Response Of Peripheral Blood Monocytes Research Articles

Single-cell profiling of peripheral blood and muscle cells reveals inflammatory features of juvenile dermatomyositis.

Introduction: Juvenile dermatomyositis (JDM) is a rare yet serious childhood systemic autoimmune condition that primarily causes skin rashes and inflammatory myopathy of the proximal muscles. Although the associated immune response involves the innate and adaptive arms, a detailed analysis of the pertinent immune cells remains to be performed. This study aims to investigate the dynamic changes of cell type, cell composition and transcriptional profiles in peripheral blood and muscle tissues, and in order to clarify the involvement of immune cells in the pathogenesis of JDM and provide a theoretical reference for JDM. Methods: Single-cell RNA sequencing combined with bioinformatic analyses were used to investigate the dynamic changes in cell composition and transcriptional profiles. Results: Analysis of 45,859 cells revealed nine and seven distinct cell subsets in the peripheral blood and muscle tissues respectively. IFITM2+ and CYP4F3+ monocytes were largely produced, and CD74+ smooth muscle cells (SMCs) and CCL19+ fibroblasts were identified as inflammatory-related cell subtypes in JDM patients, exhibiting patient-specific cell population heterogeneity.The dynamic gene expression patterns presented an enhanced type I interferon response in peripheral blood monocytes and T-cells, and SMCs and fibroblasts in muscle of untreated JDM patients. EGR1 and IRF7 may play central roles in the inflammation in both CD74+ SMCs and CCL19+ fibroblasts. Moreover, inflammatory-related monocytes could regulate T-cells, and the interaction between immune cells and SMCs or fibroblasts in muscle was enhanced under the inflammatory state. Conclusions: Immune dysregulation is one of the key pathogenic factors of JDM, and type I interferon responses are significantly enhanced in peripheral blood Monos and T cells as well as SMCs and fibroblasts. EGR1 and IRF7 may play central roles in the inflammation and are considered as potential therapeutic targets for JDM.

Typical response of CD14++CD16– monocyte to knee synovial derived mediators as a key target to overcome the onset and progression of osteoarthritis

ObjectiveSynovitis with increased infiltration of immune cells is observed in osteoarthritis (OA). Given the inflammatory condition of synovitis, we explored the protein profile of OA synovium (OAS) and its effect on circulating monocytes activation, migration, and functional commitments.MethodsKnee-synovium was acquired from end-stage OA (N = 8) and trauma patients (Trauma baseline control: TBC; N = 8) for characterization using H&E histology, IHC (iNOS), LCMS-QTOF, and MALDI-imaging. Response of peripheral blood monocytes to OAS conditioned-media (OACM) was observed using transwell (n = 6). The migrated cells were captured in SEM, quantified using phase-contrast microphotographs, and their activation receptors (CCR2, CXCR2, CX3CR1, and CD11b), pro-inflammatory genes, and phagocytic potential were studied using flow cytometry, gene expression array/qPCR, and latex beads (LB) phagocytosis assay, respectively.ResultsThe Venn diagram displayed 119 typical proteins in OAS, while 55 proteins in TBCS. The STRING protein network analysis indicated distinctive links between proteins and gene ontology (GO) and revealed proteins associated with leukocyte-mediated immunity in OAS as compared to TBC. The MALDI-imaging showed typical localized proteins at 2234.97, 2522.61, 2627.21, 3329.50, and 3539.69 m/z and IHC confirmed pro-inflammatory iNOS expression in OA synovium. CD14++CD16– classical monocytes significantly migrated in OACM and expressed CCR2, CXCR2, and CD11b receptors, TNFRSF11A, MAPK1, S100A8, HSPB1, ITGAL, NFATC1, IL13RA1, CD93, IL-1β, TNF-α, and MYD88 genes and increased LB uptake as compared to SFM.ConclusionOur findings suggest that the differential protein profile of OA synovium and the classical monocytes migrated, activated, and functionally committed in response to these mediators could be of therapeutic advantage.

IFNγ signaling response in peripheral blood monocytes: A new prognostic biomarker for breast cancer?

IFNγ signaling response in peripheral blood monocytes: A new prognostic biomarker for breast cancer?

Effects of EPA and DHA Supplementation on Plasma Specialized Pro-resolving Lipid Mediators and Blood Monocyte Inflammatory Response in Subjects with Chronic Inflammation (OR29-01-19)

ObjectivesThe role of n-3 fatty acid-derived specialized pro-resolving mediators (SPMs), including the novel docosapentaenoic acid (DPA) products, in reducing inflammation in humans has not been determined. We evaluated the differential effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation on plasma SPMs and the resulting impact on the inflammatory response of peripheral blood monocytes. MethodsIn a randomized, controlled, crossover trial, 21 subjects (9 men and 12 women, 50–75 y) with chronic inflammation (C-reactive protein > 2 μg/mL) entered a 4-week lead-in control phase (high oleic sunflower oil, 3 g/d) and then two sequential 10-week supplementation phases with pure EPA or DHA (3 g/d each), separated by a 10-week washout phase. Plasma phospholipid (PL) fatty acid composition and SPMs, including their precursors, were measured at the end of each phase. Following lipopolysaccharides (LPS) stimulation, the inflammatory response of blood monocytes was assessed by inflammatory gene expression. ResultsEPA increased PL EPA (P < 0.001) and plasma concentrations of 18-HEPE, the precursor of the E-series resolvins (RvEs) (P < 0.001). The increase in plasma 18-HEPE concentrations, was associated with the increase in PL EPA (β = 14.9 pg/ml, P < 0.01). However, RvEs were undetectable. EPA increased PL DPA (P < 0.001) but not DPA-derived SPMs. DHA increased PL DHA and plasma concentrations of 17-HDHA and 14-HDHA, the precursors of DHA-derived SPMs (P < 0.001). DHA also significantly increased PL EPA and 18-HEPE (P < 0.001), suggesting some DHA retroconversion to EPA. Interestingly, DHA lowered PL DPA (P < 0.001) but increased the DPA-derived SPMs RvD5n-3 DPA and MaR1n-3 DPA (P < 0.001). In monocytes, while both EPA and DHA lowered the LPS-induced expression of TNFA(P < 0.03 and P < 0.001, respectively), TNFA expression was inversely correlated with plasma concentrations of MaR1n-3 DPA (ρ = –0.32, P < 0.04). ConclusionsRelative to EPA, DHA supplementation increases a broader range of SPMs, with EPA and DHA differentially affecting PL DPA and DPA-derived SPMs. Plasma concentrations of MaR1n-3 DPA following EPA and DHA supplementation are associated with an attenuated inflammatory response in blood monocytes, suggesting a potential role of this SPM in reducing inflammation in humans. Funding SourcesFunded by USDA/NIFA and Cohn Student Award. Supporting Tables, Images and/or Graphs▪

A Blunted Response to the Anti-Inflammatory Cytokine IL-10 Contributes to Excessive TNF Production in Myeloproliferative Neoplasm

Rationale:Tumor Necrosis Factor-alpha (TNF) is elevated in myeloproliferative neoplasm (MPN) and plays a key role in expansion of the JAK2V617F neoplastic clone. A high TNF environment, as is the case in MPN patients, gives TNF resistant JAK2V617F mutant cells a selective advantage over their TNF sensitive non-mutant counterparts resulting in expansion of the neoplastic clone. To efficiently target TNF production therapeutically it is necessary to identify the mechanism driving this excessive TNF production. TNF is classically produced by monocytes after stimulation through Toll-like receptors (TLR), crucial pattern recognition receptors for microbial products. Because TLR signaling plays an integral role in inflammation and TNF production, we hypothesized that exaggerated signaling of the TLR pathway is the mechanism by which TNF is overproduced in MPN.Results:We compared the response of peripheral blood monocytes from MPN versus normal controls to the TLR agonists R848 (TLR7/8), LPS (TLR4), or zymosan (TLR2). After stimulation with each of these TLR agonists for 24 hours, CD14+ monocytes from MPN patients (n=18) produced increased amounts of TNF (measured by ELISA) as compared to normal controls (n=10) at all concentrations tested (p<0.05). The increased TNF production in MPN monocytes could not be explained by a higher fraction of inflammatory (CD16+ CD14+) monocytes in MPN. We next used phosflow to detect whether MPN patients have abnormal activation of downstream signaling molecules following stimulation with TLR agonists. At early time points (15min) following stimulation, MPN patients (n=6) and normal controls (n=6) phosphorylated p38 and ERK1/2 equally. At later time points (2hrs) MPN patients maintained phosphorylation of p38 and ERK1/2, whereas in normal controls phosphorylation of p38 and ERK1/2 returned to baseline (p<0.05).This suggested that the negative feedback regulation of TLR signaling may be defective in MPN patients resulting in prolonged TNF production after stimulation. To test this, we compared the tempo of TNF production following LPS stimulation in MPN versus normal monocytes using intracellular flow cytometry as well as ELISA. Monocytes from normal controls (n=12) cease production of TNF by 9 hours post LPS stimulation, whereas MPN monocytes (n=12) continue to produce TNF at 18 hours (p<0.05).LPS stimulation induces a negative feedback loop culminating in the production of IL-10, an anti-inflammatory cytokine which serves to dampen TNF production. We found that MPN monocytes produce abundant IL-10 in response to LPS stimulation. Next we measured the ability of IL-10 to dampen TNF production in LPS stimulated monocytes comparing MPN patients versus normal controls. Addition of low concentrations of rhIL-10 (1ng/ml) to LPS stimulated monocytes reduced TNF production by 50% in normal controls (n=8), whereas it reduced TNF production by only 10-20% in MPN (n=5). However, higher concentrations of IL-10 (>10ng/ml) was able to dampen TNF production in LPS stimulated MPN monocytes by 50%.Conclusions:MPN monocytes produce excessive amounts of TNF in response to TLR ligation due to a defect in the negative regulatory feedback loop which normally serves to dampen TNF production following TLR ligation. We have localized this defect to a blunted response to the anti-inflammatory cytokine IL-10. MPN monocytes are less responsive to the anti-inflammatory actions of IL-10 at low concentrations but these effects can be restored by increasing the concentration of IL-10. Together, these data suggest that administration of IL-10 may reduce excessive inflammatory cytokine production in MPN. DisclosuresNo relevant conflicts of interest to declare.

Placental Growth Factor Enhances Toll-like Receptor-Induced Inflammatory Cytokine Gene Expression Transcriptionally in Human Mononuclear Phagocytes

The production of placental growth factor (PlGF) increases during normal third trimester pregnancy, a point at which women are at an increased risk of morbid viral infections (e.g. an 8-fold increased risk of mortality during the 2009 H1N1 influenza pandemic). Pathologic increases in PlGF have also been detected in sickle cell disease patients and are associated with similarly increased morbidity (hospitalizations and acute chest syndrome). While treatment of normal donor mononuclear cells with exogenous PlGF reportedly increases cytokine transcripts (including tumor necrosis factor-alpha [TNF]), whether TNF mRNA is translated is unclear, as are: (a) the comparative activity of PlGF isoforms, (b) the scope of inflammatory cytokines involved, (c) the TNF-dependency of other induced cytokines, (d) and the potential effects of PlGF when combined with toll-like receptor (TLR) pathway activators. Consequently, we sought to test the hypothesis that PlGF could enhance pro-inflammatory responses of peripheral blood monocytes to TLR-stimulation and focused on endosomal TLRs which would be expected to be activated by the types of viral infections (ssRNA) for which pregnant women and sickle cell anemia patients are at risk.Normal primary peripheral blood CD14+ cells were exposed to the PlGF isoforms (PlGF-1, -2, -3). None induced TNF secretion. However, when CD14+ cells were exposed to both PlGF-1 (250 ng/ml) and the TLR-7/8 ligand R848 (3 μM), production of TNF mRNA (qRT-PCR) at 4-6 hours (mean 2.52-fold, p=0.05, 10 separate experiments) and TNF protein (ELISA) at 24-hours (mean 2.15-fold, p=0.0007, 24 separate experiments) was significantly enhanced. PlGF-2 and -3 had no effect on TLR-7/8 activated cells. PlGF-1 did not prolong TNF mRNA half-life (Actinomycin D chase results) but did enhance TNF gene transcription. Specifically, in kinetic studies we quantified equivalent increases in both spliced and unspliced TNF mRNA and TNF protein (Figure 1). The same experiments indicated that PlGF-1 prolongs the duration of TNF gene transcription compared to cells treated with R848 alone. The synergistic effect of PlGF with TLR-7/8 was not seen with the TLR-4 activator LPS. VEGF-A exhibited no influence on TLR-7/8 induced TNF gene expression.We sought to identify signaling pathways involved in the PlGF/TLR effect and determined by western blotting (total IKK-α and β, and phospho-IKK-α/β) that PlGF markedly enhanced R848-induced phosphorylation of IKK. Moreover, treatment of primary cells with an IKK inhibitor (BMS 345541; 0.5, 1.5, 5 μM) inhibited the enhancing effect of PlGF on R848-induced TNF production. TNF gene expression was not inhibited by small molecule inhibitors of p38 MAPK, PI3K, MEK-1/2, or ERK-1/2. Gene microarray analysis (Affymetrix HTA 2.0) revealed a PlGF/R848-specific increase in RIPK1 mRNA encoding a known IKK activator.To identify other inflammatory cytokines influenced by PlGF/TLR, and their dependency on TNF, we performed Luminex multiplex cytokine arrays of conditioned culture media from CD14+ cells cultured for 6 and 24-hours with PlGF-1 (250 ng/ml) and R848 (3 μM), and found (n=2 biological replicates) that PlGF enhanced R848-induced protein levels of: TNF, interleukin-6 (IL-6), IL-8, IL-1β, macrophage inflammatory protein 1 alpha (MIP-1α), MIP-1β, monocyte chemoattractant protein-1 (MCP-1), interferon-alpha, G-CSF, and monocyte interferon gamma inducing factor (MIG). To test the idea that the production of these factors was TNF-dependent, CD14+ cells were cultured as above in the presence and absence of soluble TNF receptor (sTNFR) (0.1, 1, 10 μg/ml). Complete inhibition of signal transduction through the TNF receptor had no effect on the production of other inflammatory cytokines (i.e. IL-8).We conclude that PlGF directly contributes to an exaggerated pro-inflammatory response in human mononuclear phagocytes incited by TLR-7/8 activation and suggest that it does so by promoting the expression or activation of an IKK kinase (e.g. RIPK1) and/or suppressing an IKK phosphatase. We also conclude that the production of inflammatory cytokines by PlGF and R848 is independent of autocrine TNF production. Clarification of the effect of PlGF on TLR-pathway hyper-reactivity may lead to the identification of key pathways that can be safely suppressed in individuals with high PlGF levels to achieve the goal of reducing virus related morbidity and mortality. [Display omitted] DisclosuresTyner:Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding; Array Biopharma: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding.

Exaggerated Response to Toll-like Receptor Agonist Contributes to Excessive TNF Production in Myeloproliferative Neoplasm

Rationale:Tumor Necrosis Factor-alpha (TNF) is elevated in myeloproliferative neoplasm (MPN) and plays a key role in expansion of the JAK2V617F neoplastic clone. A high TNF environment, as is the case in MPN patients, gives TNF resistant JAK2V617F mutant cells a selective advantage over their TNF sensitive non-mutant counterparts resulting in expansion of the neoplastic clone. To efficiently target TNF production therapeutically it is necessary to identify the mechanism driving this excessive TNF production. TNF is classically produced by monocytes after stimulation through Toll-like receptors (TLR), crucial pattern recognition receptors for microbial products. Because TLR signaling plays an integral role in inflammation and TNF production we hypothesized that exaggerated signaling of the TLR pathway is the mechanism by which TNF is overproduced in MPN. To test this hypothesis we quantified TLR responses in monocytes from MPN patients and normal controls.Results:We compared the response of peripheral blood monocytes from MPN versus normal controls to the TLR agonists R848 (TLR7/8), LPS (TLR4), or zymosan (TLR2). After stimulation with each of these TLR agonists for 24 hours, CD14+ monocytes from MPN patients (n=18) produced increased amounts of TNF (measured by ELISA) as compared to normal controls (n=10) at all concentrations tested (p<0.05). The increased TNF production in MPN monocytes could not be explained by a higher fraction of inflammatory (CD16+ CD14+) monocytes in MPN. We next used phosflow to detect whether MPN patients have abnormal activation of downstream signaling molecules following stimulation with TLR agonists. At early time points (15min) following stimulation, MPN patients (n=6) and normal controls (n=6) phosphorylated p38 and ERK1/2 equally. At later time points (2hrs) MPN patients maintained phosphorylation of p38 and ERK1/2, whereas in normal controls phosphorylation of p38 and ERK1/2 returned to baseline (p<0.05). These data suggest that the excessive production of TNF in MPN patients may be due either to persistent activation of signaling following TLR stimulation or failure to repress the activation state of these two proteins.TLR ligation induces a negative feedback loop culminating in the production of IL-10, an anti-inflammatory cytokine which serves to dampen TNF production. We next used intracellular flow cytometry to determine whether the excessive TNF production in MPN is due to a failure to dampen TNF production over time. Monocytes from normal controls (n=12) cease production of TNF by 9 hours post LPS stimulation, whereas MPN monocytes (n=8) continue producing TNF (p<0.05). This continued TNF production by MPN monocytes is coincident with a failure to produce the anti-inflammatory cytokine IL-10 in response to TLR ligation (p<0.05).Conclusions:We find that MPN monocytes produce excessive amounts of TNF in response to TLR ligation. This excessive production of TNF is due to a defect in the negative regulatory feedback loop which normally serves to dampen TNF production. Targeting the TLR pathway therapeutically in MPN may serve to reduce TNF production and neutralize the selective advantage of the JAK2V617F neoplastic clone, ultimately leading to a reduction in the JAK2V617F allele burden. DisclosuresNo relevant conflicts of interest to declare.

Two faces of vitamin e in the lung.

To the Editor: The hypothesis for tocopherol isoform-dependent effects on asthma prevalence by Cook-Mills and colleagues (1) is derived in part from their important animal work, but also their interpretation of our own work with γ-tocopherol (γT) in experimental asthma. Although we have recently demonstrated that γT-enriched supplements can reduce endotoxin-induced neutrophil responses in human lung (2), we think the authors misinterpret our results in experimental asthma models when they conclude that γT was effective against neutrophilic, but not eosinophilic, inflammation. Both our results with antigen-induced asthma and rhinitis in rats (3) and ozone-induced exacerbation of allergic rhinitis (4) demonstrate 70–100% reduction in tissue or lavage eosinophils. We saw no effect of γT on any attendant neutrophil airway inflammation. Our results were also striking for the complete abolition by γT of mucous cell metaplasia in nasal and pulmonary airways, which is a key feature of clinical rhinitis and asthma. Therefore, our results with the allergic brown Norway rats are opposed to the enhanced allergic responses associated with γT, as described in vitro and in BALB/c mice (5, 6). The use of different rodent species (brown Norway rat vs. BALB/c mouse) may contribute to the disparate observations in these animal models. Furthermore, we provided γT by oral delivery rather than by the subcutaneous route in mice, which would bypass liver metabolism. This is an important distinction considering that the anti-inflammatory metabolites of γT are known to be more potent than those of its precursor in inhibiting COX-2- and 5-LOX-mediated eicosanoids (7, 8). Epidemiologic data suggesting positive correlations between plasma γT levels and diseases including lung disease are intriguing. However, there are many interpretations that can be made from these associations. One is, as the authors suggest, that γT promotes lung and inflammatory disease. A second is that these diseases result in an increase of γT as a response to ongoing disease. A third is that γT may be a co-nutrient with other agents that promote disease. Indeed, many plant oils that are commonly used in the United States but not Europe (such as corn oil and soybean oil) not only contain high levels γT but are also high in polyunsaturated fatty acids and n-6 fatty acids, which are known to contribute oxidative stress and promote inflammation. Thus, the apparent effect of γT may be a result of the polyunsaturated fatty acids and n-6 fatty acids contained in these oils. Interestingly, safflower and olive oils, which contain relatively high α-tocopherol levels and are commonly used in Europe, are rich in monounsaturated fatty acids, which are resistant to oxidative stress and inflammation. Although animal studies and epidemiological studies provide important data regarding the actions of both γ- and α-tocopherol in inflammatory processes, only intervention studies can demonstrate the actions of these variants of vitamin E on inflammatory processes in humans. We have published two oral intervention studies in humans using a γT-enriched preparation. In these studies, we have shown that active treatment is associated with decreased ex vivo response of peripheral blood monocytes to LPS, as well as airway inflammatory response to inhaled LPS (2, 9). We have also shown that peripheral blood basophils recovered from allergic volunteers have inhibited degranulation responses to allergen ex vivo (10, 11). These observations, coupled with our animal data, strongly support the hypothesis that γT has anti-inflammatory actions against both allergic and nonallergic inflammation. We do not disagree that α-tocopherol has anti-inflammatory actions as well, especially in light of the intervention studies cited by Cook-Mills and colleagues. Overall, we agree that nutritional and nutraceutical interventions represent exciting new and inexpensive approaches to decreasing allergic disease. We also agree that careful attention to dose, route of administration, duration of therapy, analyses of serum levels of tocopherols (and their metabolites), and clinical endpoints will be important in defining the role of tocopherols in asthma therapy. Thus, extending the investigation of the roles of α- and γ-tocopherol in the modulation of allergic and inflammatory disease into human populations is needed to fully understand the roles of these agents in allergic disease.

Attenuation of toll-like receptor 2-mediated innate immune response in patients with alcoholic chronic liver disease

Alcoholic chronic liver disease (ACLD) is a common form of acquired immunodeficiency. To evaluate ex vivo toll-like receptor (TLR) 2 and TLR4 innate immune response in stable ACLD. Blood was collected from 26 males with stable ACLD and from 17 controls. Serum was used for lipopolysaccharide (LPS), sCD14, LPS-binding protein (LBP), tumour necrosis factor-alpha (TNF-alpha) and interleukin 10 (IL-10) quantification. Peripheral blood monocytes (PBM) protein expression of TLR2 and TLR4 was determined by flow cytometry. Primary cultures of anti-CD11b positive selected PBM were stimulated with the TLR2/TLR6 ligand zymosan (Zym), with TLR2/TLR1 ligand lipopeptide (Lp) and with TLR4 ligand LPS. PBM TLR1, TLR2, TLR4, TLR6, MD2, CD14, TNF-alpha and IL-10 gene expression was evaluated by reverse transcription-polymerase chain reaction. Stable ACLD patients showed increased circulating LPS (+22.5+/-4.1%), LBP (+60.6+/-12.2%) and sCD14 (+23.5+/-4.6%), with no differences in TNF-alpha and IL-10. Zym and Lp, but not LPS, induced TNF-alpha production by monocytes was blunted in ACLD (-66+/-20.4% Zym; -40.1+/-13.5% Lp; P<0.05). Basal TNF-alpha mRNA expression was decreased in PBM from ACLD patients (-50.1+/-21.0%; P<0.05), with no significant differences in the other studied genes. Results were similar in Child-Pugh A and B/C patients. Patients with stable ACLD show an attenuation of TLR2-mediated innate immune response in PBM, which may represent an important mechanism for acquired immunodeficiency. This was neither related with decreased TLR2 or its co-receptors expression nor with impaired TLR4 activation, being already present in the early stages of disease.

Peripheral blood monocytes (PBMCs) display innate antiviral response to rhinovirus (RV) infected bronchial epithelial cells (133.44)

Abstract Rhinovirus (RV) infection is an important trigger of acute asthma and COPD. Study objectives; were to characterise the response of PBMCs and CD8 lymphocytes when exposed to RV infected bronchial epithelial cells. Methods; Human Calu3 cells were used as a model for bronchial epithelial cells and infected with RV-43 and RV-1B. PBMCs were obtained from healthy individuals and separated by Ficoll-Paque centrifugation. CD8 lymphocytes were identified as CD3+, CD8+ by flow cytometry. PBMCs were exposed to infected Calu3 cells, cells exposed to UV inactive RV, PHA and TLR3 agonist PolyI:C. Response was measured by ELISA for release of interferon(IFN)-γ and TNF-α. CD8 activation with expression of perforin or proliferation measured by carboxyfluorescein succinimidyl ester assay. Results: Exposure of PBMCs to Calu3 cells infected with both RV led to significant release of IFN-γ and TNF-α, smaller release was seen in response to UV inactive RV. Exposure to PolyI:C also led to a similar release. CD8+ cells were found to be the primary source of IFN-γ release. CD8+ cells did not show an increase in perforin expression or evidence of proliferation. Conclusions; exposure of PBMCs and CD8+ cells to an RV infected bronchial epithelium alone results in an innate immune response mediated by TLR3 without evidence of a CD8 adaptive immune response.

Macrophages are a source of extracellular adenosine deaminase-2 during inflammatory responses

Serum activity of the adenosine deaminase (ADA) isozyme, ADA2, has been reported to be elevated during various disease states. Macrophages have been suggested as the cellular source of extracellular ADA activity because they are one of the only cell types in which intracellular ADA2 activity has been measured, but extracellular secretion has never been demonstrated. Rat primary peritoneal macrophages (PPMs) and peripheral blood monocytes (PBMs) were harvested and incubated for 18 h in RPMI supplemented with horse serum. PPM and PBM lysates were assayed for intracellular ADA activity (ammonia production). In vitro and in vivo extracellular ADA activities were measured in media and rat serum, respectively. Activity of ADA1 was confirmed by selective inhibition with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). ADA2 activity was inhibited by 2'-deoxycoformcin only, and was increased at a low pH (6.5). Activity of both ADA isozymes was found in PPMs and PBMs, and their media. In a separate group of rats, peritonitis was induced by ip insertion of 400 mg/kg caecal slurry. PPMs were harvested 24 h later and incubated for 18 h. In PPMs from rats with peritonitis both isozymes were elevated by a similar proportion. In contrast, media from these PPMs had a lower ADA1 and a higher ADA2 activity compared to PPMs from nonseptic rats. This resulted in a greater proportion of ADA2 in media. The isozyme proportions in serum from septic rats more closely resembled that of the PPM media. The response of PBM was small relative to that of PPM. These results suggest that macrophages are a significant source of extracellular ADA isozymes, the activity of which increases during an inflammatory response. Because extracellular isozymes profiles differ from cellular concentrations, the data also suggest differential release of each isozyme from PPMs.

T cell cytokine responses to outer membrane proteins of Haemophilus influenzae and the house dust mite allergens Der p 1 in allergic and non-allergic subjects.

Haemophilus influenzae are ubiquitous colonizers of the nasopharynx, Little is known about the T cell cytokine responses to the antigens of these bacteria and whether or not the responses may interact with responses to aeroallergen. To measure the T cell cytokine responses to conserved outer membrane protein antigens of Haemophilus influenzae and to house dust mite allergen of subjects allergic to the house dust mite and of subjects without allergic sensitization. T cell responses were measured by in vitro proliferation and cytokine release from peripheral blood monocytes (PBMC). The allergen used was Der p 1 and outer membrane proteins were recombinant polypeptides representing the OMP6 and D15 antigens. The PBMC of most subjects had proliferative responses to OMP6 and D15, which were highly correlated. The pattern of cytokine release was Th1 biased with high levels of IFN-gamma and usually little IL-5 or IL-13 although PBMC from a few subjects did release IL-5 independent of allergic status. IL-10 release was readily detected. There was no difference in the anti-OMP cytokine response of PBMC from subjects without any known allergy and the responses of PBMC from subjects who were highly allergic to house dust mite. The responses to the Der p 1 allergen showed the expected Th2 cytokine release. The outer membrane protein antigens of the ubiquitous colonizing bacteria Haemophilus influenzae induce Th1 cytokine responses which are similar for PBMC from non-allergic individuals and subjects with a high degree of allergy to the perennial house dust mite allergen and strong Th2 responses to Der p 1.

INFLUENCE OF THE MENSTRUAL CYCLE ON THE LPS-INDUCED CYTOKINE RESPONSE OF MONOCYTES

We previously observed a gender difference in the cytokine response of peripheral blood monocytes (PBM). The study was performed to find out whether the gender-related difference might be due to hormonal changes during the menstrual cycle in healthy premenopausal females. The release of tumour necrosis factor α (TNF-α) was reduced in the premenopausal females during the luteal phase compared to the follicular phase. Compared to the male controls the release of TNF-α and of interleukin 6 (IL-6) during the luteal phase was also diminished. In premenopausal females the concentration of estradiol in plasma correlated with the release of TNF-α and IL-6 during the luteal phase.

Induction of prostaglandin release from macrophages by bacterial endotoxin.

This review summarizes the role of the monocytic responses to lipopolysaccharide as it relates to periodontal disease severity. Data are presented which illustrate that the levels of prostaglandin E2 (PGE2) secreted by systemic peripheral blood monocytes in culture, in the presence of bacterial endotoxins, are highly correlated with the levels observed in the gingival crevicular fluid. Furthermore, the different periodontal diagnostic categories have varying levels of monocytic and crevicular fluid PGE2, in juxtaposition with clinical disease severity. These data are consistent with the concept that there is close synchrony between the systemic responsiveness of peripheral blood monocytes with regard to prostanoid synthesis and the local levels of mediator present within the gingival crevice.

Human monocyte/macrophage response to cobalt-chromium corrosion products and titanium particles in patients with total joint replacements.

The responses of human peripheral blood monocytes of 10 normal volunteers and 14 patients with total hip replacements to particles of commercially pure titanium and chromium orthophosphate (a corrosion product from cobalt-chromium alloy implants) were studied. In addition, these phagocytosable particles were added to cultured mononuclear cells isolated from the interfacial membrane of 14 patients with failed implants. Peripheral blood monocytes from patients who had had a total hip replacement produced significantly higher levels of interleukin-1 (both interleukin-1 alpha and interleukin-1 beta) and prostaglandin E2 following particulate stimulation than those from normal volunteers. Supernatants from both titanium and chromium orthophosphate-stimulated peripheral blood monocytes from the volunteers and patients with total hip replacement induced bone resorption (assayed in organ cultures of newborn mouse calvariae) and the proliferation of human fibroblasts. The levels of bone resorption were significantly higher (p < 0.05) in patients with implants than in normal volunteers. There were no significant differences in the responses of cells between patients with focal osteolysis and those without osteolysis. Interfacial membrane mononuclear cells also produced high levels of interleukin-1 alpha, interleukin-1 beta, and prostaglandin E2 and expressed bone resorptive activities following stimulation with either titanium or chromium orthophosphate. More importantly, interfacial membrane mononuclear cells "spontaneously" produced high levels of prostaglandin E2 that were comparable with the response of peripheral blood monocytes stimulated with particulate wear debris. The clinical relevance of this study is 2-fold. First, mononuclear cells from patients with total hip replacement were some-how "sensitized" to metal particles in comparison with mononuclear cells from individuals without an implant. Second, the chromium orthophosphate corrosion product was a potent macrophage/monocyte activator and may contribute to macrophage-mediated osteolysis and aseptic loosening.

Subcutaneously Administered Recombinant Interferon-γ in Humans

A Phase I trial was conducted to investigate the clinical toxicity, pharmacokinetics, and chemiluminescence (CL) responses of alveolar macrophages (AMs), peripheral blood neutrophils, and monocytes after subcutaneous injection of recombinant interferon-gamma (rIFN-gamma). Six patients with lung cancer received rIFN-gamma subcutaneously as single doses of 0.2, 0.6, and 1.8 mg. Bronchoalveolar lavage was performed three times: 21 h before as well as 6-7 and 27 h after injection. Serum samples were taken five times during the 27-h follow-up. IFN concentrations were measured from alveolar epithelial lining fluid (ELF) and serum by using an antiviral bioassay. IFN-gamma was not detectable in ELF after subcutaneous injection. AMs did not effect an increase in CL responses to N-formyl-methionyl-leucyl-phenylalanine or to phosphate-buffered saline. Circulating IFN-gamma was detectable at 3-12 h after an injection of 1.8 mg of rIFN-gamma, the highest dose given. CL responses of peripheral blood monocytes increased in all patients after injection, whereas the responses of neutrophils were less clear-cut. All patients developed systemic side effects such as transient fever, nausea, headaches, and flu-like symptoms. The findings suggest that rIFN-gamma passes poorly from the blood to the pulmonary alveoli. On the basis of this and our previous findings of increased CL responses in AMs and measurable IFN concentrations in ELF after inhalation of rIFN-gamma, we recommend inhalation rather than the parenteral route of IFN-gamma for the treatment of respiratory diseases.

Oxidative metabolic response of peripheral blood monocytes of monkeys during primary and chronic Plasmodium knowlesi infection

The oxygen free radical generation (as determined by cytochrome c reduction) and respiratory burst enzyme activities were measured in the peripheral blood monocytes before infecting normal animals, during primary Plasmodium knowlesi infection, after treatment of primary infection with chloroquine, after administration of various subcurative doses following reinfection and after establishment of chronicity. For all the parameters of the oxidative response, no significant difference was observed during primary infection and after curing of primary infection. However, the response was significantly increased in the reinfected monkeys given subcurative therapy; this was followed by a sharp decline after the 4th subcurative dose, after which the response continued to be low until the attainment of chronicity. The results of the present study exclude the possibility of the increase in oxidative metabolism being the effect of the drug (i.e. chloroquine), since no significant difference was observed during primary infection and after its cure. The sustained low grade parasitaemia in the chronic stage, in spite of the sharp decrease in the oxidative response, suggests that some nonoxidant parasitic killing mechanism might be playing a role in the regulation of parasitaemia.

Increased monocyte chemiluminescence in cystic fibrosis patients and in their parents.

We examined the chemiluminescence response of peripheral blood monocytes from patients with cystic fibrosis (CF) and their asymptomatic parental carriers of the CF gene to three different types of stimulation. We found that monocytes from both patients and carriers have increased luminol-dependent chemiluminescence in the first 25 min after stimulation by adherence to glass. These results are consistent with the hypothesis that monocytes from both CF heterozygotes and homozygotes respond to adhesion with increased oxygen radical formation. The increased adherence-induced monocyte chemiluminescence of the parental carriers did not vary with age or length of exposure of the parents to a child with CF. Also, repeated exposure to medications and respiratory secretions of CF patients was not associated with an increase in adherence-induced monocyte chemiluminescence of their nonbiologically related caretakers. Thus, this observed increase in chemiluminescence is not simply secondary to the medications or respiratory dysfunction seen in the patients with CF. Patients with other types of obstructive lung disease did not show increased adherence-induced monocyte chemiluminescence. We conclude that increased early phase adherence-induced monocyte chemiluminescence occurs in patients with cystic fibrosis and the obligate carriers of the CF gene independent of environmental influences.

Defective chemotactic response of human alveolar and colostral macrophages

This report describes the chemotactic response of human alveolar macrophages (AMφ) and milk macrophages (MMφ) to a panel of chemotactic agents: endotoxin (EAS) and zymosan (ZAS) activated serum, lymphocyte derived chemotactic factor (LDCF) and formylated synthetic peptides. The locomotion studies were compared with the responses of peripheral blood monocytes. Both AMφ and MMφ exhibited an extremely poor chemotactic response to all agents in comparison with the monocytic response. When monocytes were cultured for long periods, a defective response was likewise demonstrated. The chemotactic response was significantly higher in AMφ from smokers. The stimulated locomotion was not increased by the addition of a surfactant lipoprotein to the AMφ suspension. Moreover, monocytes incubated with fat- and cell-free human milk exhibited lower chemotactic responses than normal monocytes and practically in the same range as that obtained with MMφ.

Growth, Interleukin-2 Production, and Responsiveness to IL-2 in T4-Positive T Lymphocyte Populations From Malignant Cutaneous T Cell Lymphoma (Sézary’s Syndrome): The Effect of Cyclosporine A

Abstract Functional analysis and surface phenotyping using monoclonal antibodies have revealed that malignant T lymphocyte populations in the peripheral blood of patients with Sezary's syndrome resemble the T helper cell populations from normal individuals. In this article we have studied the effects of the immunosuppressive drug cyclosporine A (CsA) on growth, interleukin-2 (IL-2) production, and the induction of IL-2 responsiveness of peripheral blood monocytes (PBMs) from five patients with Sezary's syndrome in vitro, using the lectin phytohemagglutinin (PHA) and the phorbol ester phorbol myristate acetate (PMA) as stimuli. The following results were obtained: PHA-induced cell proliferation was significantly more sensitive to inhibition by CsA than that induced by PMA or a combination of PMA and PHA (P less than .005). Sezary PBMs produced only small amounts of IL-2 in response to PHA. Stimulation with PMA, however, resulted in significant IL-2 production. PMA and PHA, when given in combination, acted synergistically. The low levels of IL-2 production induced by PHA or PMA were more sensitive to CsA- mediated suppression than those induced by a combination of PHA and PMA (75% and 55% suppression, respectively). CsA-mediated growth suppression could be overcome if the cultures were supplemented with appropriate amounts of exogenous IL-2. We conclude from our data, that CsA in Sezary PBMs inhibits T cell growth indirectly as a consequence of suppression of IL-2 growth indirectly as a consequence of suppression of IL-2 production. Moreover, like normal T lymphocytes, Sezary PBMs do not express the IL-2 receptor spontaneously, but can be induced to do so. CsA does not interfere with intracellular events leading to the expression and the biologic function of the IL-2 receptor.