Fcγ receptors on aging neutrophils

Abstract Objective Neutrophils are key effector cells of the innate immune system. They recognize antigens through membrane receptors, which are expressed during their maturation and activation. Neutrophils express FcγRII (CD32), FcγRIII (CD16), and FcγRI (CD64) after being activated by different factors such as cytokines and bacterial products. These receptors are involved with phagocytosis of IgG-opsonized microbes and enhance defense mechanisms. Based on that, our study seeks to compare the expression of FcγRII, FcγRIII, FcγRI, and CD11b on neutrophils from elderly and young subjects and their expression after in vitro activation with cytokines and LPS. Methodology Neutrophils were isolated from human peripheral blood and from mice bone marrow by density gradient. After isolation, FCγRs expression was immediately analyzed by flow cytometry or after in vitro stimulation. Results In freshly isolated cells, the percentage of FcγRIIIb+ and CD11b+ neutrophils were higher in samples from young individuals; FcγRIIIa expression was more prominent on aged neutrophils; FcγRIA expression was similar in all samples analyzed. Exposure to CXCL8 and LPS resulted in a higher percentage of FcγRIa+ neutrophils on elderly individuals’ samples but lower when compared with neutrophils from young donors. We observed that LPS caused an increase in FcγRIIa expression on aging human neutrophils. In contrast, FcγRIIIb expression in response to CXCL8 and LPS stimulation was not altered in the four groups. CD11b expression was lower in neutrophils from elderly individuals even in response to LPS and CXCL8. In mice, we observed differences only regarding CD11b expression, which was increased on aged neutrophils. LPS exposure caused an increase in all FcγRs. Conclusions Our results suggest that, in humans, the overall pattern of FcγR expression and integrin CD11b are altered during aging and immunosenescence might contribute to age-related infection.


Introduction
Neutrophils are the first inflammatory cell to infiltrate inflamed or infected tissue; therefore, they are recognized as major components of the host's first line of defense. 1 They immediately migrate to the infected site, become activated, and initiate a cascade of defense mechanisms that effectively contribute to the development of the adaptive immune response. 2,3 Several cytokines, such as CXCL8 and TNF, have been described as chemoattractants and activators for neutrophils. 3,4 Basal levels of CXCL8, the most neutrophil attractant and activator, are detected in the peripheral blood of healthy subjects, but certain diseases can cause CXCL8 deregulation and affect neutrophil function. 5,6 Neutrophils motility and activity rely on the expression of specialized membrane molecules, which allows them to adhere and transmigrate through the endothelium as well as to recognize and phagocytize opsonized microorganisms. [1][2][3] Among them, the family of Fc gamma receptors for IgG (FcγRs) plays an essential role during neutrophils activation and function. FcγRs mediate antibody-dependent cellular cytotoxicity (ADCC) and the phagocytosis of opsonized invading pathogens, and elicit neutrophil recruitment. 3,7 The most well-studied FcγRs are the ones that belong to the family of type I FcγRs. 8 Specifically, the FcγRIIIb (CD16) is the most abundant FcγR; 9 the FcγRIIa (CD32) is an activating-type Fc receptor and is the second most expressed FcγR; 10 and the less abundant FcγR is the high-affinity receptor FcγRIa (CD64), which binds to IgG2a (mice) or IgG1 and IgG3 (humans). 9 Since the humoral immune response is essential for host immunity, FcγRs expression has been extensively investigated in diseases and therapies. 10

Statistical Analysis
Values are represented as mean ± SD in each figure. Comparisons between two groups were made with a t-test followed by the Mann-Whitney test for nonparametric data or with Welch´s correction for parametric data. Comparisons of three groups were made using two-way ANOVA followed by Tukey´s or Sidak's multiple comparisons test. p≤0.05 were considered as statistically significant.

Results
Altered frequency of FcγR + and CD11b + on aging human neutrophils We started evaluating the number of neutrophils in human blood. Our results demonstrated that both groups, young and elderly, displayed a similar frequency of neutrophils ( Figure 1A). However, we found lower levels of the CXCL8 chemokine, the most important activator for neutrophils, 19 in samples from elderly subjects when compared with young individuals ( Figure 1B). Next, we investigated the FcγRs expression on aging human neutrophils. In freshly isolated human cells, the percentage of FcγRIIa + neutrophils was more evident in samples from elderly individuals ( Figure 1C). In contrast, the percentage of FcγRIIIb + neutrophils was significantly higher in the young group ( Figure 1D). FcγRIa expression was similar in samples from young and elderly subjects ( Figure 1E). Since CD11b is essential for neutrophils extravasation and recruitment; 20 we also compared its expression on neutrophils from elderly and young individuals and our results showed that the frequency of CD66 + /CD11b + human neutrophils decreases with age ( Figure 1F).
Next, we investigated if LPS or CXCL8 could affect the surface expression of FcγR on neutrophils. We  Figure   4A). Moreover, LPS induced a remarkable increase of FcγRI expression on neutrophils from both groups ( Figure 4B). However, TNF did not affect this receptor expression by aged cells ( Figure 4B). LPS also could stimulate neutrophils from young mice to express CD11b ( Figure 4C). Nonetheless, TNF induced a weak decrease in CD11b expression on neutrophils from aged and young mice ( Figure 4C).   We also determined percentages of FcγRIa + neutrophils, the less abundant FcγR, before and after stimuli, and, as expected, both human neutrophils express low FcγRIa regardless of age. Although FcγRIa expression is minimal on resting neutrophils, 29 it can be upregulated after activation. 30,31 In vitro, LPS markedly increased the frequency of FcγRIa + Figure 4-Young murine samples have higher FcγRI + and CD11 + neutrophils than aged mice after in vitro activation. The expression of (A) FcγRIII/FcγRII, (B) FcγRI, and (C) CD11b on purified neutrophils from young (red bars) and aged (blue bars) mice after 18 hours of treatment with LPS or TNF were determined by flow cytometry. Control=neutrophils cultivated without treatment. Data are shown as mean±SD. The results were evaluated by two-way ANOVA followed by Tukey's and Sidak's multiple comparisons test. **p≤0.01 and ***p≤0.001.  39 The current demonstration, that elderly subjects had a lower frequency of CD11b + neutrophil, contrasts with an earlier study that reported no significant age-dependent differences in the expression of CD11b. 40 A possible explication for the difference between studies could be related to differences in cell processing and staining conditions that may result in different levels of surface CD11b and other markers of degranulation. 37 In our article, we found significant and intriguing differences regarding CD11b expression in elderly humans and aged mice during resting and upon in vitro activation, and probably reflects different effects of aging on CD11b expression on neutrophils between both species.
In short, our results suggest that, for neutrophils, immunosenescence results in an imbalance of FcγR and CD11b expression. Moreover, regarding FcγRs and CD11b expression, human and murine neutrophils respond differently to aging and such differences should be considered for the designing of further studies using experimental models for immunosenescence assessment.