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AB0133 Increased Expression of Activated Ly6g+Cd11b+Cd62l- Neutrophils in Balb/c Mice with Pristane Induced Lupus-like Disease
  1. N. Ugriumov,
  2. C. Goldenstein-Schainberg,
  3. S. Carrasco,
  4. I. Begalli Mendes,
  5. T.M. Lima,
  6. T. Vasconcelos Peixoto,
  7. S.B. Veríssimo de Mello,
  8. F.G. Soriano
  1. Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil

Abstract

Background Neutrophils are important cells of innate immunity's first line of defense and IL-8 cytokine contributes to its activation. In patients with lupus there is an increase of activated neutrophils in plasma, however data about the contribution of IL-8 on disease development are conflicting.

Objectives To analyze and quantify activated Ly6G+CD11b+CD62L- neutrophils in peripheral blood (PB) and spleen as well to evaluate the production of IL-8 in Balb/c mice with pristane induced lupus-like disease.

Methods We studied 37 mice (13 healthy controls; 24 pristane-induced mice). Pristane-induced mice (PIM) received a single intraperitoneal 0.5ml dose of pristane, while healthy controls received the same dose of saline. Samples of blood and spleen were collected after euthanasia 90 and 120 days after injections; red blood cells were lysed and remaining cells stained with monoclonal antibodies: anti-Ly6G (total neutrophils), anti-CD11b (activated neutrophils) and anti-CD62L (anergic neutrophils) (BDPharmingen™). All cell samples were evaluated by flow cytometry (Guava EasyCyte™ HT, Millipore). Plasmatic IL-8 levels were assessed by ELISA (MyBioSource™). Mann-Whitney's non-parametric test was used for statistical analysis, and P<0.05 considered significant.

Results Compared to control, PIM had higher percentage of total neutrophils 90 and 120 days after injection in PB (26.9±2.46 vs. 46.75±5.41%, P=0.002 and 26.9±2.46 vs. 38.52±4.03%, P=0.004) and spleen (15.94±1.86 vs. 33.7±6.48%, P=0.01 and 15.94±1.86 vs. 35.98±3%, P<0.0001) respectively. In PB, the percentage of activated neutrophils was similar in PIM compared to controls (P>0.05), while in the spleen, PIM had increased activated neutrophils 120 days after induction (41.83±3.63 vs. 58.73±3.94%, P=0.004). PIM and controls expressed similar percentages of anergic neutrophils in both PB and spleen (P>0.05). Controls and PIM produced similar plasmatic IL-8 levels 90 and 120 days after injection (0.65±0.18 vs. 0.2±0.05pg/ml, P≥0.05 and 0.65±0.18 vs. 1.2±0.51pg/ml, P>0.05), respectively though PIM had higher plasmatic IL8 levels 120 days compared to 90 days after induction (1.2±0.51 vs 0.2±0.05pg/ml, P=0.01).

Conclusions Increased percentage of activated neutrophils in PIM indicates innate immunity involvement. Delayed IL8 levels increments over time might suggests that this cytokine may be involved in neutrophil activation in this animal model. Therefore, additional studies are needed in order to clarify IL8 role and further pathways involved in neutrophil activation and disease development in PIM.

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Disclosure of Interest None declared

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