Effects of estradiol on lipopolysaccharide and Pam3Cys stimulation of CCL20/macrophage inflammatory protein 3 alpha and tumor necrosis factor alpha production by uterine epithelial cells in culture

We have previously demonstrated that rat uterine epithelial cells (UEC) produce CCL20/macrophage inflammatory protein 3 alpha (MIP3alpha) and tumor necrosis factor alpha (TNF-alpha) in response to live and heat-killed Escherichia coli and to the pathogen-associated molecular patterns (PAMP) lipopolysaccharide (LPS) and Pam3Cys. To determine whether estradiol (E2) modulates PAMP-induced CCL20/MIP3alpha and TNF-alpha secretion, primary cultures of rat UEC were incubated with E2 for 24 h and then treated with LPS or Pam3Cys or not treated for an additional 12 h. E2 inhibited the constitutive secretion of TNF-alpha and CCL20/MIP3alpha into culture media. Interestingly, E2 pretreatment enhanced CCL20/MIP3alpha secretion due to LPS and Pam3Cys administration. In contrast, and at the same time, E2 lowered the TNF-alpha response to both PAMP. To determine whether estrogen receptors (ER) mediated the effects of E2, epithelial cells were incubated with E2 and/or ICI 182,780, a known ER antagonist. ICI 182,780 had no effect on E2 inhibition of constitutive TNF-alpha and CCL20/MIP3alpha secretion. In contrast, ICI 182,780 reversed the stimulatory effect of E2 on LPS- and/or Pam3Cys-induced CCL20/MIP3alpha secretion as well as partially reversed the inhibitory effect of E2 on TNF-alpha production by epithelial cells. Overall, these results indicate that E2 regulates the production of TNF-alpha and CCL20/MIP3alpha by UEC in the absence as well as presence of PAMP. Since CCL20/MIP3alpha has antimicrobial activity and is chemotactic for immune cells, these studies suggest that regulation of CCL20/MIP3alpha and TNF-alpha by E2 and PAMP may have profound effects on innate and adaptive immune responses to microbial challenge in the female reproductive tract.     

Effect of oestradiol on PAMP-mediated CCL20/MIP-3 alpha production by mouse uterine epithelial cells in culture

The present study was undertaken to establish whether mouse uterine epithelial cells produce CCL20/macrophage inflammatory protein 3 alpha (CCL20/MIP-3 alpha) and to determine whether secretion is under hormonal control and influenced by pathogen-associated molecular patterns (PAMPs). In the absence of PAMPs, polarized uterine epithelial cells grown to confluence on cell culture inserts constitutively secreted CCL20/MIP-3 alpha with preferential accumulation into the apical compartment. When epithelial cells were treated with the Toll-like receptor (TLR) agonists Pam3Cys (TLR2/1), peptidoglycan (TLR2/6) or lipopolysaccharide (LPS; TLR4), CCL20/MIP-3 alpha increased rapidly (4 hr) in both apical and basolateral secretions. Time-course studies indicated that responses to PAMPs added to the apical surface persisted for 12-72 hr. Stimulation with loxoribin (TLR7) and DNA CpG motif (TLR9) increased basolateral but not apical secretion of CCL20/MIP-3 alpha. In contrast, the viral agonist Poly(I:C) (TLR3) had no effect on either apical or basolateral secretion. In other studies, we found that oestradiol added to the culture media decreased the constitutive release of CCL20/MIP-3 alpha. Moreover, when added to the culture media along with LPS, oestradiol inhibited LPS-induced increases in CCL20/MIP-3 alpha secretion into both the apical and basolateral compartments. In summary, these results indicate that CCL20/MIP-3 alpha is produced in response to PAMPs. Since CCL20/MIP-3 alpha is chemotactic for immature dendritic cells, B cells and memory T cells and has antimicrobial properties, these studies suggest that CCL20/MIP-3 alpha production by epithelial cells, an important part of the innate immune defence in the female reproductive tract, is under hormonal control and is responsive to microbial challenge.     

Effect of Escherichia coli and Lactobacillus rhamnosus on macrophage inflammatory protein 3 alpha, tumor necrosis factor alpha, and transforming growth factor beta release by polarized rat uterine epithelial cells in culture

Entry of bacteria from the vagina into the uterus raises the question of uterine epithelial cell (UEC) signaling in response to the presence of bacteria. Our model system helps to define microbially elicited UEC basolateral cytokine release, important in regulating underlying stromal immune cell protection. UECs from adult rats were grown in cell culture inserts to establish a confluent polarized monolayer as was determined by transepithelial resistance (TER). Polarized epithelial cell cultures were treated apically with live or heat-killed Escherichia coli or Lactobacillus rhamnosus prior to collection of basolateral media after 24 h of incubation. Coculture of polarized UECs with live E. coli had no effect on epithelial cell TER. In response to exposure to live E. coli, epithelial cell basolateral release of macrophage inflammatory protein 3 alpha (MIP3 alpha) and tumor necrosis factor alpha (TNF-alpha) increased at a time when basolateral release of biologically active transforming growth factor beta (TGF-beta) decreased. Incubation of UECs with heat-killed E. coli resulted in an increased basolateral release of MIP3 alpha and TNF-alpha, without affecting TER or TGF-beta. In contrast to E. coli, live or heat-killed L. rhamnosus had no effect on TER or cytokine release. These studies indicate that polarized rat UECs respond to gram-negative E. coli by releasing the cytokines MIP3 alpha and TNF-alpha, signals important to both the innate and adaptive immune systems. These findings suggest that UEC responses to bacteria are selective and important in initiating and regulating immune protection in the female reproductive tract.     

Role of MyD88 in diminished tumor necrosis factor alpha production by newborn mononuclear cells in response to lipopolysaccharide

Human newborns are more susceptible than adults to infection by gram-negative bacteria. We hypothesized that this susceptibility may be associated with a decreased response by leukocytes to lipopolysaccharide (LPS). In this study, we compared LPS-induced secretion of tumor necrosis factor alpha (TNF-alpha) by mononuclear cells (MNC) from adult peripheral blood and newborn umbilical cord blood in vitro and attempted to determine the mechanisms involved in its regulation. At a high concentration of LPS (10 ng/ml) and in the presence of autologous plasma, MNC from adults and newborns secreted similar amounts of TNF-alpha. However, in the absence of plasma, MNC from newborns secreted significantly less TNF-alpha compared to MNC from adults. Moreover, at a low concentration of LPS (0.1 ng/ml) and in the presence of plasma, TNF-alpha secretion was significantly lower for newborn MNC compared to adult MNC. Adults and newborns had similar numbers of CD14 and Toll-like receptor 4 (TLR-4)-positive cells as measured by flow cytometry. However, the intensity of the CD14 marker was greater for adult than for newborn cells. Incubation of cells with LPS led to an increase in CD14 and TLR-4 intensity for adult cells but not for newborn cells. The effect of LPS stimulation of adult or newborn cells was similar for ERK, p38, and IkappaBalpha phosphorylation, as well as IkappaBalpha degradation. Finally, we assessed levels of the TLR-4 adapter protein, the myeloid differentiation antigen 88 (MyD88). We found a direct relation between adult and newborn TNF-alpha secretion and MyD88, which was significantly decreased in newborn monocytes. Since TLR-4 signals intracellularly through the adapter protein, MyD88, we hypothesize that MyD88-dependent factors are responsible for delayed and decreased TNF-alpha secretion in newborn monocytes.     

Control of Salmonella dissemination in vivo by macrophage inflammatory protein (MIP)-3alpha/CCL20

While chemokines are clearly important in the generation of protective immunity, the role of individual chemokines in the control of bacterial infection is still poorly understood. In this study, we investigated the role of macrophage inflammatory protein (MIP)-3alpha/CCL20, a chemokine that attracts activated T and B lymphocytes and immature dendritic cells, in host responses to bacterial infection. CCL20 production was induced in subcutaneous tissue in the BALB/c mouse in response to Salmonella enteritidis, Staphylococcus aureus and zymosan, with S. enteritidis being the most potent. S. enteritidis induced CCL20 production in the spleen following either oral administration or injection into the peritoneal cavity. In contrast, no increase was observed in the Peyer's patches. In this model, following intraperitoneal injection, dose-dependent colonization of the spleen and Peyer's patches by S. enteritidis, expression of IFNgamma and IL-4, and production of antibodies against the S. enteritidis surface antigen SefA were observed. Prior treatment with neutralizing antibodies against CCL20 enhanced bacterial dissemination to the spleen and Peyer's patches and strongly biased the IFNgamma/IL-4 ratio towards a type 2 profile in the spleen, while the humoral response was unaffected. In contrast, treatment with neutralizing anti-MIP-1alpha/CCL3 antibodies enhanced the bacterial burden in the Peyer's patches but not in the spleen, had no significant effect on the cytokine ratio, but significantly inhibited anti-SefA production. Together, these results demonstrate an important role for CCL20 in the control of bacterial infection and more specifically in the regulation of cell-mediated immunity against intracellular bacteria such as S. enteritidis.     

Airway epithelial cells release MIP-3alpha/CCL20 in response to cytokines and ambient particulate matter

The initiation and maintenance of airway immune responses in Th2 type allergic diseases such as asthma are dependent on the specific activation of local airway dendritic cells (DCs). The cytokine microenvironment, produced by local cells, influences the recruitment of specific subsets of immature DCs and their subsequent maturation. In the airway, DCs reside in close proximity to airway epithelial cells (AECs). We examined the ability of primary culture human bronchial epithelial cells (HBECs) to synthesize and secrete the recently described CC-chemokine, MIP-3alpha/CCL20. MIP-3alpha/CCL20 is the unique chemokine ligand for CCR6, a receptor with a restricted distribution. MIP-3alpha/CCL20 induces selective migration of DCs because CCR6 is expressed on some immature DCs but not on CD14+ DC precursors or mature DCs. HBECs were stimulated with pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin (IL)-1beta or, because of their critical role in allergic diseases, IL-4 and IL-13. Cells were also exposed to small size-fractions of ambient particulate matter. Each of these stimuli induced MIP-3alpha/CCL20 gene and protein expression. Moreover, these agents upregulated mitogen-activated protein kinase pathways in HBECs. Inhibition of the ERK1/2 pathway or p38 reduced cytokine-induced MIP-3alpha/CCL20 expression. These data suggest a mechanism by which AEC may facilitate recruitment of DC subsets to the airway.     

Substance P regulates macrophage inflammatory protein 3alpha/chemokine C-C ligand 20 (CCL20) with heme oxygenase-1 in human periodontal ligament cells

Although substance P (SP), a potent proinflammatory peptide, is involved in inflammation and immune responses, the effect of SP on the expression of macrophage inflammatory protein 3alpha[MIP-3alpha, chemokine C-C ligand 20 (CCL20)] in periodontal ligament (PDL) cells is unknown. Equally enigmatic is the link between SP, the stress protein heme oxygenase-1 (HO-1), and CCL20 production. We investigated whether SP induces the release of chemokine CCL20 from immortalized PDL (IPDL) cells, and further clarify SP-mediated pathways. We also examined the relationship between HO-1 and CCL20 by treating PDL cells with SP. Incubating IPDL cells with SP increased expression of CCL20 mRNA and CCL20 protein in a dose-time-dependent manner. Highly selective p38 and extracellular-regulated kinase 1/2 (ERK1/2) inhibitors abrogated SP-induced expression of CCL20 in IPDL cells. SP is also responsible for initiating phosphorylation of IkappaB, degradation of IkappaB and activation of nuclear factor (NF)-kappaB. SP induced expression of HO-1 in both a concentration- and time-dependent manner, and CCL20 reflected similar patterns. The inductive effects of SP on HO-1 and CCL20 were enhanced by HO-1 inducer hemin and the membrane-permeable guanosine 3',5'-monophosphate (cGMP) analogue 8-bromo-cGMP. Conversely, this pathway was inhibited by the HO-1 inhibitor zinc protoporphyrin IX (ZnPP IX) and the selective inhibitor of guanylate cyclase, 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ). We report herein the pathway that connects SP along with other modulators of neuroimmunoregulation to the induction of HO-1 and the inflammatory mediator macrophage inflammatory protein (MIP)-3alpha/CCL20 in IPDL cells, which play an important role in the development of periodontitis or inflammation during orthodontic tooth movement.     

Production of macrophage inflammatory protein 3alpha (MIP-3alpha) (CCL20) and MIP-3beta (CCL19) by human peripheral blood neutrophils in response to microbial pathogens

Effects of bacterial pathogens on the production of macrophage inflammatory protein 3alpha (MIP-3alpha) and MIP-3beta from human peripheral blood neutrophils were investigated. Neutrophils produced both chemokines by coincubation with either gram-positive or gram-negative bacteria. Neutrophils may initiate antigen-specific immune responses through the release of these chemokines that are capable of promoting selective recruitment of dendritic cells and T-cell subsets.     

Neutrophils produce biologically active macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19.

Macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19 are members of the CC chemokine subfamily which exert their effects through specific receptors, CCR6 and CCR7, respectively. Previously, we have reported that human neutrophils have the capacity to produce a number of chemokines, including IL-8/CXCL8, GROalpha/CXCL1, IP-10/CXCL10, and MIG/CXCL9. Herein, we show that neutrophils also have the ability to express and release MIP-3alpha/CCL20 and MIP-3beta/CCL19 when cultured with either LPS or TNF-alpha. We also report that MIP-3alpha/CCL20 and MIP-3beta/CCL19 production by LPS-stimulated neutrophils is negatively modulated by IL-10. Remarkably, we found that supernatants harvested from stimulated neutrophils not only induced chemotaxis of both immature and mature dendritic cells (DC), but also triggered rapid integrin-dependent adhesion of CCR6- and CCR7-expressing lymphocytes to purified VCAM-1 and ICAM-1, respectively. Importantly, both chemotaxis and rapid integrin-dependent adhesion were dramatically suppressed by anti-MIP-3alpha/CCL20 and anti-MIP-3beta/CCL19 neutralizing antibodies, indicating that MIP-3alpha/CCL20 and MIP-3beta/CCL19 present in the supernatants were both biologically active. As these chemokines are primarily chemotactic for DC and specific lymphocyte subsets, the ability of neutrophils to produce MIP-3alpha/CCL20 and MIP-3beta/CCL19 might be significant in orchestrating the recruitment of these cell types to the inflamed sites and therefore in contributing to the regulation of the immune response.     

CCL20/macrophage inflammatory protein-3alpha production in LPS-stimulated neutrophils is enhanced by the chemoattractant formyl-methionyl-leucyl-phenylalanine and IFN-gamma through independent mechanisms

We have recently demonstrated that polymorphonuclear neutrophils (PMN), when cultured with LPS or TNF-alpha, have the capacity to release CCL20, a chemokine primarily chemotactic for immature dendritic cells and specific lymphocyte subsets. Here, we report that the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP), as well as the immunoregulatory cytokine IFN-gamma, can significantly up-modulate the production of neutrophil-derived CCL20 through entirely unrelated mechanisms. We found that fMLP dramatically up-regulates CCL20 mRNA expression and synthesis in neutrophils stimulated with LPS for 2-3 h, and that its effect takes place through CCL20 mRNA stabilization. In contrast, IFN-gamma potentiates CCL20 gene expression and production only after 21 h of LPS treatment, its effect being mediated by endogenous TNF-alpha in an autocrine fashion, as revealed using neutralizing anti-TNF-alpha antibodies added to IFN-gamma plus LPS-treated PMN. Finally, we demonstrate that activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in mediating the production of CCL20 induced by LPS (with or without IFN-gamma), whereas activation of p42/44 extracellular signal-regulated kinases (ERK) is involved in the enhancing effect of fMLP. Taken together, these findings identify novel biological actions exerted by fMLP and IFN-gamma, potentially involved in the orchestration of inflammatory and immune responses within epithelial and mucosal tissue.     

Primary human alveolar type II epithelial cell CCL20 (macrophage inflammatory protein-3alpha)-induced dendritic cell migration

Inhalation of antigenic matter stimulates rapid recruitment of dendritic cells (DCs) into the lung. Recent studies propose that the chemokine CCL20 (macrophage inflammatory protein-3alpha) may play an important role in DC recruitment. We previously showed that primary human alveolar type II epithelial (ATII) cells are a rich source of chemokines and so hypothesized that the ATII cell produces CCL20 and might therefore be a key regulator of DC recruitment into the lung. Here, we show that primary human ATII cells, but not human alveolar macrophages, produce CCL20 both constitutively (403.5 +/- 85.4 pg/ml; 24 h) and in response to endotoxin (lipopolysaccharide) exposure (1,525.0 +/- 169.4 pg/ml; 1 mug/ml lipopolysaccharide; 24 h) in a time- and dose-dependent manner. In addition, we show that peripheral blood monocyte-derived CD1a+ DCs migrate in response to conditioned media from ATII cells but not those from alveolar macrophages; DC migration was significantly correlated with the amount of CCL20 (r(2) > 0.9; P < 0.05) detected in the media but not with any other chemokine measured. We therefore conclude that the alveolar epithelium is an important source of CCL20 in the lung and that the ATII cell may play a critical role in controlling the movement of DCs through the lung both under normal and inflammatory conditions.     

Interleukin-10 inhibits proinflammatory activation of endothelium in response to Borrelia burgdorferi or lipopolysaccharide but not interleukin-1beta or tumor necrosis factor alpha

Interleukin (IL)-10 is generally regarded as an anti-inflammatory cytokine, since it acts on a variety of cell types to suppress production of proinflammatory mediators. In inflammation, endothelial cells (EC) play a crucial role in recruiting leukocytes to sites of injury or infection. In this study, the actions of IL-10 on human umbilical vein EC were investigated. IL-10 reduced migration of monocytes and T lymphocytes across endothelium stimulated by lipopolysaccharide and decreased endothelial production of chemokines in response to lipopolysaccharide and Borrelia burgdorferi, the agent of Lyme disease. However, IL-10 did not affect these responses when EC were activated by the host proinflammatory cytokines IL-lbeta or tumor necrosis factor alpha. Moreover, IL-10 did not prevent up-regulation of the adhesion molecules E-selectin and intercellular adhesion molecule-1 by EC exposed to any of these activating agents. IL-10 therefore inhibits proinflammatory activation of EC in a manner that is selective with respect to stimulus and effector response.     

Local role for tumor necrosis factor alpha in the pulmonary inflammatory response to Mycobacterium tuberculosis infection

The local intrapulmonary role of tumor necrosis factor alpha (TNF-alpha) in a protective host response during acute and chronic infection with Mycobacterium tuberculosis is incompletely understood. To directly assess its role in the intrapulmonary immune response, we compared the responses of transgenic mice with a local pulmonary blockade of TNF-alpha (SPCTNFRIIFc mice) to mice with globally inhibited TNF-alpha (TNFRKO mice) and mice with normal immune systems (control mice). Consistent with previous reports, 100% of TNFRKO mice died by 28 days after aerosol infection, and these mice had markedly increased numbers of bacteria and widespread tissue necrosis in their lungs compared to controls. The median survival time of the SPCTNFRIIFc mice was 142 days, and 75% died by 180 days. Even though the numbers of bacteria in the lungs of the SPCTNFRIIFc mice were marginally increased compared to controls, these mice had a persistent neutrophilic inflammatory response and increased expression of proinflammatory cytokines (interleukin-1 alpha/beta [IL-1 alpha/beta], IL-18, gamma interferon, IL-6, and macrophage migration inhibitory factor) and chemokines (eotaxin, macrophage inflammatory protein 1 alpha/beta, gamma interferon-inducible protein 10, macrophage chemotaxic protein 1, and TCA-3) in their lungs. These studies with the SPCTNFRIIFc mice provide direct evidence for the local importance of TNF-alpha in the proper regulation of host defense to M. tuberculosis. The studies also suggest that when the local actions of TNF-alpha are selectively impaired in the lungs, tissue destruction and death ensue, at least in part, due to persistent expression of proinflammatory mediators that would normally be downregulated.     

Effect of lung surfactant collectins on bronchoalveolar macrophage interaction with Blastomyces dermatitidis: inhibition of tumor necrosis factor alpha production by surfactant protein D

Alveolar surfactant modulates the antimicrobial function of bronchoalveolar macrophages (BAM). Little is known about the effect of surfactant-associated proteins in bronchoalveolar lavage fluid (BALF) on the interaction of BAM and Blastomyces dermatitidis. We investigated BALF enhancement or inhibition of TNF-alpha production by BAM stimulated by B. dermatitidis. BAM from CD-1 mice were stimulated with B. dermatitidis without or with normal BALF, surfactant protein A-deficient (SP-A-/-) or surfactant protein D-deficient (SP-D-/-) BALF, or a mixture of SP-A-/- and SP-D-/- BALF. An enzyme-linked immunosorbent assay was used to measure tumor necrosis factor alpha (TNF-alpha) in culture supernatants. BALFs were standardized in protein concentration. BAM plus B. dermatitidis (BAM-B. dermatitidis) TNF-alpha production was inhibited > or = 47% by BALF or SP-A-/- BALF (at 290 or 580 microg of protein/ml, P < 0.05 to 0.01); in contrast, SP-D-/- BALF did not significantly inhibit TNF-alpha production. If SP-A-/- BALF was mixed in equal amounts with SP-D-/- BALF, TNF-alpha production by BAM-B. dermatitidis was inhibited (P < 0.01). Finally, pure SP-D added to SP-D-/- BALF inhibited TNF-alpha production by BAM-B. dermatitidis (P < 0.01). B. dermatitidis incubated with BALF and washed, plus BAM, stimulated 63% less production of TNF-alpha than did unwashed B. dermatitidis (P < 0.05). SP-D was detected by anti-SP-D antibody on BALF-treated unwashed B. dermatitidis in an immunofluorescence assay (IFA). The BALF depleted by a coating of B. dermatitidis lost the ability to inhibit TNF-alpha production (P < 0.05). 1,3-beta-Glucan was a good stimulator of BAM for TNF-alpha production and was detected on B. dermatitidis by IFA. beta-Glucan incubated with BALF inhibited the binding of SP-D in BALF to B. dermatitidis as demonstrated by IFA. Our data suggest that SP-D in BALF binds beta-glucan on B. dermatitidis, blocking BAM access to beta-glucan, thereby inhibiting TNF-alpha production. Thus, whereas BALF constituents commonly mediate antimicrobial activity, B. dermatitidis may utilize BALF constituents, such as SP-D, to blunt the host defensive reaction; this effect could reduce inflammation and tissue destruction but could also promote disease.     

Preexposure of murine macrophages to CpG oligonucleotide results in a biphasic tumor necrosis factor alpha response to subsequent lipopolysaccharide challenge

Bacterial DNA and synthetic oligonucleotides containing CpG sequences (CpG-DNA and CpG-ODN) provoke a proinflammatory cytokine response (tumor necrosis factor alpha [TNF-alpha], interleukin-12 [IL-12], and IL-6) and increased mortality in lipopolysaccharide (LPS)-challenged mice via a TNF-alpha-mediated mechanism. It was hypothesized that preexposure of macrophages to CpG-ODN would result in an increased TNF-alpha response to subsequent LPS challenge in vitro. Using the murine macrophage cell line RAW 264.7, we demonstrated both a rapid proinflammatory cytokine response (TNF-alpha) and a delayed inhibitory cytokine response (IL-10) with CpG-ODN. Preexposure of macrophages to CpG-ODN for brief periods (1 to 3 h) augmented TNF-alpha secretion and mRNA accumulation following subsequent LPS challenge (1 microg/ml). However, prolonged preexposure to CpG-ODN (6 to 9 h) resulted in suppression of the TNF-alpha protein and mRNA response to LPS. The addition of anti-IL-10 antibody to CpG-ODN during preexposure resulted in an increase in the LPS-induced TNF-alpha response over that induced by CpG-ODN preexposure alone. Thus, while brief preexposure of macrophages to CpG-ODN augments the proinflammatory cytokine response to subsequent LPS challenge, prolonged preexposure elicits IL-10 production, which inhibits the TNF-alpha response. Although the initial proinflammatory effects of CpG-DNA are well established, the immune response to CpG-DNA may also include autocrine or paracrine feedback mechanisms, leading to a complex interaction of proinflammatory and inhibitory cytokines.     

Augmentation of the neutrophil response to Naegleria fowleri by tumor necrosis factor alpha.

Conditioned medium from phytohemagglutinin-stimulated human mononuclear leukocytes, previously shown to activate neutrophils for amoeba killing, was found to contain high levels of tumor necrosis factor alpha (TNF-alpha) by an enzyme-linked immunosorbent assay. The effects of human recombinant TNF-alpha on the response of human neutrophils to the pathogenic free-living amoeba Naegleria fowleri was studied in vitro. The data showed that recombinant human TNF-alpha augmented the neutrophil respiratory burst (assessed by the cytochrome c reduction assay and lucigenin-dependent chemiluminescence assay) in response to amoebae opsonized with human serum. The priming effects of TNF-alpha were transient; marked enhancement was found with short 5- to 30-min preincubations of neutrophils with the cytokine. The enhancement of oxygen radical production was evident with 20 U of TNF-alpha per 10(6) neutrophils and continued to increase with up to 100 U. TNF-alpha also augmented the neutrophil lysosomal enzyme release in response to N. fowleri. The results support previous reports suggesting an important role of neutrophil cytokine activation for effective immunity against free-living amoebae.     

Interleukin-18 and gamma interferon production by oral epithelial cells in response to exposure to Candida albicans or lipopolysaccharide stimulation

Oral candidiasis is a collective name for a group of disorders caused by the dimorphic fungus Candida albicans. Host defenses against C. albicans essentially fall into two categories: specific immune mechanisms and local oral mucosal epithelial cell defenses. Since oral epithelial cells secrete a variety of cytokines and chemokines in response to oral microorganisms and since C. albicans is closely associated with oral epithelial cells as a commensal organism, we wanted to determine whether interleukin-18 (IL-18) and gamma interferon (IFN-gamma) were produced by oral epithelial cells in response to C. albicans infection and lipopolysaccharide (LPS) stimulation. Our results showed that IL-18 mRNA and protein were constitutively expressed by oral epithelial cells and were down-regulated by Candida infections but increased following LPS stimulation. Both C. albicans and LPS significantly decreased pro-IL-18 (24 kDa) levels and increased active IL-18 (18 kDa) levels. This effect was IL-1beta-converting-enzyme dependent. The increase in active IL-18 protein levels promoted the production of IFN-gamma by infected cells. No effect was obtained with LPS. Although produced only at an early stage, secreted IFN-gamma seemed to be a preferential response by oral epithelial cells to C. albicans growth. These results provide additional evidence for the contribution of oral epithelial cells to local (direct contact) and systemic (IL-18 and IFN-gamma production) defense against exogenous stimulation such as C. albicans infection or LPS stimulation.