Evidence of dysregulated cytokine production by sinus lavage and peripheral blood mononuclear cells in patients with treatment-resistant chronic rhinosinusitis.

BACKGROUND: Treatment-resistant chronic rhinosinusitis (CRS) imposes a clinical challenge. Its pathogenesis may be associated with dysregulated immune/inflammatory responses in the sinus. OBJECTIVE: To evaluate production of types 1 and 2 T cytokines (interferon gamma [IFN-gamma] and interleukin [IL] 5/IL-4, respectively) and regulatory/inflammatory cytokines (IL-10, IL-12, and IL-18) by sinus lavage (SL) cells and peripheral blood mononuclear cells (PBMCs) in patients with treatment-resistant CRS. METHODS: Sample SL cells and PBMCs obtained from 19 patients with treatment-resistant CRS were cultured with or without stimuli, and cytokine levels in the supernatant were measured using enzyme-linked immunosorbent assay. Control PBMC samples were obtained from 26 children. RESULTS: Chronic otitis media was found in 15 patients. Neutrophils and/or epithelial cells were dominant in SL cells. IFN-gamma, IL-12p40, and IL-10 (>100 pg/mL) were detected in SL cell cultures from 12, 9, and 8 patients, respectively. Production of IL-12p40 and IL-18 by SL cells correlated positively with phytohemagglutinin and IL-12p70 stimuli. In 12 patients, we detected IL-18 (>100 pg/mL) in SL cell cultures without stimuli, whereas PBMCs produced little IL-18, irrespective of stimuli. There was no correlation between cytokine levels produced by SL cells and PBMCs, except for IL-12p40 produced using IL-18. Decreased IFN-gamma production by PBMCs was observed in 6 patients with CRS compared with controls, but 4 of them had elevated IFN-gamma production by SL cells. Production of IL-12p40 by PBMCs was higher in 10 patients with CRS than in controls, and 7 of these patients had lower IL-10 production, resulting in an increased IL-12p40/IL-10 ratio. CONCLUSIONS: There is a role for locally produced regulatory cytokines in IFN-gamma production in the sinus in patients with treatment-resistant CRS. However, aberrant cytokine production patterns by PBMCs can be detected at high frequency in these patients, indicating that this can be used as a prognostic marker for patients with CRS.

Proinflammatory and regulatory cytokine production associated with innate and adaptive immune responses in children with autism spectrum disorders and developmental regression.

We determined innate and adaptive immune responses in children with developmental regression and autism spectrum disorders (ASD, N=71), developmentally normal siblings (N=23), and controls (N=17). With lipopolysaccharide (LPS), a stimulant for innate immunity, peripheral blood mononuclear cells (PBMCs) from 59/71 (83.1%) ASD patients produced >2 SD above the control mean (CM) values of TNF-alpha, IL-1beta, and/or IL-6 produced by control PBMCs. ASD PBMCs produced higher levels of proinflammatory/counter-regulatory cytokines without stimuli than controls. With stimulants of phytohemagglutinin (PHA), tetanus, IL-12p70, and IL-18, PBMCs from 47.9% to 60% of ASD patients produced >2 SD above the CM values of TNF-alpha depending on stimulants. Our results indicate excessive innate immune responses in a number of ASD children that may be most evident in TNF-alpha production.

Polycyclic aromatic hydrocarbon diol epoxides increase cytosolic Ca(2+) of airway epithelial cells.

Polycyclic aromatic hydrocarbons (PAHs) increase cytosolic Ca(2+) concentration ([Ca(2+)](i)) in lymphocytes and mammary epithelial cells, but little is known regarding their effects on [Ca(2+)](i) in airway epithelium. We hypothesized that benzo[a]pyrene (BP) and/or anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), a carcinogenic BP metabolite, increases [Ca(2+)](i) in untransformed human small airway epithelial (SAE) cells and that their effects on [Ca(2+)](i) are directly proportional to carcinogenicity. SAE [Ca(2+)](i) was determined by a ratiometric digital Ca(2+) imaging system. BPDE increased SAE [Ca(2+)](i) within 20 s in media with high (1 mM) and low (10 nM) Ca(2+) at a threshold concentration of 0.2 nM. Elevation of [Ca(2+)](i) persisted longer with high Ca(2+). Neither BP nor solvent altered [Ca(2+)](i). Thapsigargin and inositol 1,4,5- phosphate receptor (InsP(3)R) antagonists inhibited this BPDE action with low Ca(2+). We conclude that BPDE but not BP increases [Ca(2+)](i) partly by mobilizing Ca(2+) from cytosolic stores through an InsP(3)R. The most potent carcinogenic PAH diol epoxide increased in SAE [Ca(2+)](i) at the lowest threshold concentration, suggesting that carcinogenicity is directly proportional to the action of PAHs on SAE [Ca(2+)](i). Short-term exposure to BPDE 36 to 48 h before the study rendered SAE cells less sensitive to BPDE, suggesting that BPDE may also induce persistent changes in Ca(2+) signaling pathways.

Dietary ribonucleotides modulate type 1 and type 2 T-helper cell responses against ovalbumin in young BALB/cJ mice.

Dietary ribonucleotides have been shown to augment type 1 T-helper cell (Th1) responses to a protein antigen (Ag) in Th1-prone C57BL/6 mice, but their effects on type 2 Th (Th2)-prone mice are unknown. BALB/cJ mice have skewed Th2 responses against ovalbumin (OVA), characterized by augmented production of Th2 cytokines and immunoglobulin (Ig)G1/IgE antibodies (Ab); Th1 responses augment IgG2a Ab production, whereas Th2 responses augment IgG1/IgE Ab production. In this study, we determined the effects of dietary ribonucleotides obtained from yeast on the balance of Th1/Th2 responses against OVA in young BALB/cJ mice. Mice were fed a ribonucleotide-free (NF) or ribonucleotide-supplemented (NS) diet (4.74 g nucleotides/kg diet) and given OVA (10 microg/dose) with incomplete Freund's adjuvant (IFA) at 3 and 6 wk. We assessed T-cell responses in the regional draining lymph nodes (LN) by measuring production and expression of Th1/Th2 cytokines, interferon-gamma (IFN-gamma) and interleukin-5 (IL-5), respectively. Anti-OVA IgG subclass and IgE Ab levels were determined 3 wk after the first OVA challenge and 5 d and 2 wk after the second OVA challenge. Dietary ribonucleotides significantly augmented OVA-specific IFN-gamma production by the regional draining LN cells after the first and second OVA challenges. The NS diet increased anti-OVA IgG2a Ab levels after the first OVA challenge and both anti-OVA IgG2a and anti-OVA IgG2b after the second challenge. OVA-specific IgG1 and IgE Ab levels were lower (P < 0.05) after the second OVA challenge in mice fed the NS diet. Dietary ribonucleotides did not affect production or expression of IL-5. Our findings thus indicate that in Th2-prone BALB/c J mice, dietary ribonucleotides modulated skewed Th2 responses against OVA toward Th1 as measured by production of IFN-gamma, a Th1 cytokine, and changes in anti-OVA Ab isotype levels.

Impairment of STAT activation by IL-12 in a patient with atypical mycobacterial and staphylococcal infections.

IL-12 plays a pivotal role in the stimulation of immune responses against intracellular infections. This role is manifested in the increased susceptibility to atypical mycobacterial and salmonella infections among individuals whose lymphocytes lack expression of IL-12Rbeta1. Here, we report on a patient with Mycobacterium avium infection, recurrent Staphylococcus aureus sinusitis, and multiple adverse drug reactions whose T cells were unable to produce IFN-gamma or proliferate in response to IL-12 despite the expression of wild-type IL-12Rbeta1 and IL-12Rbeta2. The defect in these functional responses to IL-12 was selective, as cytolytic activity induced by IL-12 was intact, and lymphocytes were responsive to stimulation by IL-2. An examination of cytokine signaling revealed that STAT4 and extracellular regulated kinase 1 (ERK1) activation by IL-12 was intact, whereas the activation of STAT1, -3, and -5 by IL-12 was lost. This impairment of STAT activation was specific for IL-12, as STAT activation by IL-2, IL-15, and IFN-gamma was unaffected. These findings demonstrate that the activation of STAT4 alone is not sufficient for IL-12-induced IFN-gamma production and proliferation and suggest that other STATs play a role in these responses to IL-12. While the etiology of the impaired IL-12 signaling in this patient has not yet been elucidated, the absence of mutations in IL-12Rbeta1 or IL-12Rbeta2 and the preservation of STAT4 activation raise the possibility that there may be a mutation in an as yet undiscovered component of the IL-12 signaling complex that is normally required for the recruitment and activation of STAT1, -3, and -5.

Dietary nucleotides modulate antigen-specific type 1 and type 2 t-cell responses in young c57bl/6 mice.

Mice fed a nucleotide-free (NF) diet have impaired antibody (Ab) responses. The mechanisms responsible for this effect are not understood but may be related to specific changes in T-cell functions. The objective of this study was to examine the effects of dietary nucleotides on serum immunoglobulin-G (IgG) subclass Ab levels and T-cell cytokine production by cells from the lymph nodes draining the site of antigen challenge. C57BL/6 (B6) mice were fed an NF diet or the same diet supplemented with nucleotides (NS diet; 4.74 g nucleotides/kg). Keyhole limpet hemocyanin (KLH; 25 microg/dose), a T-dependent protein neoantigen, was given with incomplete Freund's adjuvant. We administered KLH at 3, 6, and 9 wk to determine primary and secondary responses. Anti-KLH IgG subclass Ab levels were measured 3 wk after the first KLH challenge and 2 wk after the last KLH challenge. T-cell responses in lymph nodes draining the site of KLH challenge were assessed 5 d after the primary and 14 d after the final KLH challenge. We measured mRNA expression and production of interferon-gamma and interleukin-5, type-1 and type-2 T-cell cytokines, respectively. Anti-KLH IgG2a and IgG2b Ab levels were higher in the NS diet group than in the NF diet group after the last KLH challenge. The NS diet group had higher interferon-gamma production and mRNA expression than did the NF diet group after the first KLH challenge. Because increased levels of interferon-gamma and IgG2a/IgG2b Ab reflect a shift toward type-1 responses to antigen stimuli, our results show that dietary nucleotides preferentially enhance type-1 responses to KLH given with incomplete Freund's adjuvant.

Antitumor activity of astaxanthin and its mode of action.

Astaxanthin, a carotenoid without vitamin A activity, may exert antitumor activity through the enhancement of immune responses. Here, we determined the effects of dietary astaxanthin on tumor growth and tumor immunity against transplantable methylcholanthrene-induced fibrosarcoma (Meth-A tumor) cells. These tumor cells express a tumor antigen that induces T cell-mediated immune responses in syngenic mice. BALB/c mice were fed astaxanthin (0.02%, 40 micrograms/kg body wt/day in a beadlet form) mixed in a chemically defined diet starting zero, one, and three weeks before subcutaneous inoculation with tumor cells (3 x 10(5) cells, 2 times the minimal tumorigenic dose). Three weeks after inoculation, tumor size and weight were determined. We also determined cytotoxic T lymphocyte (CTL) activity and interferon-gamma (IFN-gamma) production by tumor-draining lymph node (TDLN) and spleen cells by restimulating cells with Meth-A tumor cells in culture. The astaxanthin-fed mice had significantly lower tumor size and weight than controls when supplementation was started one and three weeks before tumor inoculation. This antitumor activity was paralleled with higher CTL activity and IFN-gamma production by TDLN and spleen cells in the astaxanthin-fed mice. CTL activity by TDLN cells was highest in mice fed astaxanthin for three weeks before inoculation. When the astaxanthin-supplemented diet was started at the same time as tumor inoculation, none of these parameters were altered by dietary astaxanthin, except IFN-gamma production by spleen cells. Total serum astaxanthin concentrations were approximately 1.2 mumol/l when mice were fed astaxanthin (0.02%) for four weeks and appeared to increase in correlation with the length of astaxanthin supplementation. Our results indicate that dietary astaxanthin suppressed Meth-A tumor cell growth and stimulated immunity against Meth-A tumor antigen.

Cytokine production by sinus lavage, bronchial lavage, and blood mononuclear cells in chronic rhinosinusitis with or without atopy.

BACKGROUND: Chronic sinus inflammation may be determined partly by a balance of proinflammatory and counterregulatory cytokines and other mediators in the sinus. However, their mechanistic roles in chronic rhinosinusitis (CRS) are not well understood. OBJECTIVE: To evaluate production of proinflammatory (interferon gamma [IFN-gamma] and interleukin [IL] 12) and counterregulatory cytokines (IL-10 and IL-4) by sinus lavage (SL), bronchial lavage (BL), and peripheral blood mononuclear (PBMN) cells in patients with CRS. METHODS: We analyzed SL, BL, and PB samples obtained at surgery from 26 patients with CRS. Cytokine production was determined by culturing cells with or without stimuli. The results were evaluated in comparison with other inflammatory variables (cytologic findings, total protein, IgG, and lactose dehydrogenase), bacterial cultures, and clinical features. RESULTS: Production of IFN-gamma by SL cells was variable and did not correlate with other inflammatory variables, microbes grown, IL-10/IL-12p40 production by SL cells, or IFN-gamma production by BL or PBMN cells. Production of IL-4 by lavage cells was undetectable. None of 10 patients with elevated IFN-gamma production (>800 pg/10(6) SL cells with mitogen stimuli) had allergic rhinitis, whereas 12 of 16 patients with low IFN-gamma production (<500 pg/10(6) SL cells) had allergic rhinitis with positive reactivity to common aeoroallergens. There was no significant difference in other variables measured between low and high IFN-gamma production groups. CONCLUSIONS: Elevated IFN-gamma production by SL cells may indicate much less possibility of allergic rhinitis in patients with CRS, but other variables measured did not differ in patients with high or low IFN-gamma production by SL cells.

Interferon gamma levels in the sinus, ear, and airway in a rabbit sinusitis model induced by Bacteroides inoculation.

BACKGROUND: Previously, we found minimal bacterial dissemination and no evidence of systemic inflammation in a rabbit sinusitis model in which the left maxillary sinus was inflamed by Bacteroides inoculation with the ostium closed. However, we observed an increase in anti-Bacteroides IgG antibodies in the contralateral sinus, lower airway, and middle ear, with an apparent increase in interferon gamma (IFN-gamma) messenger RNA expression in the ear and sinus mucosa. OBJECTIVE: To evaluate how IFN-gamma production in the upper and lower airway is associated with localized bacterial sinusitis. DESIGN: Interferon gamma levels were measured in lavage solutions from the sinus, airway, and middle ear and in serum at 1, 2, 3, and 4 weeks following bacterial inoculation. SUBJECTS: The subjects were 6 rabbits at each time point. The controls were untreated (n = 5) and sham-operated (n = 4-5) rabbits at 2 and 4 weeks. INTERVENTION: Bacteroides fragilis (10(8) plaque-forming units) was inoculated into the left maxillary sinus. RESULTS: Interferon gamma levels in the ear and sinus were less than 0.2 microg/g protein in controls. Following bacterial inoculation into the left sinus, IFN-gamma levels increased up to 10-fold in both sinuses and even more in the middle ear at 3 weeks, independent of bacterial dissemination. Mean +/- SD IFN-gamma levels in the airway (0.3+/-0.28 microg/g protein in controls) were not altered by bacterial inoculation into the sinus. Serum IFN-gamma levels were very low (<0.05 microg/g protein) in most rabbits and were unchanged by bacterial inoculation. CONCLUSIONS: Interferon gamma levels increase in the ear and contralateral sinus in response to localized sinus inflammation, indicating concerted mucosal proinflammatory immune responses in the upper airway. Such responses may lead to the aseptic middle ear inflammation often observed in patients with chronic sinusitis.

Juzen-taiho-to, a Japanese herbal medicine, modulates type 1 and type 2 T cell responses in old BALB/c mice.

Aging is associated with skewed type 2 (T2) T cell responses that may be modulated by herbal medicines. A group of Japanese herbal medicines, so-called "Hozai," have been used to improve the physical condition of the elderly. One representative "Hozai," Juzen-Taiho-To (JTX) appears to have beneficial effects on cancer patients. In this study we hypothesized that JTX modulated skewed T2 responses in the elderly. T1 and T2 responses against ovalbumin (OVA) were examined in old BALB/c mice fed JTX (0.2% w/w). We measured anti-OVA IgG1, IgG2a, and IgG2b antibody (Ab) levels after the primary and secondary OVA challenges; T1 and T2 responses augment IgG2a/IgG2b Ab and IgG1/IgE Ab production, respectively. We also assessed production of T1 and T2 cytokines (IFN-gamma and IL-5, respectively), and co-stimulatory molecule expression by regional draining lymph node cells. JTX-fed mice had higher IgG2b Ab and IFN-gamma production than controls along with lower IgG1 Ab. JTX did not alter IL-5 production or co-stimulatory molecule expression. Hoelen, an herbal component, induced similar changes. Our results indicate that JTX and Hoelen modulate T cell responses against OVA toward more balanced T1/T2 responses in old BALB/c mice. Such effects of JTX may help prevent the development of diseases associated with immunodisregulation in the elderly.

Localized sinus inflammation in a rabbit sinusitis model induced by Bacteroides fragilis is accompanied by rigorous immune responses.

We evaluated inflammatory and immune responses against Bacteroides fragilis in a rabbit sinusitis model. Bacteroides was inoculated into the left maxillary sinus, and inflammatory (histology, cell number/cytology, lactose dehydrogenase, and apoptosis) and immune responses in the sinus, airway, and peripheral blood (PB) were determined for up to 4 weeks. In the inflamed sinus, the lactose dehydrogenase level was markedly elevated, with neutrophilic infiltration, severe tissue inflammation, and increased apoptosis. Low-grade tissue inflammation was present in the contralateral and sham-operated sinuses, but other parameters remained unchanged, and so did those in the airway and PB in the inoculated rabbits. Serum IgG antibody levels increased rapidly, were highest at 3 weeks, and began to decline at 4 weeks. Cellular immune responses (proliferation and interferon-gamma mRNA expression) against Bacteroides were detected in the PB of all inoculated rabbits. Vigorous immune responses against Bacteroides may have localized but failed to terminate inflammation in the sinus, indicating importance of microenvironmental factors.

Middle ear pathologic changes associated with chronic anaerobic sinusitis in rabbits.

OBJECTIVE/HYPOTHESIS: To study the histopathologic changes in association with the inflammatory/immune response present in the middle ears of a rabbit model of unilateral chronic anaerobic sinusitis. STUDY DESIGN: New Zealand white rabbits, two at each experimental time point. Normal rabbits and sham-operated animals served as controls. METHODS: Left maxillary sinusitis was induced by inoculating Bacteroides fragilis surgically after closure of the ostium. Cultures, lavages, and mucosa were harvested from bilateral middle ear and sinus cavities at 1, 2, 3, and 4 weeks following inoculation. Parameters analyzed include tissue for histopathologic study, immunoglobulin G antibody (IgG Ab) against B fragilis, and lactate dehydrogenase (LDH) levels in lavage samples, interferon gamma (IFN gamma) messenger RNA (mRNA) expression in mucosal tissue, and bacterial culture. RESULTS: Despite closure of the ostium of the left sinus, mild to moderate dissemination of B fragilis into the right sinus and left and right ears were observed in some but not all rabbits (2/8, 5/7, and 2/8, respectively). Histopathologic changes in the right sinus and middle ears were much less severe in contrast to the severe inflammatory changes in the left sinus. An immune response against B fragilis appeared to occur in the sinuses and ears bilaterally independent of bacterial dissemination, as evidenced by a rise of IgG Ab in lavage fluid and detection of IFNg mRNA. Neither control nor sham-operated animals had detectable levels of IFNg mRNA or IgG Ab. In B fragilis-inoculated rabbits, the magnitude of IgG Ab responses was equivalent in the right and left ear, independent of B fragilis dissemination; IgG Ab levels in the middle ear positively correlated to each other (P < .01) and to the levels in the sinuses (P < .01 and P < .01). LDH levels were closely associated with bacterial growth and degree of tissue inflammation. CONCLUSION: This reproducible model of chronic sinusitis provides an opportunity to study the middle ear infection and inflammatory/immune responses occurring with sinusitis. Our results indicate bilateral middle ear mucosal immune responses to an elicited sinus infection, independent of B fragilis dissemination.

Effects of anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene on human small airway epithelial cells and the protective effects of myo-inositol.

Benzo[a]pyrene (B[a]P), a tobacco-derived carcinogen, induces lung tumors in rodents through its carcinogenic metabolite, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE). Tumorigenesis is inhibited by dietary myo-inositol in the post-initiation phase. However, little is known about how B[a]PDE and myo-inositol affect normal human lung cells. We addressed this question using untransformed human small airway epithelial (SAE) cells. SAE cell viability decreased <50% in parallel to an increase of apoptotic cells (>20%) 2 days after the cells were treated for 1 h with B[a]PDE (>100 nM). In contrast, the cell number and viability were not altered in A549 human lung cancer cells by B[a]PDE treatment up to 10 microM with <5% apoptotic cells and <10 U/l LDH in the medium. SAE cells retain the features of basal cells in serum-free, low Ca2+ (4 nM) medium up to 4-5 passages, but in serum-supplemented or serum-free, high Ca2+ (1 mM) cultures, they differentiate into non-ciliated epithelial cells expressing Clara cell secretory protein (CCSP). A non-toxic, physiologically relevant dose of B[a]PDE (1 nM) partially inhibited serum and Ca2+-induced SAE cell differentiation. This effect was abolished by wortmannin, a phosphatidylinositol-3 kinase (PI-3K) inhibitor, and PD98059, a mitogen activated protein kinase (MAPK) kinase-1 (MEK1) inhibitor, but not by SB202190, a p38 MAPK inhibitor, or melittin, a protein kinase C inhibitor. Myo-inositol (10-100 microM) did not alter growth or differentiation of untreated SAE or A549 cells, but reversed the inhibitory effect of B[a]PDE on serum and Ca2+-induced SAE cell differentiation when supplemented to the culture after B[a]PDE treatment. This myo-inositol action was not altered by PD98059, wortmannin or melittin, but was partially suppressed by SB202190. Collectively, these results indicate that B[a]PDE inhibits serum-induced SAE cell differentiation, possibly involving activating signals through a PI-3K/MEK1 mediated MAPK pathway, whereas myo-inositol protects SAE cells against this inhibitory effect of B[a]PDE perhaps through both PI-3K/MEK1 and p38 MAPK pathways.

Airway epithelium and apoptosis.

Recent advances revealed that airway epithelium possesses versatile functions and plays a vital role in the mucosal defense and inflammatory responses. A maintenance of airway epithelium integrity is thus important and appears to be tightly regulated by a balanced cell proliferation and apoptosis. However, homeostasis of airway epithelium is likely affected by multiple environmental pathogens, irritants (reactive oxygen species, allergens, etc.), and toxins that may lead to various lung diseases. This review briefly summarizes airway epithelium apoptosis/proliferation in physiologic and pathological conditions along with various factors influencing airway epithelium homeostasis.

The effects of hyperoxic injury and antioxidant vitamins on death and proliferation of human small airway epithelial cells.

Previously it was reported that hyperoxia induced death of the human lung adenocarcinoma cell line (A549 cells) by necrosis, not by apoptosis. This study examined proliferation and death of untransformed human small airway epithelial (SAE) cells in normoxia or hyperoxia in comparison with A549 cells. We tested the hypothesis that SAE cells respond differently to hyperoxic injury than do A549 cells. We measured total cell number and viability, thymidine incorporation (SAE cells only), lactate dehydrogenase (LDH) release, and apoptotic changes as markers for cell proliferation and death. Protective effects of antioxidant vitamins also were examined in SAE cells. In normoxia, subconfluent SAE cells had less apoptosis and fewer detached cells, but higher thymidine incorporation than did near-confluent cells. Hyperoxia suppressed thymidine incorporation and augmented apoptosis in both subconfluent and near-confluent SAE cells. Hyperoxia decreased the total cell number only in subconfluence, whereas SAE cell viability declined with hyperoxia in near confluence, but not in subconfluence. For SAE cells, necrosis assessed by LDH release was minimal in all conditions and was not augmented by hyperoxia in SAE cells. In contrast, normoxic A549 cells proliferated more rapidly than did SAE cells with a large number of cells detached during the culture. A549 cells underwent necrotic cell death under confluent or in hyperoxic conditions, but had much less apoptotic cell death. In SAE cells, vitamin E partially prevented the decline of thymidine incorporation with hyperoxia in subconfluence and protected against apoptotic changes with hyperoxia in both subconfluent and near-confluent conditions. Vitamin C prevented apoptosis with hyperoxia only in near-confluent SAE cells. Thus, SAE cells maintained balanced apoptosis and cell proliferation that were altered by cell density and hyperoxia and demonstrated very little necrosis with hyperoxia. Although A549 cells underwent cell death mainly by necrosis, they also were influenced by cell density and hyperoxia. Cell density also determined specific antioxidant vitamin protection in SAE cells.

IgG antibody levels in the sinus, ear, and airway in a rabbit model of sinusitis with Bacteroides.

OBJECTIVE: To evaluate distribution of IgG antibodies (Ab) in the airway, ear, and sinuses in association with inflammatory changes in a rabbit sinusitis model. DESIGN: We measured IgG Ab and lactate dehydrogenase levels in solutions from sinus, airway, and middle ear lavage and in serum, and determined interferon y messenger RNA expression in sinus and ear mucosa at 1, 2, 3, and 4 weeks after inoculation with Bacteroides fragilis. SUBJECTS: Six rabbits at each time point; controls were untreated (n=5) and sham-operated rabbits at 2 and 4 weeks (n=4-5). INTERVENTION: Bacteroides fragilis was inoculated into the left maxillary sinus with ostium closed. RESULTS: IgG Ab was undetectable in all controls. IgG Ab (>50 microg/g protein) was present at 2, 3, and 4 weeks in most bilateral sinus lavage samples and in 2 of 6, 5 of 6, and 6 of 10 ear lavage samples at 2, 3, and 4 weeks, respectively, following inoculation. Inflammatory changes (histological and lactate dehydrogenase) were much greater in the inflamed sinus. IgG Ab (>50 microg/g protein) was also detected in most bronchoalveolar lavage samples after 2 weeks. Interferon gamma mRNA was undetectable in all untreated and most sham-operated controls but was detected in the bilateral sinus mucosa at 1 to 2 weeks, and remained detectable up to 4 weeks in most rabbits. Serum IgG Ab levels positively correlated with those in lavage samples, with highest correlation with right sinus lavage IgG Ab levels (r=0.56, P<.001). CONCLUSION: IgG Ab levels in the upper airway mucosa likely increase within 2 weeks following bacterial inoculation as a part of mucosal immune responses independent of tissue necrosis.

The actions of polynucleotides on effector stage cloned murine T-helper cells differ in each subset and depend on antigen concentration.

Polynucleotides enhance T-helper (Th) cell-mediated humoral immune responses in naive resting Th cells, B cells, and antigen-presenting cells (APC) from unprimed mouse spleen. If polynucleotides augment Th cell functions independent of the activation stage of Th cells, then polynucleotides may cause hyperimmune responses. In this study we examined the effects of polynucleotides on effector-stage murine Th cell clones in vitro. The A.E7 clone (primed with pigeon cytochrome C, origin: B10.A mice) and CDC35 clone (primed with rabbit gamma-globulin, origin: DBA/2 mice) were used as representative type 1 (Th1) and type 2 (Th2) Th cells, respectively. Th clones were stimulated with antigen (Ag) in polynucleotide-supplemented or control cultures in the presence of syngeneic spleen cells (either CD4- or irradiated). The number of antibody (Ab)-secreting cells was counted to measure T-dependent Ab production. Production of interferon-gamma (IFNgamma) for the Th1 clone and interleukin-5 (IL-5) for the Th2 clone were measured. Without Ag stimulation, cytokine production and the number of Ab-secreting cells formed were very low and not altered by polynucleotides. With suboptimal Ag challenges provided by Ag-primed spleen cells, polynucleotides enhanced IFNgamma production by the Th1 clone, while they suppressed Th1 clone-mediated Ab production and IL-5 production by the Th2 clone. Polynucleotides did not alter Th2 clone-mediated Ab production. These actions of polynucleotides appeared to be dose-dependent. With optimal Ag challenges, polynucleotides did not affect our measures of Th cell activation. Polynucleotide action in vitro on effector-stage Th cell clones differed in each Th cell subset and depended on Ag concentration.

Cell density and antioxidant vitamins determine the effects of hyperoxia on proliferation and death of MDCK epithelial cells.

Epithelial cells are prone to oxidant injury, which could change epithelial cell homeostasis and lead to degenerative diseases. We examined the effects of hyperoxia on death and proliferation off Madin-Darby canine kidney (MDCK) epithelial cells and antioxidant vitamin protection. Subconfluent and near-confluent MDCK cells were cultured under normoxia or hyperoxia for two days. We measured cell number and viability, mitochondria enzymatic activity, thymidine incorporation, necrosis [lactate dehydrogenase (LDH) release], and apoptosis (DNA fragmentation and morphological changes). When the cells were subconfluent, hyperoxia decreased the number of adherent cells, mitochondrial enzymatic activity, and thymidine incorporation, but neither LDH release nor apoptotic changes increased compared with normoxic controls. In normoxia, near-confluent cells had lower nonadherent cell numbers, mitochondrial enzymatic activity, and thymidine incorporation than subconfluent cells; hyperoxia further decreased the latter two parameters and increased apoptotic changes and LDH release in near-confluent cells. Vitamin E protected mitochondrial enzymatic activity, apoptotic changes, and LDH release against hyperoxic injury but did not affect changes in thymidine incorporation with hyperoxia. Vitamin C partially protected the mitochondrial enzymatic activity and thymidine incorporation in subconfluence, but not in near confluence. These results indicate that cell density is a major determinant of the effects of hyperoxic injury and the profile of antioxidant vitamin protection.

Nucleotide-free diet suppresses antigen-driven cytokine production by primed T cells: effects of supplemental nucleotides and dietary fatty acids.

Our previous studies suggest that nucleotides modulate T-helper (Th) cell-mediated antibody (Ab) production. This nucleotide action is influenced by dietary fatty acids. Herein, we report the effects of nucleotide-free (NF) diets normal or high in saturated fatty acid on antigen-driven Th cell activation by using cytokine production as an indicator. C57BL/6 mice were fed a NF diet, a NF diet plus mononucleotide and nucleoside mixture (OG-VI), a NF diet high in saturated fatty acid (NF-SFA), or a NF-SFA diet plus OG-VI. Mice were then challenged with neoantigen, a keyhole limpet hemocyanin (KLH). Regional draining lymph nodes were collected 5-7 d following antigen (AG) priming, rechallenged with KLH in the culture, and resultant cytokine production was measured. IFN gamma and IL-5 production was lower in mice fed a NF diet at protein and mRNA levels. IL-4 and IL-2 mRNA expression was also lower in mice fed a NF diet. IFN gamma protein levels were higher in mice fed a NF-SFA diet than in mice fed a NF diet, but production of other cytokines was equally suppressed in those fed a NF-SFA diet. In vivo OG-VI supplementation prevented aberrant cytokine production in mice fed a NF or NF-SFA diet. Polynucleotides added to the culture restored impaired IFN gamma and IL-5 production in mice fed a NF diet but did not further augment cytokine production in mice of other diet groups. These results indicate a potential role of nucleotides in Ag-driven Th-cell activation, and this nucleotide action is partly under the influence of dietary fatty acids.

An orally supplemented mononucleotide mixture prevents the decrease in T cell-dependent humoral immunity in C57BL/6 mice fed a nucleotide-free diet.

T-cell-dependent humoral immune responses are lower in mice fed a nucleotide-free (NF) diet. In a previous study, a mononucleotide and nucleoside mixture prevented the decrease in humoral immune responses in mice fed a NF diet when a total of seven doses [2100 micromol/(kg x dose)] were administered intraperitoneally. In the present study, C57BL/6 (B6) mice were fed a NF diet for 3 wk with or without mononucleotide mixture (MM) supplementation. The MM was given at the levels of 14 or 70 micromol/(kg x d) by daily gavage feeding for 3 wk. Control mice were fed a NF diet without supplements (negative control) or a NF diet plus the mononucleotide/nucleoside mixture administered intraperitoneally (positive control). Both doses of MM prevented the decrease in T-dependent antibody (Ab) production in mice fed a NF diet as effectively as positive controls. T-helper (Th) spleen cells from mice of each diet group were enriched, mixed with Th cell-depleted spleen cells from each diet group, and antigen-primed in the culture. The number of Ab-secreting cells formed was higher with Th cells from mice with oral MM supplements or from positive controls than with those from mice without nucleotide supplement. The source of Th cell-depleted spleen cells did not influence the number of Ab-secreting cells. Thus, orally supplemented nucleotides can prevent the suppression of Th cell-dependent humoral immunity in mice fed a NF diet with doses likely to be provided by dietary sources.