Modulation of cytokine and nitric oxide production by keratinocytes, epithelial cells, and mononuclear phagocytes in a co-culture model of inflammatory acne.

INTRODUCTION: Ultraviolet B (UVB, 290 nm to 320 nm) has been reported to modulate the cytokine-mediated inflammatory process in various inflammatory skin conditions, including production of TNF-α, IL-1α, IL-6, IL-8, and IL-10. We constructed an in vitro model system involving co-culture of different cell types to study the effect of UVB on the inflammatory process using nitric oxide (NO) and tumor necrosis factor (TNF)-α as markers of inflammation. OBJECTIVE: This study was conducted to quantitatively assess the products secreted by human epithelial keratinocytes in the presence and absence of macrophages/monocytes. METHODS: Cells were exposed to UVB radiation (50 mJ to 200 mJ per cm2) or treated with bacterial lipopolysaccharide (LPS) as stimulator of inflammatory response. Nitric oxide (NO) was measured by modified Griess assay and TNF-α was measured by quantitative ELISA. For the co-culture system, SC monocytes were seeded in a 24-well Transwell tissue culture plate whereas irradiated keratinocytes were seeded in the individual baskets subsequently placed on top of the monocyte cultures, and samples of culture supernatants were collected at 1 to 6 days. RESULTS: When primary human epidermal keratinocytes (NHEK) were irradiated with UVB, a dose-dependent stimulation of TNF-α production was observed (33% to 200% increase). TNF-α production was not changed significantly in SC monocytes/NHEK co-culture. In contrast, when macrophages were irradiated with UVB, significant inhibition of NO production (40% suppression, P<0.001) was seen. CONCLUSION: This improved model of cutaneous inflammation could use multiple cells to study their interactions and to offer convenience, reproducibility, and a closer approximation of in vivo conditions.

Tetracycline-mediated IgE isotype-specific suppression of ongoing human and murine IgE responses in vivo and murine memory IgE responses induced in vitro.

We previously reported that minocycline treatment of allergic asthmatic patients had oral steroid sparing effects and improved their clinical status and that minocycline suppressed in vitro induction of IgE responses by their PBMC. The effect of minocycline on human or animal IgE responses in vivo has not been studied. Allergic asthmatics (serum IgE: 505 +/- 535 IU ml(-1)) were given minocycline (150 mg po to 250 mg po BID) as add-on therapy to standard care for up to 10 months; control subjects (IgE: 405 +/- 472 IU ml(-1)) received standard care (n = 6 per group). Serum immunoglobulin (IgM, IgG, IgE and IgA) levels were determined monthly (Nephelometry, Unicap Total IgE Fluoroenzyme immunoassay). BALB/c mice (n = 6 per group) were injected intraperitoneally with benzylpenicilloyl(14)-Keyhole limpet hemocyanin (BPO(14)-KLH) in alum on days 0, 21 and 42, fed with minocycline or doxycycline (10-100 mg kg(-1)) on day 44 and numbers of BPO-specific IgG(1), IgE and IgA antibody-forming cell (AFC) in mesenteric LN and spleen and serum immunoglobulin levels were determined on days 46-70 (enzyme-linked immunosorbent spot assay, ELISA). The ability of minocycline or doxycycline to suppress in vitro induction of murine memory IgE responses also was investigated. Minocycline strongly suppressed serum IgE levels of allergic asthmatics (9% per month) (P = 0.012). Minocycline (and doxycycline) also strongly suppressed peak murine IgE AFC and serum IgE responses (>95, approximately 75%, respectively) and in vitro induction of memory IgE responses by murine mesenteric LN and spleen cells (>95%). Tetracycline suppression of all human and murine IgE responses was IgE isotype specific. Suppression of murine IgE responses in vivo was dose dependent and lasted 5-7 days.

Correlation of plasma complement split product levels with allergic respiratory disease activity and relation to allergen immunotherapy.

BACKGROUND: Allergens, including dust mite and grass pollen, and mast cell tryptase are known to generate the complement split products (CSPs) C5a and C3a, which can then trigger allergic inflammation. The relation of these anaphylatoxin levels to clinical allergic disease responses is not known. OBJECTIVE: To evaluate the relationship of plasma CSP levels to allergic respiratory disease variables in an adult cohort. METHODS: A cross-sectional survey was used to assess the association of plasma C5a desArg and C3a desArg levels with clinical allergic respiratory disease variables. Furthermore, a time course of the effect of routine allergen immunotherapy on plasma CSP levels and cutaneous and pulmonary responses was determined. RESULTS: Adult plasma C5a desArg levels correlate with asthma severity as determined by a physician (P = .01) and by Asthma Quality of Life Questionnaire scores (P < .01). Change in plasma C5a desArg levels 1 hour after immunotherapy is associated with baseline rhinoconjunctivitis symptom severity (P = .03), change in total mean wheal diameter (P = .05), and total dust mite dosage (P = .04). Change in plasma C3a desArg levels 3 hours after immunotherapy correlates with change in total mean wheal diameter induced by dust mite (P = .01). Change in plasma CSP levels after immunotherapy did not correlate with change in spirometric outcome. CONCLUSIONS: Plasma C5a desArg levels reflect allergic respiratory disease severity as assessed by physicians and correlate with Asthma Quality of Life Questionnaire scores. Changes in CSP levels after immunotherapy reflect cutaneous allergic responses, especially to dust mite allergen.

Long Term Persistence of IgE Anti-Varicella Zoster Virus in Pediatric and Adult Serum Post Chicken Pox Infection and after Vaccination with Varicella Virus Vaccine.

The production of IgE specific to different viruses (HIV-1, Parvovirus B19, RSV), and the ability for IgE anti-HIV-1 to suppress HIV-1 production in vitro, strongly suggest an important role for IgE and/or anti viral specific IgE in viral pathogenesis. Previous studies in our laboratory were the first to report the presence of IgE anti-varicella zoster virus (VZV) in an adolescent patient with shingles. However, the presence and long term persistence of IgE anti VZV antibodies has not been studied in adults. The presence of serum IgE in addition to IgE and IgG anti-VZV antibody in sera were studied in children (N=12) (0-16 y/o) and adults (N=9) (32-76 y/o) with either a past history of (wild type) chicken pox (N=7 children, 9 adults) or 5 years after vaccination with varicella zoster (N=2 children) (Varicella virus vaccine live, Oka/Merck), as well as in non-infected subjects (N=3 children). Of the patients who had a positive history of chicken pox 13 of 16 (81%) contained IgE anti-VZV antibodies; they were both serum IgEHi (>100 IU/ml) and IgELo (<100 IU/ml). Of the patients who were vaccinated, IgE anti-VZV antibodies were undetected. In contrast, serum from the patients without a history of chicken pox or vaccination did not make either IgE or IgG anti-VZV antibodies. This is the first demonstration of the existence of IgE anti-VZV antibodies, and its long-term persistence in serum of previously infected subjects. Future studies regarding the functional role of anti-viral IgE and its relationship to VZV are warranted.

Two distinct T cell subsets, CD4+ and CD8+CD60+, and their cytokines are required for in vitro induction of human ragweed-specific memory IgE responses.

CD8(+)CD60(+) T cells (80-98% CD45RO(+); 20% CD23(+)) are significantly increased in the blood of serum IgE(+) ragweed-sensitized (RS) compared with serum IgE-nonatopic humans (p = 0.001). CD8(+)CD60(+) T cells of the RS patients produced IL-2, IL-4, IL-10, IL-12, IFN-alpha. and IFN-gamma, but not IL-6 or IL-13. When their PBMC were cultured with ragweed Ag (RA), peak IgE responses occurred on day 10; none was induced with non-cross-reacting or without Ag; nonatopic PBMC did not respond to any stimulant. When either CD4(+) or CD8(+)CD60(+) T cells were depleted from RS PBMC before culture with RA, no IgE responses were induced. If purified CD4(+) T cells or low numbers of CD8(+)CD60(+) T cells were added back to the depleted PBMC, IgE responses were restored. However, higher numbers of CD8(+)CD60(+) T cells totally suppressed IgE responses. Total suppression also was obtained when RS PBMC were cultured with RA and either anti-IL-2, IL-4, IL-10, IL-12, IFN-gamma (all concentrations), or IFN-alpha (low concentrations), but not anti-IL-6 or IL-13. Higher concentrations of anti-IFN-alpha potentiated IgE responses.

Detection of IgE anti-parvovirus antibodies in human breast milk.

Breast milk is a complex fluid, rich in nutrients and non-nutritional bioactive components, including antimicrobial factors, immunoglobulins, cytokines, and anti-inflammatory substances. Although IgE is implicated in viral immunity, its role in breast milk in parvovirus B19 immunity has not been studied. Total immunoglobulin levels of IgE, IgG, and IgE anti-parvovirus B19 antibodies were determined by ELISA and Western blot analysis in breast milk and in sera from a mother and her nursing infant (female, 10 mo). For specific IgE protein determination, breast milk was fractionated by chromatography on G-100 Sephadex; 3 peaks were collected and separated by SDS PAGE. The levels of total IgE in breast milk and its fractions were low (<2.4 ng/ml), and those of maternal and infant serum were negligible (18 and 4.3 IU/ml, respectively). Nevertheless, the breast milk and maternal and infant sera contained IgE anti-parvovirus B19 antibodies, even though the infant was never infected with parvovirus B19. Total serum levels of maternal IgG were within the normal range and those of infant IgG were low (473 mg/dl); total IgG in breast milk was not determined. Maternal serum contained some detectable IgG anti-parvovirus antibodies that were not present in infant serum or breast milk. Total maternal and infant serum levels of IgM and IgA were within the normal ranges. The presence of IgE anti-parvovirus B19 antibodies in breast milk suggests that IgE anti-viral antibodies are transmitted in breast milk and may provide protective responses in nursing children.

Sophorolipids improve sepsis survival: effects of dosing and derivatives.

INTRODUCTION: Sophorolipids, a family of natural and easily chemo-enzymatically modified microbial glycolipids, are promising modulators of the immune response. We have previously demonstrated that sophorolipids mediate anti-inflammatory effects, including decreasing sepsis-related mortality at 36 h in vivo in a rat model of septic peritonitis and in vitro by decreasing nitric oxide and inflammatory cytokine production. Here we assessed the effect of sophorolipids on sepsis-related mortality when administered as a (1) single bolus versus sequential dosing and (2) natural mixture versus individual derivatives compared with vehicle alone. METHODS: Intra-abdominal sepsis was induced in male, Sprague Dawley rats, 200 to 240 g, via cecal ligation and puncture. Sophorolipids (5-750 mg/kg) or vehicle (ethanol/sucrose/physiological saline) were injected intravenously (i.v.) via tail vein or inferior vena cava at the end of the operation either as a single dose or sequentially (q24 h x 3 doses); natural mixture was compared with select sophorolipid derivatives (n = 10-15 per group). Sham-operated animals served as nonsepsis controls. Survival rates were compared at 1 through 6 d post sepsis induction and tissue was analyzed by histopathology. Significance was determined by Kruskal-Wallis analysis with Bonferroni adjustment and Student's t-test. RESULTS: Sophorolipid treatment at 5 mg/kg body weight improved survival in rats with cecal ligation and puncture-induced septic shock by 28% at 24 h and 42% at 72 h for single dose, 39% at 24 h and 26% at 72 h for sequential doses, and 23% overall survival for select sophorolipid derivatives when compared with vehicle control (P < 0.05 for sequential dosing). Toxicity was evident and dose-dependent with very high doses of sophorolipid (375-750 mg/kg body weight) with histopathology demonstrating interstitial and intra-alveolar edema with areas of microhemorrhage in pulmonary tissue when compared with vehicle controls (P < 0.05). No mortality was observed in sham operated controls at all doses tested. CONCLUSIONS: Administration of sophorolipids after induction of intra-abdominal sepsis improves survival. The demonstration that sophorolipids can reduce sepsis-related mortality with different dosing regimens and derivatives provides continuing evidence toward a promising new therapy. Toxicity is evident at 75 to 150x the therapeutic dose in septic animals.

Sophorolipids block lethal effects of septic shock in rats in a cecal ligation and puncture model of experimental sepsis.

OBJECTIVE: Sophorolipids, a family of natural and easily chemoenzymatically modified microbial glycolipids, are promising modulators of the immune response. The potential of the therapeutic effect of sophorolipids was investigated in vivo in a rat model of sepsis and in vitro by analysis of nitric oxide and cytokine production. DESIGN: Prospective, randomized animal study. SETTING: Experimental laboratory. SUBJECTS: Male Sprague-Dawley rats, 200-240 g. INTERVENTIONS: Intra-abdominal sepsis was induced in vivo in 166 rats via cecal ligation and puncture (CLP); 60 rats were used to characterize the model. The remaining rats were treated with sophorolipids or vehicle (dimethylsulfoxide [DMSO]/physiologic saline) by intravenous (iv) tail vein or intraperitoneal (IP) injection immediately post-CLP (25/group). Survival rates were compared at 36 hrs after surgery. In vitro, macrophages were cultured in lipopolysaccharide (LPS) +/- sophorolipid and assayed for nitric oxide (NO) production and gene expression profiles of inflammatory cytokines. In addition, splenic lymphocytes isolated from CLP rats +/- sophorolipid treatment (three per group) were analyzed for cytokine production by RNase protection assay. MEASUREMENTS AND MAIN RESULTS: CLP with 16-gauge needles optimized sepsis induction and resultant mortality. Sophorolipid treatment improved rat survival by 34% (iv) and 14% (IP) in comparison with vehicle controls (p < .05 for iv treatment). Sophorolipids decreased LPS-induced macrophage NO production by 28% (p < .05). mRNA expression of interleukin (IL)-1beta was downregulated by 42.5 +/- 4.7% (p < .05) and transforming growth factor (TGF)-beta1 was upregulated by 11.7 +/- 1.5% (p < .05) in splenocytes obtained 6 hrs postsophorolipid treatment. LPS-treated macrophages cultured 36 hrs with sophorolipids showed increases in mRNA expression of IL-1alpha (51.7%), IL-1beta (31.3%), and IL-6 (66.8%) (p < .05). CONCLUSIONS: Administration of sophorolipids after induction of intra-abdominal sepsis significantly decreases mortality in this model. This may be mediated in part by decreased macrophage NO production and modulation of inflammatory responses.

IgE, CD8(+)CD60+ T cells and IFN-alpha in human immunity to parvovirus B19 in selective IgA deficiency.

Although IgE is implicated in viral immunity, its role in parvovirus B19 immunity and its relationship to other immunological states has not been studied. Total serum immunoglobulin levels, IgG and IgE anti-parvovirus B19, blood lymphocyte numbers, and epsilon and cytokine specific mRNA were determined in pediatric patients with normal serum IgA levels (IgA+) and selective IgA deficiency (IgA-) on days 0 (initial diagnosis) and 14, and 3 years after recovery (nephelometry, Western blot test, flow cytometry, reverse transcriptase-polymerase chain reaction). We found that both patients had serum IgM, IgG, IgE, and IgA levels within normal ranges on day 0 to 3 years, excluding IgG(1) and IgA in the IgA- patient, which were elevated and negative, respectively, and IgE in the IgA+ patient, which was elevated (>100 IU/ml). The serum IgA+ and IgA- patients made IgE (and IgG) anti-parvovirus B19 at all time points. Excluding CD8(+)CD60+ T cells, determinations of T, B, and NK lymphocyte subsets always were within normal ranges. In both patients, CD8(+)CD60+ T-cell numbers were within normal ranges on day 0, but dramatically increased on day 14 (more than fivefold). At 3 years, they had returned to normal in the IgA+ patient, but remained high in the IgA- patient. On day 0 to 3 years, peripheral blood mononuclear cells of both patients expressed epsilon- and interferon (IFN)-alpha-specific mRNA. On day 0, the IgA+ patient expressed interleukin (IL)-4 and IL-10, but not IL-2, IFN-gamma, or IL-6 mRNA; the IgA- patient expressed IL-6 and IL-10 mRNA, but not IL-4, IL-2, or IFN-gamma mRNA. At 3 years, the IgA+ patient expressed mRNA for all cytokines, but the IgA- patient did not express mRNA for any of these cytokines. Our results suggest that IgE is important in parvovirus B19 immunity, and that IFN-alpha and CD8(+)CD60+ T cells may regulate IgE memory responses and isotype switching.

Detection of IgE anti-parvovirus B19 and increased CD23+ B cells in parvovirus B19 infection: relation to Th2 cytokines.

The immune profile of a parvovirus B19-infected patient (male, 8 years old) was studied on day 0 (initial presentation) and on days 14 and 210 post symptom presentation (psp). Before infection, the patient was skin test positive to various allergens, including ragweed and tree and grass pollens, and had a serum IgE level of 150 IU/mL. On day 0, the patient was diagnosed as parvovirus B19 infected, as judged by the presence of IgG anti-parvovirus Abs in serum (EIA) and presentation of "slap cheek" rash. The patient's serum IgE level increased from 150 IU/mL before infection to 256 IU/mL on day 0, was 233 IU/mL on day 14, and returned to preinfection levels on day 210. In contrast, there was little change in the levels of serum IgM, IgG, or IgA (nephelometry). IgE anti-parvovirus B19 protein (VP-N) was detected in serum (Western blot) on days 0, 14, and 210, despite the decrease in total IgE on day 210. Although there was no increase in total numbers of blood CD23+ B cells on day 0, by day 14 the numbers of these cells increased dramatically (93%), remaining high on day 210. In contrast, there were virtually no changes in total numbers of CD4+ and CD8+ T cells or CD16/56+ NK precursor cells on days 0-210. On day 0, when IgG and IgE anti-parvovirus were detected in serum, patient's peripheral blood mononuclear cells (PBMC) expressed mRNA for the Th2 cytokines IL-4 and IL-10, but not for the Th1 cytokines IFN-gamma or IL-2. However, by day 14 psp, PBMC expressed mRNA for the Th1 cytokines IFN-gamma and IL-2, as well as for IL-4 and IL-10. This is the first demonstration of the existence of IgE anti-parvovirus B19 Ab. The presence of IgE anti-parvovirus B19 Ab in serum on day 0 and its persistence in serum 7 months psp suggests that IgE anti-parvovirus may be useful in prognosis of parvovirus B19 infection. Our results reinforce the idea that IgE, in general, may play a major role in anti-viral immunity, perhaps in conjunction with CD23+ cells. The results further suggest that clearance of this infection is accompanied by a switch to Th1 cytokines.

Effect of minocycline and doxycycline on IgE responses.

BACKGROUND: We have recently found that the tetracycline minocycline suppresses inflammatory responses in serum immunoglobulin (Ig)E-positive asthmatic patients, and that IgE levels can decrease in these patients. The mechanism by which minocycline suppresses these responses is unknown. OBJECTIVE: We have now investigated the ability of the tetracyclines, minocycline and doxycycline, to regulate IgE responses of peripheral blood mononuclear cells (PBMC) obtained from serum IgE-positive asthmatic patients. METHODS: The distributions of CD3+, CD4+, CD8+, and CD19+ lymphocytes in peripheral blood of serum IgE-positive asthmatic patients and IgE-negative nonasthmatic controls, and cytokine-specific mRNA expression by their PBMC were determined by flow cytometry (reverse transcriptase-polymerase chain reaction). Serum Ig levels also were determined (nephelometry, fluoroenzymeimmunoassay, enzyme-linked immunoadsorbent assay; n = 7/group). PBMC (1.5 x 10(6)/mL) were cultured with anti-CD40 monoclonal antibody and recombinant human interleukin-4 in the presence/absence of minocycline or doxycycline (0.1 to 10 microg/mL), and IgE levels in supernatants determined on days 0, 3, and 10 (enzyme-linked immunoadsorbent assay). RESULTS: Asthmatic and nonasthmatic subjects had similar numbers of blood CD4+ T cells (779/mm3 +/- 73 and 766 +/- 115, respectively) and CD19+ B-cells (239/mm3 +/- 35 and 379 +/- 95, respectively); however, CD8+ T cell numbers were decreased in asthmatic compared with nonasthmatic subjects (378/mm3 +/- 66 and 568 +/- 53, respectively; P = 0.045). High IgE levels were detected in supernatants of asthmatic PBMC on day 10 (28 ng/mL +/- 12), whereas control IgE levels did not change (<2.5 ng/mL). When either minocycline or doxycycline was included in culture, IgE production by asthmatic PBMC was strongly suppressed in dose-dependent fashion on day 10 (>80% with 10 microg/mL); control IgE did not change (<2.5 ng/mL). CONCLUSIONS: The results are consistent with the idea that the therapeutic benefits obtained by asthmatic patients from minocycline may, in part, result from IgE suppression.

HIV type 1-specific IgE in serum of long-term surviving children inhibits HIV type 1 production in vitro.

We previously identified a group of long-term pediatric survivors who had acquired HIV-1 through maternal transmission; had not received antiretroviral therapy; are now >8 years old, in good health, and with no opportunistic infections; and have not failed to thrive, although they have greatly decreased numbers of blood CD4+ T cells (<500/mm(3)). All the children have elevated total serum IgE levels (210-2475 IU/ml) and make anti-HIV-1 IgE or IgE directed against non-HIV-1 specificities (radioimmunoassay, Western blot assay); they have no detectable antigenemia. We have now studied the ability of anti-HIV-1 IgE in serum obtained from these children to regulate (1) production of HIV-1 by interleukin 2/phytohemagglutinin (IL-2/PHA)-stimulated peripheral blood mononuclear cells (PBMCs) taken from HIV-1-seronegative donors and infected with a T cell-tropic clone of HIV-1, and (2) transmission of a primary HIV-1 strain from adult AIDS patients to uninfected IL-2/PHA-stimulated PBMCs (p24 core antigen production). High levels of HIV-1 production were observed when PBMCs were cultured for 5 days in the presence of HIV-1-seronegative donor serum that was either IgE positive or IgE negative (IgE, >100 or <100 IU/ml, respectively). HIV-1 production also was observed when PBMCs were cultured with HIV-1-infected donor serum that either contained IgE directed against non-HIV-1 specificities or was IgE negative; these levels were 40% less than those seen with sera from the HIV-1-seronegative donors. Far greater inhibition of virus production was observed if the serum in culture contained anti-HIV-1 IgE (>95%). Virus neutralization did not appear to account for the inhibition obtained with anti-HIV-1 IgE-containing serum because virus production was not suppressed in cultures to which serum was added immediately preinfection (<10%), but was strongly suppressed when serum was added 1.5 hr postinfection (>95%). The inhibition of virus production obtained with serum containing anti-HIV-1 IgE was reversed when (1) serum was depleted of IgE (immunoaffinity), but not when it was depleted of IgG (protein G-Sepharose) before inclusion in culture postinfection, (2) anti-IgE, but not anti-IgG, was included in culture, or (3) serum was heat treated before culture. The results indicate that serum from certain HIV-1-infected pediatric long-term survivors contains agents that inhibit HIV-1 production in vitro, and that these agents include anti-HIV-1 IgE. They suggest that a cytotoxic event, rather than virus neutralization, plays an important role in anti-HIV-1 IgE-mediated inhibition of virus production.