Abstract
Abstract Neutrophils modulated Ag presentation following immunization with Ags in CFA or IFA or alum. The neutrophils had an important negative role in the CD4 T cell and B cell responses to three protein Ags: hen egg white lysozyme, OVA, and listeriolysin O. In their absence (by depleting with Abs for only the first 24 h, or using genetically neutropenic mice), the cellular responses increased several-fold. The CD8 response was not affected or slightly decreased. Competition for Ag between the presenting cells and the neutrophils, as well as an effect on the response to Ag-bearing dendritic cells (DCs), was documented. Neutrophils entered the draining lymph nodes rapidly and for a brief period of several hours, localizing mainly to the marginal sinus and superficial cortex. There they established brief contact with DCs and macrophages. Moreover, neutrophils imprinted on the quality of the subsequent DCāT cell interactions, despite no physical contact with them; by intravital microscopy, the clustering of Ag-specific T cells and DCs was improved in neutropenic mice. Thus, neutrophils are obligate cells that briefly enter sites of immunization and set the level of Ag presentation. A brief depletion may have a considerably positive impact on vaccination.
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📋 Methods
Mice
All mice were bred and maintained under pathogen-free conditions at Washington University in St. Louis in accordance with institutional animal care guidelines. C57.BL/6 (B6) B10.BR and C.B-17 mice (H-2 d haplotype) were obtained from The Jackson Laboratory. B10.BR mice having NOX2 ā/ā or iNOS ā/ā were generated from B6 mutant mice ( 21 ). C57.BL/6, G-CSFR ā/ā mice were generated and obtained from the laboratory of Daniel Link (Washington University in St. Louis, MO). LysM-eGFP were generated by Faust et al. ( 22 ) and CD11c-eYFP by Lindquist et al. ( 23 ). All mice were crossed to B10.BR background. LysM-eGFP/CD11c-eYFP double reporter mice were the F1 heterozygote. B10.BR mice bearing a membrane form of HEL, mHEL, were generated in our laboratory ( 24 ). C.B-17 mice were originally purchased from Taconic and maintained at the animal facility of Washington University. Antigen HEL was obtained from Sigma and purified by affinity chromatography to remove about 3% of contaminant proteins. Purified HEL contained
📊 Figures
Figure 1
Influx of neutrophils into draining lymph nodes after immunization
B10.BR mice were immunized with 10nmole HEL (A, Cu2013E) or Hilyte Fluor 555-labeled HEL (B) in complete Freund's adjuvant. Popliteal lymph nodes were harvested at the indicated time. (A, D) 5u03bcm f...
Figure 2
T cell responses after neutrophil depletion
(A) Fold increase of HEL recalls response from neutrophil-depleted and control Ig-treated mice at different doses of HEL immunization in B10.BR mice. Data were obtained from a total of 31 independent ...
Figure 3
Neutrophil effect in mice deficient in G-CSFR, ROS, NO u02d9 or B cells
(A) G-CSFR u2212/u2212 , (D) NOX2 u2212/u2212 , iNOS u2212/u2212 or (E) u03bcMT mice were immunized with 10nmole HEL in complete Freund's adjuvant, groups were treated with either control Ig or neutro...
Figure 4
Antibody responses after neutrophil depletion
Control Ig or neutrophil-depleting Ab treated B10.BR mice were immunized with 10nmole of HEL in the various adjuvants as indicated in panels A, B and C. At day 14 of immunization, sera were collected ...
Figure 5
Antigen presentation after neutrophil depletion
B10.BR mice were immunized with 10nmole HEL in complete Freund's adjuvant after injection of control Ig or neutrophil depleting antibody. Popliteal lymph nodes were harvested at 3 hours (A), 16 hours ...
Figure 6
Effect of neutrophils on antigen bearing APC
Splenic DC or macrophages were isolated from mHEL or B10.BR mice by MACS and transferred into footpad of neutrophil depleted- or undepleted-B10.BR recipients. At 3 hours after cell transfer, incomplet...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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