Triple-Color FluoroSpot Analysis of Polyfunctional Antigen-Specific T Cells by Quantification of Spot-Forming Units and Relative Spot Volumes.

Switching from ELISpot to FluoroSpot enables the analysis of spot-forming units representing cells producing different cytokines as well as the frequencies of spots derived from cells co-secreting multiple cytokines. Due to the fluorescent read-out signal, sophisticated reader instruments can also measure the relative spot volume, making it possible to differentiate between spots generated by cells secreting different levels of one or more cytokines. Here we describe how triple FluoroSpot assays can be used to define polyfunctional T cells secreting multiple cytokines and how different T-cell populations can differ in the levels of cytokines they secrete.

Memory B-Cell Responses Against Merozoite Antigens After Acute Plasmodium falciparum Malaria, Assessed Over One Year Using a Novel Multiplexed FluoroSpot Assay.

Memory B cells (MBCs) are believed to be important for the maintenance of immunity to malaria, and these cells need to be explored in the context of different parasite antigens and their breadth and kinetics after natural infections. However, frequencies of antigen-specific MBCs are low in peripheral blood, limiting the number of antigens that can be studied, especially when small blood volumes are available. Here, we developed a multiplexed reversed B-cell FluoroSpot assay capable of simultaneously detecting MBCs specific for the four Plasmodium falciparum blood-stage antigens, MSP-119, MSP-2, MSP-3 and AMA-1. We used the assay to study the kinetics of the MBC response after an acute episode of malaria and up to one year following treatment in travelers returning to Sweden from sub-Saharan Africa. We show that the FluoroSpot assay can detect MBCs to all four merozoite antigens in the same well, and that the breadth and kinetics varied between individuals. We further found that individuals experiencing a primary infection could mount and maintain parasite-specific MBCs to a similar extent as previously exposed adults, already after a single infection. We conclude that the multiplexed B-cell FluoroSpot is a powerful tool for assessing antigen-specific MBC responses to several antigens simultaneously, and that the kinetics of MBC responses against merozoite surface antigens differ over the course of one year. These findings contribute to the understanding of acquisition and maintenance of immune responses to malaria.

Activation of Neutrophils via IP3 Pathway Following Exposure to Demodex-Associated Bacterial Proteins.

Rosacea is a chronic inflammatory condition that predominantly affects the skin of the face. Sera from rosacea patients display elevated reactivity to proteins from a bacterium (Bacillus oleronius) originally isolated from a Demodex mite from a rosacea patient suggesting a possible role for bacteria in the induction and persistence of this condition. This work investigated the ability of B. oleronius proteins to activate neutrophils and demonstrated activation via the IP3 pathway. Activated neutrophils displayed increased levels of IP1 production, F-actin formation, chemotaxis, and production of the pro-inflammatory cytokines IL-1ß and IL-6 following stimulation by pure and crude B. oleronius protein preparations (2 µg/ml), respectively. In addition, neutrophils exposed to pure and crude B. oleronius proteins (2 µg/ml) demonstrated increased release of internally stored calcium (Ca(2+)), a hallmark of the IP3 pathway of neutrophil activation. Neutrophils play a significant role in the inflammation associated with rosacea, and this work demonstrates how B. oleronius proteins can induce neutrophil recruitment and activation.

ApoE production in human monocytes and its regulation by inflammatory cytokines.

The apoE production by tissue macrophages is crucial for the prevention of atherosclerosis and the aim of this study was to further elucidate how this apolipoprotein is regulated by cytokines present during inflammation. Here we studied apoE production in peripheral blood mononuclear cells (PBMC) and analysis was made with a newly developed apoE ELISpot assay. In PBMC, apoE secretion was restricted to monocytes with classical (CD14(++)CD16(-)) and intermediate (CD14(+)CD16(+)) monocytes being the main producers. As earlier described for macrophages, production was strongly upregulated by TGF-ß and downregulated by bacterial lipopolysaccharide (LPS) and the inflammatory cytokines IFN-γ, TNF-α and IL-1ß. We could here show that a similar down-regulatory effect was also observed with the type I interferon, IFN-α, while IL-6, often regarded as one of the more prominent inflammatory cytokines, did not affect TGF-ß-induced apoE production. The TNF-α inhibitor Enbrel could partly block the down-regulatory effect of IFN-γ, IFN-α and IL-1ß, indicating that inhibition of apoE by these cytokines may be dependent on or synergize with TNF-α. Other cytokines tested, IL-2, IL-4, IL-12, IL-13, IL-17A and IL-23, had no inhibitory effect on apoE production. In contrast to the effect on monocytes, apoE production by primary hepatocytes and the hepatoma cell line HepG2 was more or less unaffected by treatment with cytokines or LPS.

Circulating monocytes are not the major source of plasma cytokines in patients with sepsis.

In sepsis, large quantities of inflammatory cytokines are released into the bloodstream. The cellular source of these cytokines is unclear, and we have here investigated to what extent circulating cells in blood contributed to this production. We used the enzyme-linked immunospot technique to study the spontaneous as well as the lipopolysaccharide (LPS)-induced secretion of the proinflammatory cytokines interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), granulocyte-macrophage colony-stimulating factor, IL-1ß, IL-12p40, and the anti-inflammatory cytokine IL-10 from whole-blood cells. The study comprised 32 septic patients (24 with septic shock) and 30 healthy controls. Despite significantly increased plasma cytokine levels in the septic patients, the number of spontaneous cytokine-secreting cells was small or nonexistent and did not differ between the two groups. Lipopolysaccharide stimulation of cells from the same samples triggered substantially increased numbers of cytokine-producing cells in both patients and controls. However, although the numbers of IL-6- and tumor necrosis factor α-secreting monocytes were very similar in both groups, significantly fewer IL-1ß-, IL-10-, IL-12p40-, and granulocyte-macrophage colony-stimulating factor-secreting monocytes were seen in samples from septic patients as compared with healthy controls. The reduced number of cytokine-secreting cells in response to LPS stimulation correlated with disease severity, as expressed by Sequential Organ Failure Assessment score and the stage of sepsis. In summary, circulating leukocytes did not appear to be responsible for the increased plasma levels of cytokines observed in sepsis. A selective sepsis-induced downregulation of cytokine secretion in response to LPS underscores the complexity of cytokine regulation in sepsis.

Efficient internalization of mesoporous silica particles of different sizes by primary human macrophages without impairment of macrophage clearance of apoptotic or antibody-opsonized target cells.

Macrophage recognition and ingestion of apoptotic cell corpses, a process referred to as programmed cell clearance, is of considerable importance for the maintenance of tissue homeostasis and in the resolution of inflammation. Moreover, macrophages are the first line of defense against microorganisms and other foreign materials including particles. However, there is sparse information on the mode of uptake of engineered nanomaterials by primary macrophages. In this study, mesoporous silica particles with cubic pore geometries and covalently fluorescein-grafted particles were synthesized through a novel route, and their interactions with primary human monocyte-derived macrophages were assessed. Efficient and active internalization of mesoporous silica particles of different sizes was observed by transmission electron microscopic and flow cytometric analysis and studies using pharmacological inhibitors suggested that uptake occurred through a process of endocytosis. Moreover, uptake of silica particles was independent of serum factors. The silica particles with very high surface areas due to their porous structure did not impair cell viability or function of macrophages, including the ingestion of different classes of apoptotic or opsonized target cells. The current findings are relevant to the development of mesoporous materials for drug delivery and other biomedical applications.

ELISpot analysis of LPS-stimulated leukocytes: human granulocytes selectively secrete IL-8, MIP-1beta and TNF-alpha.

Granulocytes and monocytes/macrophages represent key effector cells of the innate immune system. While human monocytes have been recognized as capable of secreting a broad spectrum of cytokines, the situation has been less clear in granulocytes with studies often showing conflicting results. In this study, lipopolysaccharide (LPS)-induced cytokine secretion from polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC) was analyzed at the single cell level with the enzyme-linked immunospot (ELISpot) assay. This method allowed us to establish the cytokine profiles for both PBMC and PMN based on the frequency and pattern of cytokine secreting cells, rather than on the amount of produced cytokine detectable in solution by ELISA. As a result, low levels of contaminating mononuclear cells present in our PMN preparations could be discriminated from granulocytes. Using this technique, neutrophils were found to secrete the two chemokines, IL-8 and MIP-1beta in response to LPS. Also TNF-alpha was secreted but in lower amounts and by significantly fewer cells. However, and as opposed to several other reports, we were unable to detect secretion of IL-1beta, IL-6, IL-10, IL-12 and GM-CSF. In contrast to the limited cytokine production by PMN, PBMC secreted considerably larger amounts of the investigated cytokines with CD14(+) monocytes being the primary source of production. Finally, we believe that the cytokine ELISpot technique may provide a powerful tool by which cells of the innate immune system can be studied from a functional perspective at the single cell level.