     

Factors affecting Caco-2 intestinal epithelial cell interleukin-6 secretion

Intestinal epithelial cells (IEC) have previously been shown to produce several cytokines including interleukin-6 (IL-6). However, many factors which may regulate IL-6 secretion by human IEC still remain a mystery due in part to the lack of appropriate model cell lines and the difficulty of culturing human IEC over long periods of time. We have determined that the human colonic carcinoma cell line Caco-2 is capable of secreting IL-6 when stimulated by the inflammatory cytokines IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and stimulation of these cells with IL-1beta plus TNF-alpha induced a synergistic enhancement of IL-6 secretion. The inflammatory cytokine-induced enhancement in IL-6 secretion was greatest when the cells were cultured in a 10% CO2 atmosphere as compared to cells grown in 5% CO2, suggesting that environmental CO2 levels may affect IEC cytokine secretion. Finally, long-term culture of the Caco-2 cells to induce cellular differentiation had no effect on the capacity of these cells to produce IL-6, indicating that the regulation of IL-6 secretion was not affected by differentiation. Taken together, these studies provide important information on the factors which regulate IL-6 secretion by human IEC as they may contribute to the cytokine network during a mucosal inflammation. The results also suggest that the Caco-2 cell line is an appropriate model for further studies on the regulation of cytokine secretion by human IEC.     

Tumor-specific cytokine release by donor T cells induces an effective host anti-tumor response through recruitment of host naive antigen presenting cells

We recently reported that tumor eradication induced by immunotherapy (IT) in a congenic mouse model using tumor infiltrating lymphocytes (TIL) + recombinant interleukin-2 (rIL-2) is dependent on recruitment of naive host immune cells at the tumor sites. The recruitment of host immune cells was induced mainly through a local secretion of interferon-gamma (IFN-gamma) produced by donor T cells. We now further investigated how a non-specific inflammatory response progresses to a host T-cell-mediated tumor-specific response. In cross-over experiments using MCA-105 and MCA-205 sarcoma tumors, pulmonary metastatic disease was eradicated only in mice treated with tumor-matched TIL + rIL-2. In vitro, TIL stimulated with the tumor of origin secreted relatively high levels of IFN-gamma and granulocyte-macrophage colony stimulating factor (GM-CSF) compared to TIL stimulated with mismatched tumor cells. In lungs of tumor-bearing mice treated with matched TIL + rIL-2, significant increases in the percentages of IFN-gamma, GM-CSF and tumor necrosis factor-alpha (TNF-alpha) positive cells were detected, as well as of macrophages, natural killer (NK) cells and dendritic cells. Depletion of macrophages or NK cells did not inhibit the efficacy. In contrast, depletion of dendritic cells partially inhibited the efficacy of the treatment. Combined depletion of dendritic cells and macrophages abrogated more than 80% of the efficacy. Our data suggest that successful IT may require 3 steps: (1) release of inflammatory cytokines by donor TIL after restimulation by tumor cells; (2) infiltration of host immune cells in response to local cytokine production; and (3) activation of tumor-specific host immune cells by dendritic cells and to a lesser extent by macrophages.     

IL-4 enhances IEC-6 intestinal epithelial cell proliferation yet has no effect on IL-6 secretion

Intestinal epithelial cells (IEC) form an important line of defence at the intestinal mucosa by providing a barrier to lumenal contents and also by their ability to secrete various inflammatory cytokines. Recently, several T cell-derived cytokines have been shown to regulate specific IEC functions. In this study, the effect of IL-4 on IEC proliferation and secretion of the inflammatory cytokine IL-6 was investigated using the non-transformed rat IEC-6 intestinal epithelial cell line. Recombinant rat (rr)IL-4 was found to enhance IEC-6 cell proliferation over 4 days of culture, and this enhancement was dose-dependent. Further studies using specific antibodies confirmed that IL-4 induced the effect and that the effect was not mediated by autocrine-produced transforming growth factor-alpha. However, IL-4 did not induce IL-6 secretion by the IEC-6 cells, nor did it alter IL-1 beta-induced IL-6 secretion. These results indicate that T cells may be capable of regulating IEC proliferation via the secretion of IL-4 without altering the capacity of the IEC to function in the inflammatory response by secreting IL-6.     

MU-opioid receptor-knockout mice: role of mu-opioid receptor in morphine mediated immune functions

The role of the mu-opioid receptor in immune function was investigated using mu-opioid receptor knockout mice (MOR-KO). Morphine modulation of several immune functions, including macrophage phagocytosis and macrophage secretion of TNF-alpha, was not observed in the MOR-KO animals, suggesting that these functions are mediated by the classical mu-opioid receptor. In contrast, morphine reduction of splenic and thymic cell number and mitogen-induced proliferation were unaffected in MOR-KO mice, as was morphine inhibition of IL-1 and IL-6 secretion by macrophages. These latter results are consistent with morphine action on a naloxone insensitive morphine receptor, a conclusion supported by previous studies characterizing a nonopioid morphine binding site on immune cells. Alternatively, morphine may act either directly or indirectly on these cells, by a mechanism mediated by either delta or kappa opioid receptors.     

Examinations with the Cardiff Acuity Test

Intestinal epithelial cells (IEC) can exist as polarized cells and are capable of secreting interleukin-6 (IL-6), yet it has not been determined if this IL-6 is secreted in a polarized fashion. Using the non-transformed rat IEC-6 intestinal epithelial cell line grown on microporous membrane inserts, we have determined that these cells were capable of secreting IL-6 preferentially to the basal surface when stimulated basally with IL-1 beta. In contrast, stimulation of the cells with TNF-alpha resulted in an equal level of IL-6 secretion to the apical and basal surfaces, regardless of whether the cells were stimulated by the apical or basal route. Experiments designed to test the permeability of the IEC-6 cell layer to apically added sodium fluorescein confirmed that neither IL-1 beta nor TNF-alpha altered the integrity of the cell layer after three days. These results suggest that IEC may have the capacity to secret IL-6 in different patterns depending upon the stimulation received. This would allow communication between the IEC and lamina propria cells via basal secretion and rapid communication between IEC via apical secretion.     

Macrophages expressing a fusion protein derived from bactericidal/permeability-increasing protein and IgG are resistant to endotoxin

OBJECTIVES: To generate a recombinant fusion protein (FP) based on the endotoxin-binding domain of bactericidal/permeability-increasing protein (BPI) and the constant domain of IgG and to test its ability to inhibit lipopolysaccharide (LPS)-induced macrophage tumor necrosis factor alpha (TNF-alpha) secretion. DESIGN: A murine macrophage cell line, RAW 264.7, was transfected with a BPI-IgG FP before incubation with LPS. The amount of LPS-induced TNF-alpha protein secreted was measured and compared with that secreted by cells transfected with a control construct. SETTING: Basic science research laboratory. MAIN OUTCOME MEASURES: Secreted TNF-alpha protein concentration. RESULTS: After transfection, RAW 264.7-cell FP expression was detected in cell lysates and supernatants. At each LPS dose tested, cells transfected with the FP gene secreted less TNF-alpha than did cells transfected with a control construct. CONCLUSIONS: The FP possesses substantial antiendotoxin activity, as delineated by inhibition of LPS-induced TNF-alpha secretion by murine macrophages transfected with the fusion gene construct. In the future, such FP may be used as a clinical reagent to reduce the morbidity and mortality associated with serious gram-negative bacterial infections in surgical patients.     

Neurokinin-1 (NK-1) receptor is required in Clostridium difficile- induced enteritis

Toxin A, a 308,000-Mr enterotoxin from Clostridium difficile, mediates antibiotic-associated diarrhea and colitis in humans. Injection of toxin A into animal intestine triggers an acute inflammatory response characterized by activation of sensory neurons and immune cells of the intestinal lamina propria, including mast cells and macrophages, and migration of circulating neutrophils in the involved intestinal segment. In this study we show that mice genetically deficient in the neurokinin-1 receptor are protected from the secretory and inflammatory changes as well as from epithelial cell damage induced by toxin A. The protective effect of neurokinin-1R deletion correlates with diminished intestinal levels of the cytokine TNF-alpha and its mRNA and the leukocyte enzyme myeloperoxidase. These results demonstrate a major requirement for substance P receptors in the pathogenesis of acute inflammatory diarrhea.     

Intramedullary pressure reduction in the femur shaft in total hip endoprosthesis. Effect on the rate of thrombosis]

PROBLEM: Factors in seminal plasma stimulate an intense but transient inflammatory response in the murine endometrium at mating. The aim of our current studies is to delineate the cytokine-leukocyte interactions comprising this response and to elucidate the significance of these events in changes in the maternal immune system and as determinants of pregnancy outcome. METHOD: We have reviewed our recent findings. RESULTS: Transforming growth factor (TGF)-beta1 has been identified as the inflammation-inducing moiety in seminal plasma. Seminal TGFbeta1 initiates endometrial leukocyte infiltration by up-regulating epithelial cell expression of granulocyte-macrophage colony-stimulating factor. Other cytokines and chemokines including regulated and normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and monocyte chemotactic protein-1 are also implicated as mediators of macrophage and granulocyte recruitment and activation. One consequence of this inflammatory response is the induction of a transient state of hyporesponsiveness to paternal major histocompatibility class I antigens. CONCLUSION: Our studies suggest that semen may play a critical role in providing the antigenic and environmental signals necessary to initiate an appropriate maternal immune response to the conceptus during pregnancy.     

HindIII liposomes suppress delayed-type hypersensitivity responses in vivo and induce epidermal IL-10 in vitro

The purpose of this study was to evaluate the effects of resident islet macrophage activation on beta cell function. Treatment of freshly isolated rat islets with TNF-alpha and LPS results in a potent inhibition of glucose-stimulated insulin secretion. The inhibitory actions of TNF + LPS are mediated by the intraislet production and release of IL-1 followed by IL-1-induced inducible nitric oxide synthase (iNOS) expression by beta cells. The IL-1R antagonist protein completely prevents TNF + LPS-induced nitrite production, iNOS expression and the inhibitory effects on glucose-stimulated insulin secretion by rat islets. Resident macrophages appear to be the source of IL-1, as a 7-day culture of rat islets at 24 degrees C (conditions known to deplete islets of lymphoid cells) prevents TNF + LPS-induced iNOS expression, nitrite production, and the inhibitory effects on insulin secretion. In addition, macrophage depletion also inhibits TNF + LPS-induced IL-1alpha and IL-1beta mRNA expression in rat islets. Immunocytochemical colocalization of IL-1beta with the macrophage-specific marker ED1 was used to provide direct support for resident macrophages as the islet cellular source of IL-1. IL-1beta appears to mediate the inhibitory actions of TNF + LPS on beta cell function as TNF + LPS-induced expression of IL-1beta is fourfold higher than IL-1alpha, and Ab neutralization of IL-1beta prevents TNF + LPS-induced nitrite production by rat islets. These findings support a mechanism by which the activation of resident islet macrophages and the intraislet release of IL-1 may mediate the initial dysfunction and destruction of beta cells during the development of autoimmune diabetes.     

Polyethylene particles of a 'critical size' are necessary for the induction of cytokines by macrophages in vitro

Particulate wear debris from total hip prosthetic components can stimulate macrophages to produce mediators of osteolysis which may cause aseptic implant loosening. This study evaluated the in vitro response of murine peritoneal macrophages to polyethylene particles of definitive size distributions at varying volume doses. Ceridust 3615 polyethylene particles with a mean size of 0.21, 0.49, 4.3 and 7.2 microm and GUR 120 polyethylene resin with a mean size of 88 microm were co-cultured with C3H murine peritoneal macrophages at volume (microm)3 to cell number ratios of 100:1, 10:1, 1:1 and 0.1: 1. The secretion of IL-6, IL-1beta and TNF-alpha was determined by ELISA. Significantly elevated levels of TNF-alpha and IL-1beta were determined at 100:1 ratios when the macrophages were challenged with particles with a mean size of 0.49, 4.3 and 7.2 microm, and at 10:1 ratios for particles with a mean size of 0.49 and 4.3 microm. IL-6 production was significantly elevated at 100:1 ratios for mean particle sizes of 0.49 and 4.3 microm. Particles outside this range produced considerably less cytokine suggesting that both the size and volume (or number) of polyethylene particles are critical factors in macrophage activation. Therefore particles in the phagocytosable size range of 0.3-10 microm appear to be the most biologically active.     

Local and systemic effects of macrophage cytokines in intestinal inflammation

The activation of macrophages and newly recruited monocytes appears to be common to both Crohn's disease and ulcerative colitis, despite different inductive stimuli. Similar activation occurs acutely during the course of invasive intestinal infections such as shigellosis, but is then usually downregulated. The macrophage cytokines tumor necrosis factor-alpha and interleukin-1 (IL-1) are centrally involved in the local inflammatory response, and blockade of either cytokine greatly attenuates the inflammatory lesion. Induction of focal vascular thrombosis and matrix degradation are thought to be an important component of this focal damage. Both cytokines and IL-6 are now recognized to contribute to the systemic effects of intestinal disease, including growth suppression, anorexia, and chronic anemia. Disturbance of sleep patterns, mood, and affect may also occur, and recent evidence points towards bidirectional interplay between macrophage cytokines and central nervous system function.     

The role of mononuclear phagocytes and dendritic cells in allergic inflammation

Mononuclear-phagocytic system is a diffuse network of cells which includes monoblasts and promonocytes of the bone marrow, blood monocytes, as well as free and fixed tissue macrophage cells. In different tissues and organs macrophages acquire different morphological and functional properties under the influence of the local tissue factors. Interaction of macrophages with other cells and molecules is performed via the large number of different receptors resulting in activation of the macrophage cell, accompanied by a series of morphological and metabolic changes which potentiate all its functions. Activated macrophage cells were found in certain diseases. Macrophages and dendritic cells are associated with all aspects of immunity. Owing to their capacity to undergo phagocytosis they are of the utmost importance for unspecific defense from microorganisms. As accessory cells they also participate in cellular and humoral immunity, being at the same time effector cells owing to their capacity of antigen presentation. Moreover, they also participate in immune response regulation owing to their influence on the function of other cells, including mast cells, basophilic leukocytes and T lymphocytes, in which they may influence differentiation toward Th1 or Th2 and cytokine milieu favorable for allergic reaction. Dendritic cells are the most important antigen-presenting cells and thus, they play a major role in activation of helper T lymphocytes, and mode of antigen presentation is significant for regulation of the nature and intensity of the immune response. Pulmonary macrophage cells have been most thoroughly studied, and the observed changeability of their functional and morphological characteristics is of the utmost importance for studying of the pathogenetic properties and regulation of the chronic inflammatory response in bronchial asthma.     

Particle digestibility is required for induction of the phosphatidylserine recognition mechanism used by murine macrophages to phagocytose apoptotic cells

One of the characteristic features of programmed cell death in vivo is the rapid recognition and removal of apoptotic cells by macrophages. Although there are several potential mechanisms by which the macrophage can identify a cell as apoptotic, it has been shown recently that murine-elicited macrophages stereospecifically recognize phosphatidylserine (PS) exposed on the surface of apoptotic cells. The particulate stimulus, beta-1, 3-glucan, stimulates bone marrow-derived macrophages to express several characteristics of inflammatory macrophages, and induced these cells to recognize PS on apoptotic cells; this activity was correlated with the ability to form rosettes with PS-expressing RBC. Induction of PS recognition in bone marrow-derived macrophages was associated with digestibility of the stimulus, because L, but not D amino acid particles or latex, were able to stimulate macrophage recognition of PS. The requirement for digestibility could be bypassed by the addition of exogenous TGF-beta, which induced macrophage recognition of PS after stimulation with either latex or D amino acid particles. That endogenously produced TGF-beta played a role in the glucan-stimulated response was indicated by the ability of anti-TGF-beta antibodies to inhibit digestible particle-induced recognition of PS. The induction of the PS recognition mechanism correlated well with the expression of other markers for the inflammatory phenotype. These studies indicate that the PS receptor may be a marker for the inflammatory phenotype, which appears to be induced by the phagocytosis of particulate digestible stimuli. Endogenously produced TGF-beta is suggested to play an autocrine or paracrine priming role in the induction of the PS receptor.     

In vitro and in vivo dependency of chemokine generation on C5a and TNF-alpha

Under a variety of conditions, alveolar macrophages can generate early response cytokines (TNF-alpha, IL-1), complement components, and chemotactic cytokines (chemokines). In the current studies, we determined the requirements for TNF-alpha and the complement activation product C5a in chemokine production in vitro and in vivo. Two rat CXC chemokines (macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC)) as well as three rat CC chemokines (MIP-1alpha, MIP-1beta, and monocyte chemoattractant protein (MCP)-1) were investigated. Chemokine generation in vitro was studied in rat alveolar macrophages stimulated with IgG immune complexes in the absence or presence of Abs to TNF-alpha or C5a. The rat lung injury model induced by IgG immune complex deposition was employed for in vivo studies. Abs to TNF-alpha or C5a were administered intratracheally or i.v., and effects on chemokine levels in bronchoalveolar lavage fluids were quantitated by ELISA. Both in vitro and in vivo studies demonstrated the requirements for TNF-alpha and C5a for full generation of CXC and CC chemokines. In vitro and in vivo blockade of TNF-alpha or C5a resulted in significantly reduced production of chemokines. Supernatant fluids from in vitro-stimulated macrophages revealed by Western blot analysis the presence of C5a/C5adesArg, indicating intrinsic generation of C5a/C5adesArg by alveolar macrophages and explaining the higher efficiency of intratracheal vs i.v. blockade of C5a in reducing chemokine production. These results underscore the central role of both TNF-alpha and C5a, which appear to function as autocrine activators to promote CXC and CC chemokine generation by alveolar macrophages.