Mononuclear Cell Function Research Articles

Method of birth alters interferon-gamma and interleukin-12 production by cord blood mononuclear cells.

Many uncertainties exist regarding the capability of cord blood mononuclear cells (CBMC) to produce cytokines. A number of conflicting reports led us to examine the effects of method of birth on CBMC production of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and interleukin-12 (IL-12). While constitutive production of IL-4 was found in both vaginally and cesarean-delivered infants, constitutive IFN-gamma or IL-12 production was found in neither. CBMC from vaginally delivered infants responded to stimulation with concanavalin A/phorbol 12-myristate 13-acetate (Con A/PMA), phytohemagglutinin (PHA), and lipopolysaccharide (LPS) with significantly higher levels of IFN-gamma than CBMC from unlabored cesarean section (CS) infants. Production of IL-12 was increased in the vaginally delivered group in response to LPS and PHA but not to ConA/PMA. In contrast, mode of delivery was not associated with differences in IL-4 production. These results indicate that mode of delivery significantly alters the capability of CBMC to produce some cytokines and therefore should be taken into account in interpreting fetal/neonatal mononuclear cell function studies.

Immune response in asymptomatic smokers.

It has been demonstrated that cigarette smoking affects the immune system. Impairment of alveolar mononuclear cell function, described previously, may contribute to the higher rate of postoperative respiratory infections. However, increased susceptibility of smokers to infections of other origin (e.g. wound-related) implies that tobacco effect is not restricted to the respiratory immune competent cells. The present study was designed to investigate the systemic effect of tobacco smoking as it exerted on blood-derived immune cells. We measured systemic cytotoxic activity of natural killer cells, production of pro- and anti-inflammatory cytokines by blood mononuclear cells and their proliferation in response to mitogens. To minimize the immunosuppressive effect of other smoke-related factors, the smokers with chronic obstructive pulmonary disease (COPD) were excluded from this study. Peripheral blood mononuclear cells (PBMC) from 24 chronic asymptomatic smokers, and 28 controls, age and gender matched, were isolated and incubated in vitro with lipopolysaccharide (LPS) or phytohemagglutinin (PHA) to induce secretion of IL-1beta, IL-1ra, IL-6, IL-10, TNFalpha and IL-2, respectively, from mononuclear cells. The level of the cytokines in the supernatants was measured using ELISA kits. The proliferative response to the mitogens PHA and concanavalin A (ConA) was evaluated by 3H-thymidine incorporation and NK cell cytotoxicity by 51Cr release assay. Mononuclear cells from smokers showed increased production of the pro-inflammatory cytokines IL-1beta, IL-6 and TNFalpha and enhanced proliferative response to mitogens as compared to non-smoking population. The secretion of IL-2 and the anti-inflammatory cytokines IL-1ra and IL-10 was similar in both groups. NK cell cytotoxic activity was suppressed in the smokers. Cigarette smokers without chronic obstructive pulmonary disease (COPD) exhibit impaired NK cytotoxic activity in peripheral blood and unbalanced systemic production of pro- and anti-inflammatory cytokines. These changes may serve as predisposing factors for respiratory and systemic infections in the postoperative period and should alert an anesthetist during perioperative management.

Vitamin E-modified filters modulate Jun N-terminal kinase activation in peripheral blood mononuclear cells

The generation during hemodialysis of activated complement fragments and reactive oxygen species, including nitric oxide (NO), may affect peripheral blood mononuclear cell (PBMC) function. Currently, little is known about signal transduction pathways involved in PBMC activation. Jun N-terminal kinase (JNK) is a novel mitogen-activated protein (MAP) kinase phosphorylated and activated in response to oxidative stress and directly involved in cell activation. The present study evaluated the activation of JNK in PBMCs isolated from eight uremic patients undergoing, in a randomized manner, three month-subsequent periods of hemodialysis with a low-flux cellulose acetate (CA) and a vitamin E-modified cellulose membrane (CL-E). After each period of treatment, PBMCs were harvested before (T0), during (T15) and after three hours (T180) of dialysis. At the indicated time points, plasma C5b-9 generation by ELISA and inducible NO synthase (iNOS) gene expression by in situ hybridization were evaluated also. The activation of JNK was studied by Western blotting using a specific monoclonal anti-phospho-JNK antibody, which recognizes the activated form of JNK. At T0, a significant increase in plasma C5b-9 levels was found in CA patients compared to CL-E-treated patients. During hemodialysis, C5b-9 levels rose more significantly in CA patients than in CL-E patients and returned to baseline values only in CL-E patients. At the same time, in CA patients an increased iNOS gene expression was observed at T180 together with a striking activation of JNK. By contrast, PBMC from CL-E-treated patients showed undetectable levels of phospho-JNK and a significant reduction in iNOS expression. Interestingly, incubation of PBMCs with normal human plasma (10%), activated by contact with a cellulosic membrane, induced a time-dependent increase in JNK phosphorylation that was completely inhibited by blocking complement cascade activation. Our data suggest that JNK phosphorylation is strikingly increased in PBMCs obtained from CA-treated patients and may represent a key cellular event in PBMC activation during dialysis with bioincompatible membranes. The activation of this signaling enzyme, mediated by active complement fragments and PBMC-dialyzer interaction, can be significantly reduced by the use of vitamin E-coated membrane.

Differential expression and tissue compartmentalization of the inflammatory response following thermal injury.

Studies have shown that both animal tissue-fixed immune cells and human peripheral blood mononuclear cell (PBMC) functions are altered after burn injury. Additional studies suggest that the burn injury-induced alterations in these divergent cell populations from different species are similar. It remains unknown, however, whether the observed changes in animal tissue-fixed immune cell function following thermal injury also occurs to a similar extent in the PBMC population. The aim of our study was to compare PBMC and tissue-fixed immune cell functions from the same animal using a murine burn model. At 7 days post-burn, mice were more susceptible to sepsis and delayed type hypersensitivity responses were suppressed. Splenocytes isolated from injured mice displayed suppressed proliferation and increased IL-10 production. In contrast, PBMC from injured mice displayed suppressed proliferation, IL-2 and IFN-gamma production. Splenic macrophage nitric oxide, PGE(2), TNF-alpha, IL-6 and IL-10 production was enhanced post-burn and IL-12 production was suppressed. PBMC from such animals displayed enhanced PGE(2) production and suppressed IL-6 and IL-12 production. These results indicate that while an immunosuppressive Th(2) phenotype (increased IL-10 and/or suppressed IL-2, IFN-gamma) was induced in both the splenic and PBMC compartments post-injury, differential expression and dimorphism in the response also exists. Thus, the assessment of only PBMC function in burn patients may not accurately reflect the patient's actual immune status at the tissue level.

Redox imbalance and immune functions: opposite effects of oxidized low-density lipoproteins and N-acetylcysteine.

This study investigates the in vitro effects of oxidized low-density lipoproteins (ox-LDL), 'physiological' pro-oxidants, N-acetylcysteine (NAC), a free radical scavenger and glutathione precursor, and their combination on human peripheral blood mononuclear cell functions. We found that treatment with ox-LDL induced a significant down-regulation of proliferative response to mitogens, antigens and interleukin-2. Lipid extracts from ox-LDL were able to reproduce the same effect as the lipoprotein. On the other hand, NAC exposure induced a significant up-regulation of proliferative responses to all the stimuli used. Moreover, we showed that natural killer (NK) cell-mediated cytotoxic activity was significantly down-regulated by ox-LDL while treatment with NAC induced a significant up-regulation of NK-cell activity. Finally, we found that ox-LDL and NAC exerted opposite effects on the cytokine network, interfering both at the protein secretion level and the messenger RNA synthesis level. More importantly, when NAC was used in combination with ox-LDL the proliferative responses, NK-cell-mediated cytotoxic activity and cytokine production were restored to values comparable to controls. These data indicate that ox-LDL and NAC modulate immune functions, exerting opposite effects reflecting their pro-oxidant and antioxidant behaviours. Our results add new insights to the key role played by redox imbalance as a modulator of immune system homeostasis and suggest that an antioxidant drug such as NAC could be useful against pathologies associated with an increase in lipid peroxidation.

Carotenoid photodegradation products and human peripheral blood mononuclear cell function

The effects of UV-photodegraded β-carotene (φβC) and lycopene (φLyc) on immune function parameters of human peripheral blood mononuclear cells (PBMC) was investigated. φβC (8.6–10.0 μg/ml) inhibited 3H-thymidine incorporation by PBMC an average of 81%, 64% and 66%, when stimulated with Concanavalin A (Con A), phytohemagglutinin (PHA) and lipopolysaccharide (LPS), respectively, without affecting cell viability. φβC and φLyc inhibited Con A and LPS-stimulated proliferation similarly, indicating activity due to nonretinoid products. φβC inhibited IL-2 synthesis by stimulated PBMC, and rIL-2 addition partially reversed inhibition. φβC exposure only during the initial 6 hrs inhibited proliferation, and inhibition was maximal even if φβC addition occurred 8 hrs after mitogen addition. φβC treatment resulted in arrest of cells in G 0 + G 1. Antiproliferative activity was associated with a fraction soluble in ethanol but not water. These results suggest that inhibition of proliferation by φβC occurs downstream of mitogen binding. β-carotene and lycopene undergo photodegradation in human skin in vivo, and these findings suggest that phenomenon may influence aspects of immune function.

Immune Response of Bangladeshi Children With Acute Diarrhea Who Subsequently Have Persistent Diarrhea

ABSTRACTBackgroundBecause altered immune responses may be a risk factor for persistent diarrhea, various aspects of the immune response were examined to elucidate the underlying immune mechanisms that may be involved in the development of persistent diarrhea.MethodsChildren (7–12 months of age) with watery diarrhea for 6 to 8 days from the Dhaka Hospital of the International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), were enrolled. Children were classified as having acute diarrhea (AD) or persistent diarrhea (PD) if diarrhea resolved within 14 days or persisted for more than 14 days, respectively. Uninfected control children (n = 13), from the Nutrition Follow‐Up Unit of ICDDR,B were also enrolled. Of the 123 children with diarrhea who were enrolled, 85 had AD and 38 had PD. Comparisons were performed for clinical features, nutritional status (weight for age, plasma transferrin, and serum albumin levels), and immune responses: neutrophil function; peripheral blood mononuclear cell function, delayed‐type hypersensitivity (DTH) responses, plasma levels of immunoglobulins, tumor necrosis factor‐α, and interferon‐γ. Univariate analyses were conducted to assess differences among the three groups of children and between children with AD and PD. Logistic regression was performed to determine risk factors for PD.ResultsThere were no differences in clinical features and nutritional status among the groups of children studied. More children in whom PD developed had a negative DTH response to tuberculin than those with AD (P = 0.021). Also, a negative DTH response to tuberculin was a significant risk factor for PD (odds ratio [OR] = 3.8, 95% confidence interval [CI] = 1.5–9.9).ConclusionsChildren with acute diarrhea with a negative DTH response to tuberculin are more likely to have development of persistent diarrhea.

A Biochemical and Functional Assessment of Monocyte Phosphodiesterase Activity in Healthy and Asthmatic Subjects

The aim of this study was to investigate whether cyclic adenosine 3′5-monophosphate (cAMP) phosphodiesterase (PDE) activity is altered in monocytes from mild asthmatic subjects. Total cAMP PDE activity (pmol/min per mg protein) was significantly greater in homogenates prepared from monocytes from asthmatic subjects (68.3 ± 7.0, n=9) compared to healthy individuals (46.3 ± 3.3, n=14, P<0.05). The PDE inhibitors siguazodan (PDE3-selective), rolipram (PDE4-selective) and theophylline (non-selective) produced a concentration-dependent inhibition of cAMP PDE activity in homogenates from monocytes from normal and asthmatic subjects. However, siguazodan produced significantly greater (P<0.05), and rolipram significantly less (P<0.05), inhibition of total cAMP PDE activity in monocytes from asthmatics (n=4) than from healthy individuals (n=5). cAMP PDE activity was inhibited with equal potency by theophylline in monocytes from healthy and asthmatic subjects. We also investigated the functional consequences of the changes in PDE activity in mononuclear cells obtained from asthmatic subjects. There was no significant difference in the ability of PDE4 inhibitors to attenuate TNF α release from monocytes obtained from asthmatic compared with healthy subjects (P>0.05). Despite a significant increase in the biochemical activity of PDE3 in monocytes from asthmatic subjects, the PDE3 inhibitor siguazodan, failed to significantly reduce TNF α release from human monocytes. Thus, total cAMP PDE activity is increased in monocytes taken from mild asymptomatic asthmatics compared to healthy subjects and is reflected by an increase in the proportion of PDE3 and a decrease in the proportion of PDE4. This augmented enzyme activity was not associated with an alteration in the ability of PDE4 inhibitors to attenuate mononuclear cell function from asthmatics compared to healthy individuals.

Interaction of Mycobacterium tuberculosis-induced transforming growth factor beta1 and interleukin-10.

Mycobacterium tuberculosis is associated with the activation of cytokine circuits both at sites of active tuberculosis in vivo and in cultures of mononuclear cells stimulated by M. tuberculosis or its components in vitro. Interactive stimulatory and/or inhibitory pathways are established between cytokines, which may result in potentiation or attenuation of the effects of each molecule on T-cell responses. Here we examined the interaction of transforming growth factor beta1 (TGF-beta1) and interleukin-10 (IL-10) in purified protein derivative (PPD)-stimulated human mononuclear cell cultures in vitro. TGF-beta1 induced monocyte IL-10 (but not tumor necrosis factor alpha) production (by 70-fold, P < 0.02) and mRNA expression in the absence but not in the presence of PPD. Both exogenous recombinant (r) IL-10 and rTGF-beta1 independently suppressed the production of PPD-induced gamma interferon (IFN-gamma) in mononuclear cells from PPD skin test-positive individuals. Synergistic suppression of IFN-gamma in cultures containing both rTGF-beta1 and rIL-10 was only seen when the responder cell population were peripheral blood mononuclear cells (PBMC) and not monocyte-depleted mononuclear cells and when PBMC were pretreated with rTGF-beta1 but not with rIL-10. Suppression of PPD-induced IFN-gamma in PBMC containing both rTGF-beta1 (1 ng/ml) and rIL-10 (100 pg/ml) was 1.5-fold higher (P < 0.05) than cultures containing TGF-beta1 alone and 5.7-fold higher (P < 0.004) than cultures containing IL-10 alone. Also, neutralization of endogenous TGF-beta1 and IL-10 together enhanced PPD-induced IFN-gamma in PBMC in a synergistic manner. Thus, TGF-beta1 and IL-10 together potentiate the downmodulatory effect on M. tuberculosis-induced T-cell production of IFN-gamma, and TGF-beta1 alone enhances IL-10 production. At sites of active M. tuberculosis infection, these interactions may be conducive to the suppression of mononuclear cell functions.

Monocyte stimulation by reactive oxygen species: role of superoxide and intracellular Ca2+.

Production of reactive oxygen species has generally been linked to inflammatory processes. Whether the presence of superoxide and hydrogen peroxide affects mononuclear cell function was investigated with an in vitro model using isolated human mononuclear cells. Human mononuclear cells were isolated from buffy coat. Toxicity was measured with Trypan blue exclusion, secreted TNF-alpha was measured with an ELISA, reactive oxygen species within mononuclear cells were quantified with dichlorodihydrofluorescein diacetate fluorescence, intracellular TNF-alpha was measured with flow cytometry and fluorescence microscopy. The enzymatic production of superoxide caused a dose-dependent increase in TNF-alpha synthesis, whereas hydrogen peroxide was ineffective. Flow cytometric measurements and immunofluorescence microscopy demonstrated that monocytes were the main TNF-alpha producing population. This proinflammatory reaction was further characterized by pharmacologically investigating Ca2+ signalling pathways. Superoxide stimulated TNF-alpha secretion was inhibited by intracellular Ca2+-buffers (MAPT-AM and BAPT-AM) or VOC operating antagonists (diltiazem and verapamil) and only to a small extent by pharmacological inhibitors of ligand-gated pathways (TMB-8 and SKF 96368). We propose that superoxide activates human mononuclear cells in a Ca2+-dependent manner.

Canarypox-CMV-pp65 recombinant immunization of seronegative subjects elicits pp65 specific CTL precursors with a frequency comparable to that of naturally seropositive individuals

It is well-established that central nervous system activation affects peripheral blood mononuclear cell (PBMCs) function through the release of the catecholamines (Epi) and norepinephrine (NE), which act on ß2-adrenergic receptors (ß2AR). However, most studies have used non-specific stimulation of cells rather than antigen-specific responses. Likewise, few studies have parsed out the direct effects of ß2AR stimulation on T cells versus indirect effects via adrenergic stimulation of antigen presenting cells (APC). Here we report the effect of salmeterol (Sal), a selective ß2AR agonist, on IFN-γ+ CD4 and IFN-γ+ CD8 T cells following stimulation with Cytomegalovirus lysate (CMVL-strain AD169) or individual peptides spanning the entire region of the HCMV pp65 protein (pp65). Cells were also stimulated with Staphylococcal enterotoxin B. Additionally, we investigated the effect of Epi and Sal on cytotoxic cell killing of transfected target cells at the single cell level using the CD107a assay.The results show that Sal reduced the percentage of IFN-γ+ CD4 and IFN-γ+ CD8 T cells both when applied directly to isolated T cells, and indirectly via treatment of APC. These inhibitory effects were mediated via a ß2 adrenergic-dependent pathway and were stronger for CD8 as compared to CD4 T cells. Similarly, the results show that Sal suppressed cytotoxicity of both CD8 T and NK cells in vitro following stimulation with Chinese hamster ovary cell line transfected with MICA*009 (T-CHO) and the human erythromyeloblastoid leukemic (K562) cell line. The inhibitory effect on cytotoxicity following stimulation with T-CHO was stronger in NK cells compared with CD8 T cells.Thus, targeting the ß2AR on lymphocytes and on APC leads to inhibition of inflammatory cytokine production and target cell killing. Moreover, there is a hierarchy of responses, with CD8 T cells and NK cells inhibited more effectively than CD4 T cells.

Effect of two-chambered bicarbonate lactate-buffered peritoneal dialysis fluids on peripheral blood mononuclear cell and polymorphonuclear cell function in vitro

Low pH, high osmolality, increasing glucose concentration, and glucose degradation products (GDP) formed during heat sterilization of conventional peritoneal dialysis (PD) fluids have been shown to have a detrimental effect on cells involved in peritoneal host defense. The two-chambered PD fluid bag in which glucose at pH ∼ 3 is separated from a bicarbonate (25 mmol/L)-lactate (15 mmol/L) buffer during heat sterilization permits PD fluids with lower GDP to be delivered to the patient at neutral pH. To establish the possible benefit of two-chambered bag PD fluids on peripheral blood mononuclear cell (PBMC) and polymorphonuclear (PMN) cell function, we compared conventional 1.5% Dianeal (1.5%D) with 1.5% two-chambered bag bicarbonate-lactate (1.5%D-B), and conventional 4.25% Dianeal (4.25%D) with 4.25% two-chambered bag bicarbonate-lactate (4.25%D-B). Furthermore, to study the effect of the sterilization process on PBMC and PMN function, we compared filter-sterilized 4.25%D (4.25%D-F) with 4.25%D and 4.25%D-B. PBMC were harvested by Ficoll-Hypaque separation, and 2.5 × 10 6 cells in RPMI were incubated with an equal volume of the test fluids for 4 hours, palleted, and resuspended in RPMI containing 10 ng endotoxin for a further 20 hours. Tumor necrosis factor alpha (TNFα) production by endotoxin-stimulated PBMC was not significantly different ( P = 0.10) between 1.5%D-B and 1.5%D, but was significantly higher ( P = 0.01) with 4.25%D-B compared with 4.25%D. PBMC exposed to filter-sterilized fluid (4.25%D-F) showed significantly higher endotoxin-stimulated TNFα production compared with 4.25%D ( P = 0.02), but was not significantly different from 4.25%D-B ( P = 0.40). PMN were harvested by Ficoll-Hypaque separation and 10 × 10 6 cells incubated with test fluids for 30 minutes. After incubation, phagocytosis (phagocytosis index) was determined by the uptake of 14C-labeled Staphylococcus aureus, oxidative burst by reduction of ferricytochrome C to ferrocytochrome C on stimulation with PMA, and enzyme release by measurement of endotoxin-stimulated bactericidal/permeability increasing protein (BPI). Bicarbonate-lactate two-chambered fluids of similar osmolality and glucose concentration conferred a significant improvement in phagocytosis ( P = 0.02 for 1.5%D-B and P < 0.001 for 4.25%D-B). Oxidative burst and BPI release were significantly higher in 4.25%D-B compared with 4.25%D ( P < 0.001). Filter-sterilized 4.25%D-F conferred a significant improvement in phagocytosis and oxidative burst compared with 4.25%D ( P < 0.001) or 4.25%D-B ( P < 0.001). Furthermore, conventional 4.25%D was associated with significantly lower BPI release compared with 4.25%D-F ( P = 0.01). GDP's acetaldehyde and 5-HMF were analyzed in 4.25%D-B, 4.25%D, and 4.25%D-F. Acetaldehyde was below the lower limit (0.79 ppm) of the standard curve in 4.25%D-B and 4.25%D-F fluids but was detected (3.76 to 5.12 ppm) in all of the 4.25%D fluids. Relative levels of 5-HMF in the 4.25%D-B (0.032 to 0.041 Abs @ 284 nm) and 4.25%D (0.031 to 0.036 Abs @ 284 nm) were similar. The lowest levels (0.001 Abs @ 284 nm) were observed in the filter-sterilized 4.25%D-F. The beneficial effects of two-chambered bicarbonate lactate-buffered PD fluids on PBMC and PMN function are probably related to reduction of GDP from heat sterilization of glucose in a separate chamber at a lower pH. This improvement in biocompatibility could have a beneficial effect on peritoneal defenses.

Thermal Injury Functionally Alters Bone Marrow-Derived Macrophages

Thermal injury quantitatively and qualitatively alters hematopoiesis, including monocyte-macrophage lineage changes, resulting in altered mononuclear cell function. These bone marrow cells (BMCs) ultimately become fixed tissue macrophages (e.g., Kupffer cells). To study the effects of thermal injury on macrophage-hepatocyte interactions, rat BMCs were isolated 24 hours after burn injury, and myelopoiesis was induced by 7-day culture in granulocyte-macrophage colony-stimulating factor. Separate cultures included inflammatory mediators with growth factor function (IL-6 or PGE2). Cultured cells were incubated up to 96 hours with isolated normal hepatocytes (+/- lipopolysaccharide stimulation). The 96-hour exposure to postburn BMCs produced less of the acute phase proteins (APPs), C3 and transferrin, but more cytotoxicity as measured by 1-lactate dehydrogenase release. Sham BMCs cultured with added IL-6 caused higher APP release and minimal cytotoxicity, whereas burn BMCs stimulated lower APP release and retained cytotoxicity. In conclusion, myeloid cells regulate APP synthesis differently after thermal injury and may become more cytotoxic to hepatocytes.

Mononuclear cell functions in healthy volunteers fed borage oil ( Borago officinalis) : ∗#Harbige LS, ∗Fisher B and ∗Fadlon EJ. #School of Chemical and Life Sciences, University of Greenwich, London SE18 6PF UK ∗Department of Immunology, UMDS. St Thomas' Hospital, London SE1 7EH UK

Mononuclear cell functions in healthy volunteers fed borage oil ( Borago officinalis) : ∗#Harbige LS, ∗Fisher B and ∗Fadlon EJ. #School of Chemical and Life Sciences, University of Greenwich, London SE18 6PF UK ∗Department of Immunology, UMDS. St Thomas' Hospital, London SE1 7EH UK

Modification of mononuclear cell function after incubation with albumin-bound unsaturated fatty acids or soybean oil emulsion.

Administration of total parenteral nutrition (TPN) with soybean oil emulsion leads to a linoleic acid enrichment of the plasma membrane that may explain an in vivo activation of mononuclear cells (MNC) seen in our previous studies. Fatty acids from the lipid emulsion may have been accessible to MNC after endocytosis of lipid particles, or by direct uptake of fatty acids after lipoprotein lipase hydrolyzation of the emulsion triglycerides. To resemble the incorporation of fatty acids in vivo, we have modified MNC membrane lipid composition by incubation with different albumin-bound unsaturated fatty acids (UFA) or soybean oil emulsion. After incubation with albumin-bound linoleic and oleic acid, the unstimulated release of superoxide anion was unchanged, while zymosan-stimulated release was 140% (n.s) and 112% (p < 0.05) and phorbol-myristate-acetate (PMA)-stimulated release 148% (p < 0.05) and 124% (p < 0.05) of controls, respectively. Incubation with other UFAs or emulsion did not change superoxide anion release. Unstimulated lymphocyte proliferation increased 3 to 13-fold (p < 0.05) after incubation with all UFAs compared to controls, while UFA incubation did not change phytohemagglutinin (PHA) or PMA-stimulated proliferation. Unstimulated lymphocyte proliferation was decreased after incubation with emulsion, while PHA/PMA-stimulated proliferation was unchanged. Increase in membrane fluidity was detectable only after incubation with emulsion. The increased reactivity may have been caused by changes in the lipid environment surrounding membrane-bound enzymes important for signal transduction through the plasma membrane.

Alterations in IL-6, IL-8, GM-CSF. TNF-α, and IFN-γ Release by Peripheral Mononuclear Cells in Patients with Active Vitiligo

The purpose of this study was to clarify the relationship between the cellular and humoral immune components in the pathogenesis of vitiligo vulgaris. By using cytokines as indicators of peripheral mononuclear cell (MNC) function, we compared the effects of phytohemagglutinin (PHA) and purified IgG on MNCs derived from patients suffering from active vitiligo with those from normal controls. The results revealed (i) a significant increase in spontaneous production of IL-6 and IL-8 in patients; (ii) PHA, purified IgG from patients (IgG-anti-MC), or IgG from normal controls (N-IgG) induced a significant increase in IL-6 but diminished GM-CSF, TNF-alpha, and IFN-gamma release in patients; and (iii) IgG-anti-MC brought about a significantly higher stimulatory effect on IL-1beta and IFN-gamma production than N-IgG in normal controls. Immunologically, IL-6 can enhance melanocyte ICAM-1 expression, which may increase leukocyte-melanocyte attachment and cause melanocyte damage in vitiligo. A decrease in GM-CSF (an intrinsic growth factor for melanocyte) production may retard recovery from vitiligo by checking the proliferation of surviving melanocytes. A significant decrease in TNF-alpha and IFN-gamma production may partially explain the reduced inflammatory reaction in vitiliginous lesions. That IgG-anti-MC stimulates an increase in IL-1beta and IFN-gamma production in controls suggests that IgG-anti-MC may play a role in melanocyte destruction mediated by monocytes.

4-Hydroxynonenal inhibits interleukin-1 beta converting enzyme.

Lipid peroxidation results from the interaction of reactive oxygen species and polyunsaturated fatty acids. Metabolites generated from oxidative stress play an important role in the pathogenesis of a variety of diseases and biologic processes. One such product generated from lipid peroxidation in 4-hydroxynonenal (HNE). HNE is thiol reactive and exhibits numerous cellular effects. In this study, the inhibition of the cysteine protease, interleukin-1 beta (IL-1 beta) converting enzyme (ICE), by HNE in human blood mononuclear cells was investigated. HNE blocked the release of lipopolysaccharide (LPS)-stimulated IL-1 beta (EC50 5 microM) and IL-10 (EC50 2 microM) in a dose-dependent manner and, to a lesser extent, tumor necrosis factor-alpha (TNF-alpha) (EC50 15 microM) release. However, LPS-stimulated elevation of intracellular proIL-1 beta levels was not affected by HNE treatment. HNE inhibited ICE activity in lysed cells in a similar dose-dependent manner, measured by hydrolysis of the fluorogenic substrate YVAD-AMC and recombinant proIL-1 beta. To confirm that the inhibition of ICE activity by HNE was not an indirect effect, ICE activity was examined using purified recombinant human ICE (rHu-ICE). HNE inhibited rHu-ICE activity in a dose-dependent manner. Thus, low levels of HNE can suppress mononuclear cell release of IL-1 beta, probably by interacting with the active site cysteine of ICE. These results have implications for modulating mononuclear cell function during oxidative stress conditions.

Effects of prednisolone on bone turnover in patients with corticosteroid resistant asthma

Although corticosteroid resistant (CR) bronchial asthma is associated with impaired in vitro an din vivo glucocorticoid (GC) responsiveness in mononuclear cell function, it is not known whether this is a cell specific phenomenon or whether these patients are at risk of glucocorticoid side-effects as corticosteroid-sensitive (CS) subjects. In order to address this question we have examined the effects of GCs on biochemical indices of bone turnover in vivo. Six CS and six CR subjects received prednisolone 40 mg orally at 09.00 daily for 5 days. At 08.30 on day 1 and day 6 a fasting blood was taken for estimation of serum osteocalcin, tartrate resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP; total and bone isoenzyme), and a urine sample taken for estimation of free deoxy-pyridinoline crosslinks. TRAP and ALP were measured by a colorimetric method and osteocalcin and deoxy-pyridinoline crosslinks by ELISA. Serum osteocalcin (nmol/L) decreased from 1.39 +/- 0.09 (mean +/- SEM) to 1.14 +/- 0.07 (P = 0.023) and from 1.19 +/- 0.05 to 0.96 +/- 0.1 (P = 0.037) in the CS and CR groups respectively. There was no difference between baseline and post-treatment levels in either group. Serum total ALP (units/L) decreased from 178 +/- 7 to 168 +/- 4 (P = 0.02) and from 195 +/- 22 to 175 +/- 16 (P = 0.04) in the CS and CR groups, respectively. There was no difference between baseline and post-treatment levels in either group. There was no significant elevation of deoxy-pyridinoline crosslinks or TRAP and no suppression of bone ALP in either group. We suggest that CR asthmatics may be equally at risk from the metabolic side-effects of glucocorticoids.

Alterations in lymphocyte phenotype and function in children with shigellosis who develop complications.

This study was designed to see whether alterations occur in peripheral blood mononuclear cell phenotype and function in children with Shigella dysenteriae 1 infection with complications (leukemoid reaction and/or hemolytic-uremic syndrome) and whether there are any alterations prior to the development of complications. The following groups of children (ages, 12 to 60 months) were compared: children without any infection (n = 51), children with uncomplicated shigellosis (n = 65), children admitted with complicated shigellosis (leukemoid reaction and/or hemolytic-uremic syndrome) (n = 29), and children with shigellosis who developed complications after enrollment (subsequently complicated shigellosis) (n = 12). Tests for the peripheral blood mononuclear cell phenotype (CD3, CD4, CD8, CD57 [corrected], CD20, and CD25), spontaneous proliferation, and the proliferative response to phytohemagglutinin, pokeweed mitogen, and the lipopolysaccharide of S. dysenteriae 1 were performed, as were skin tests for delayed-type hypersensitivity (DTH). Children who subsequently developed complications differed from other groups of children as follows: (i) the numbers of CD3+ and CD4+ cells were lower than in uninfected children (P < 0.05), (ii) the CD4/CD8 ratio was lower than in children with uncomplicated shigellosis (P < 0.05) and in uninfected children (P < 0.05), and (iii) the levels of spontaneous proliferation of peripheral blood mononuclear cells were higher and DTH responses were lower than those in children with uncomplicated shigellosis (P < 0.05 and P < 0.017, respectively). Children with complications differed by having (i) increased numbers of CD3- CD57- [corrected] CD20- cells (P < 0.05) compared with those in other groups of children and (ii) lower CD4/CD8 ratios (P < 0.05), higher levels of spontaneous proliferation (P < 0.05), and lower DTH responses (P = 0.005) than children with uncomplicated shigellosis. Three to five days after enrollment, the number of CD4+ cells increased in children who subsequently developed complications (P = 0.025), i.e., when they developed complications and at this time their CD4+ cell number was similar to that of other groups of children. Thus, lymphocyte phenotype and function are altered prior to the development of complications in children with shigellosis, and once complications develop, the pattern of alterations changes. Whether these alterations have a role in precipitating complications or whether they reflect early events underlying the development of complications remains to be elucidated.

The effects of prednisolone on the cutaneous tuberculin response in patients with corticosteroid-resistant bronchial asthma

We have used the classical tuberculin cutaneous delayed hypersensitivity immune response to investigate the effects of corticosteroids on mononuclear cell function in nine subjects with corticosteroid-resistant asthma (CRA) and six subjects with corticosteroid-sensitive asthma (CSA), who demonstrated sensitivity to intradermal purified protein derivative (PPD) of Mycobacterium tuberculosis. In a double-blind, crossover, placebo-controlled study, patients were given oral prednisolone or placebo, starting on day 0, and a predetermined intradermal dose of PPD on day 7. On day 9 the site of the induration was measured and biopsied for immunohistochemical analysis. There was no difference in skin induration between the CSA and CRA groups during the placebo arm of the study ( p = 0.38). Prednisolone significantly suppressed the cutaneous induration ( p < 0.003) in the CSA group but not in the CRA group. As compared with placebo, the number of macrophages ( p = 0.018), eosinophils ( p = 0.009), and T memory cells ( p = 0.009) was suppressed by prednisolone in the CSA group but not in the CRA group. No significant suppression of the number of neutrophils or monocytes/immature macrophages was induced by prednisolone in either group. There was no difference in intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial leukocyte adhesion molecule-1 expression in blood vessels or epidermis between the CSA and CRA groups, with no suppression by prednisolone in either group. These findings suggest a defect in the responsiveness of cellular immune mechanisms to the suppressive effects of corticosteroids in steroid-resistant asthma. The differential suppressive effects of corticosteroids on cellular recruitment in the PPD response between the individuals with CSA and those with CRA are not due to modulation of expression of endothelial adhesion molecules. (J A LLERGY C LIN I MMUNOL 1996;97:698-706.)