Individual cells simultaneously produce both IL-4 and IL-6 in vivo

Shirai, Akira; Sierra, Víctor Parra; Kelly, Christopher I.; Klinman, Dennis M.

doi:10.1016/1043-4666(94)90030-2

Cited by 29 publications

(23 citation statements)

References 42 publications

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“…This CTL activity was de- Figure 3. Cytokine ELIspot assays were used to detect individual IL-4 and IFN␥ secreting BALB/c spleen cells (34,35). Panel A examines the number of spleen cells actively secreting cytokine in vivo in mice 3 wk after pCSP or CS.1 treatment when compared with age-matched controls (averageϮSD of three individually studied mice/group).…”

Section: Resultsmentioning

confidence: 99%

Induction of neonatal tolerance by plasmid DNA vaccination of mice.

G¹,

Yamshchikov²,

Sedegah³

et al. 1996

J. Clin. Invest.

123

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Plasmid DNA vaccines capable of preventing viral, bacterial, and parasitic infections are currently under development. Our labs have shown that a plasmid DNA vaccine encoding the circumsporozoite protein of the malaria parasite elicits protective immunity against live sporozoite challenge in adult BALB/c mice. We now find that the same DNA vaccine induces tolerance rather than immunity when administered to 2-5 d-old mice. Neonatally tolerized animals were unable to mount antibody, cytokine or cytotoxic responses when rechallenged with DNA vaccine in vitro or in vivo. Tolerance was specific for immunogenic epitopes expressed by the vaccine-encoded, endogenously produced antigen. Mice challenged with exogenous circumsporozoite protein produced antibodies against a different set of epitopes, and were not tolerized. These findings demonstrate important differences in the nature and specificity of the immune response elicited by DNA vaccines versus conventional protein immunogens. ( J. Clin. Invest. 1996. 98: 2700-2705.)

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Section: Resultsmentioning

confidence: 99%

Induction of neonatal tolerance by plasmid DNA vaccination of mice.

G¹,

Yamshchikov²,

Sedegah³

et al. 1996

J. Clin. Invest.

123

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“…These include interleukin (IL) 6, which contributes to T-and B-cell activation (17-20), IFN-y, which (25)(26)(27).…”

Section: Introductionmentioning

confidence: 99%

“…IL-6 knockout mice are extremely susceptible to infection (15,32 (26,33). Indeed, the background levels of IL-6, IL-12, and IFN-y production in untreated mice has been attributed to spontaneous immune activation caused by environmental and self-antigens in our specific pathogen-free colony (26,27,33 …”

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confidence: 99%

“…Plates were overlaid with secondary biotinylated anticytokine antibody at 1 gtg/ml (25,26), washed, and treated with a 1:2000 dilution of avidin-conjugated alkaline phosphatase (Vector Laboratories) or phosphatase-conjugated anti-IgM antibody (Southern Biotechnology Associates). Individual cytokine or IgM-secreting cells were visualized and quantitated as described (25)(26)(27).…”

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confidence: 99%

“…Supernatants were serially diluted in PBS/1% bovine serum albumin and incubated on anti-cytokine-coated microtiter plates for 2 hr. Biotinylated anti-cytokine antibody followed by avidin-conjugated alkaline phosphatase were used in a colorimetric assay to detect the presence of bound cytokine (27 Table 2), or DNA at 100 jig/ml for 10 hr. Stimulation index is calculated as fold increase in number of cytokinesecreting cells over background.…”

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confidence: 99%

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CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma.

Klinman

Beaucage

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

1,375

840

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Bacterial infection stimulates the host to mount a rapid inflammatory response. A 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines was shown to contribute to this response by inducing polyclonal B-cell (3,4,(7)(8)(9)11). Recent work from our laboratory suggested that this B-cell stimulation was mediated by a 6-base nt motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines (12). This motif is expressed nearly 20 times more frequently in bacterial than vertebrate DNA (12)(13)(14).In addition to B-cell activation, the innate immune response to bacterial infection is characterized by the production of immunomodulatory cytokines (15,16). These include interleukin (IL) 6, which contributes to T-and B-cell activation (17-20), IFN-y, which (25)(26)(27).The concentration of cytokine in culture supernatants was determined by using a modification of the assay system described above. Supernatants were serially diluted in PBS/1% bovine serum albumin and incubated on anti-cytokine-coated microtiter plates for 2 hr. Biotinylated anti-cytokine antibody followed by avidin-conjugated alkaline phosphatase were used in a colorimetric assay to detect the presence of bound cytokine (27).Cell Purification. Mononuclear cells were incubated with biotinylated anti-CD4, anti-CD8, anti-CD11, anti-CD14, or

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ELISPOT Assay to Detect Cytokine‐Secreting Murine and Human Cells

Klinman

Nutman

1994

CP in Immunology

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The filter immunoplaque assay, otherwise called the enzyme-linked immunospot assay (ELISPOT), was initially developed to detect and quantitate individual antibody-secreting B cells. Recent modifications have improved the sensitivity of the ELISPOT assay such that cells producing as few as 100 molecules of specific protein per second can be detected. The ELISPOT assay utilizes two high-affinity cytokine-specific antibodies directed against different epitopes on the same cytokine molecule: either two monoclonal antibodies or a combination of one monoclonal antibody and one polyvalent antiserum. ELISPOT generates spots based on a colorimetric reaction that detects the cytokine secreted by a single cell. The spot represents a "footprint" of the original cytokine-producing cell. Spots are permanent and can be quantitated visually, microscopically, or electronically.

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Individual cells simultaneously produce both IL-4 and IL-6 in vivo

Cited by 29 publications

References 42 publications

Induction of neonatal tolerance by plasmid DNA vaccination of mice.

Induction of neonatal tolerance by plasmid DNA vaccination of mice.

CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma.

ELISPOT Assay to Detect Cytokine‐Secreting Murine and Human Cells

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