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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.

Receptors, IgG; Neutrophils; Aging

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.11 - Faurschou M, Borregaard N. Neutrophil granules and secretory vesicles in inflammation. Microbes Infect. 2003;5(14):1317-27. doi: 10.1016/j.micinf.2003.09.008 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.22 - Segal AW. How neutrophils kill microbes. Annu Rev Immunol. 2005;23:197-223. doi: 10.1146/annurev.immunol.23.021704.115653,33 - Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 2011;11(8):519-31. doi: 10.1038/nri3024 Several cytokines, such as CXCL8 and TNF, have been described as chemoattractants and activators for neutrophils.33 - Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 2011;11(8):519-31. doi: 10.1038/nri3024,44 - Futosi K, Fodor S, Mocsai A. Neutrophil cell surface receptors and their intracellular signal transduction pathways. Int Immunopharmacol. 2013;17(3):638-50. doi: 10.1016/j.intimp.2013.06.034 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.55 - Bauer JW, Baechler EC, Petri M, Batliwalla FM, Crawford D, Ortmann WA, et al. Elevated serum levels of interferon-regulated chemokines are biomarkers for active human systemic lupus erythematosus. PLoS Med. 2006;3(12):e491. doi: 10.1371/journal.pmed.0030491,66 - Kruger P, Saffarzadeh M, Weber AN, Rieber N, Radsak M, von Bernuth H, et al. Neutrophils: between host defence, immune modulation, and tissue injury. PLoS Pathog. 2015;11(3):e1004651. doi: 10.1371/journal.ppat.1004651

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.11 - Faurschou M, Borregaard N. Neutrophil granules and secretory vesicles in inflammation. Microbes Infect. 2003;5(14):1317-27. doi: 10.1016/j.micinf.2003.09.008 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.33 - Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 2011;11(8):519-31. doi: 10.1038/nri3024,77 - Tsuboi N, Asano K, Lauterbach M, Mayadas TN. Human neutrophil Fcgamma receptors initiate and play specialized nonredundant roles in antibody-mediated inflammatory diseases. Immunity. 2008;28(6):833-46. doi: 10.1016/j.immuni.2008.04.013 The most well-studied FcγRs are the ones that belong to the family of type I FcγRs.88 - Pincetic A, Bournazos S, DiLillo DJ, Maamary J, Wang TT, Dahan R, et al. Type I and type II Fc receptors regulate innate and adaptive immunity. Nat Immunol. 2014;15(8):707-16. doi: 10.1038/ni.2939 Specifically, the FcγRIIIb (CD16) is the most abundant FcγR;99 - Nimmerjahn F, Ravetch JV. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol. 2008;8(1):34-47. doi: 10.1038/nri2206the FcγRIIa (CD32) is an activating-type Fc receptor and is the second most expressed FcγR;1010 - Anania JC, Chenoweth AM, Wines BD, Hogarth PM. The Human FcγRII (CD32) Family of Leukocyte FcR in Health and Disease. Front Immunol. 2019;10:464. doi: 10.3389/fimmu.2019.00464 and the less abundant FcγR is the high-affinity receptor FcγRIa (CD64), which binds to IgG2a (mice) or IgG1 and IgG3 (humans).99 - Nimmerjahn F, Ravetch JV. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol. 2008;8(1):34-47. doi: 10.1038/nri2206

Since the humoral immune response is essential for host immunity, FcγRs expression has been extensively investigated in diseases and therapies.1010 - Anania JC, Chenoweth AM, Wines BD, Hogarth PM. The Human FcγRII (CD32) Family of Leukocyte FcR in Health and Disease. Front Immunol. 2019;10:464. doi: 10.3389/fimmu.2019.00464,1111 - Kerntke C, Nimmerjahn F, Biburger M. There is (scientific) strength in numbers: a comprehensive quantitation of fc gamma receptor numbers on human and murine peripheral blood leukocytes. Front Immunol. 2020;11:118. doi: 10.3389/fimmu.2020.00118 However, the mechanisms of how immunosenescence affect these receptors’ expression and function, especially on neutrophils, remains poorly investigated. It is well recognized that the aging process hampers the host’s ability to develop robust and effective immune response.1212 - Gruver AL, Hudson LL, Sempowski GD. Immunosenescence of ageing. J Pathol. 2007;211(2):144-56. doi: 10.1002/path.2104,1313 - Goronzy JJ, Weyand CM. Understanding immunosenescence to improve responses to vaccines. Nat Immunol. 2013;14(5):428-36. doi: 10.1038/ni.2588 Studies have identified differential expression of several receptors on neutrophils from elderly adults as well as distinct activation patterns that were associated with Candida-related denture stomatitis.1414 - Gasparoto TH, Vieira NA, Porto VC, Campanelli AP, Lara VS. Differences between salivary and blood neutrophils from elderly and young denture wearers. J Oral Rehabil. 2011;38(1):41-51. doi: 10.1111/j.1365-2842.2010.02126.x,1515 - Gasparoto TH, Sipert CR, Oliveira CE, Porto VC, Santos CF, Campanelli AP, et al. Salivary immunity in elderly individuals presented with Candida-related denture stomatitis. Gerodontology. 2012;29(2):e331-9. doi: 10.1111/j.1741-2358.2011.00476.x Since alterations in FcγRs and CD11b expression might contribute to immunosenescence and age-related diseases, we aimed at investigating if age affects FcγRII, FcγRIII, FcγRI, and CD11b expression on human and murine neutrophils.

Methodology

Subjects

Thirty healthy subjects from two age groups were recruited in Bauru, state of São Paulo, Brazil, and enrolled in our study: elderly group (62 - 75 years; mean ± SD 66.9±4.2; n=15, 08 women and 07 men); and young group (20 - 49 years; mean ± SD 26.4±10.6; n=15, 07 women and 08 men). The age range was determined based on a previous study.1616 - Wenisch C, Patruta S, Daxbock F, Krause R, Horl W. Effect of age on human neutrophil function. J Leukoc Biol. 2000;67(1):40-5. doi: 10.1002/jlb.67.1.40 Exclusion criteria for all subjects included the existence of any systemic infectious disease, cancer, endocrine, immune or hematologic alterations and tobacco consumption. We also dismissed volunteers that were under chemo- or radiotherapy, under antibiotics or antifungal medications; or psychotic, heavy metal, anticonvulsant, or cardiotonic treatment. The study was approved by the local Research Ethics Committee of Bauru School of Dentistry (CAAE 41907015.2.0000.5417) and written informed consent was obtained from each patient.

Animals

Procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (CONCEA) and approved by the Institutional Animal Care and Use Committee of the Bauru School of Dentistry, University of São Paulo (Protocol #0042/2013). In total, 6-8-week-old female and male mice aged (young) and 8-month-old (middle-aged) C57BL/6 were provided by the School of Medicine of Ribeirão Preto, University of São Paulo. Each mouse was housed in an isolated cage, and food and water were provided ad libitum.

Isolation and culture of human neutrophils

Human neutrophils were purified from the peripheral blood of healthy donors immediately after collection by density gradient centrifugation using the Histopaque 1119 and 1083 gradients (Sigma-Aldrich) as previously described.1717 - Gasparoto TH, Oliveira CE, Vieira NA, Porto VC, Cunha FQ, Garlet GP, et al. Activation pattern of neutrophils from blood of elderly individuals with Candida-related denture stomatitis. Eur J Clin Microbiol Infect Dis. 2012;31(6):1271-7. doi: 10.1007/s10096-011-1439-z. Neutrophils were identified by staining with Turk’s solution and positivity for FITC-conjugated anti-human CD66b (clone G10F5) was established by flow cytometry. Neutrophil stimulation was performed with 100 ng/mL LPS from Escherichia coli (Sigma) and/or 10 ng/mL CXCL8 (BD Biosciences Corp., San Diego, CA).

Cytokine assay

CXCL8 levels were measured using ELISA kits (BD Biosciences), according to the manufacturer’s instructions

Isolation of murine neutrophils

Murine neutrophils were purified from the bone marrow. Femurs and tibiae of C57Bl/6 mice were dissected, decontaminated with 70% ethanol for 2 min, and then washed in PBS. Then, the bone marrow was flushed with RPMI 1640 medium (Gibco, Grand Island, NY), and layered on the Histopaque 1017 and 1119 gradients (Sigma-Aldrich) and centrifuged as previously described.1818 - Chiarella P, Reffo V, Bruzzo J, Bustuoabad OD, Ruggiero RA. Therapeutic anti-tumor vaccines: from tumor inhibition to enhancement. Clin Med Oncol. 2008;2:237-45. doi: 10.4137/cmo.s538 Neutrophil stimulation was performed with 100 ng/mL LPS from Escherichia coli and/or 10 ng/mL TNF (Sigma-Aldrich).

FACS analysis of neutrophils

FACS assays were also used to measure Fcγ receptors on neutrophils. For immunostaining of human neutrophils, primary antibodies used were FITC-conjugated anti-human CD66b (clone G10F5), APC-conjugated anti-human CD16 (FcγRIIIb, clone 3G8), PE-conjugated anti-human CD32 (FcγRIIa, 3D3), PE-conjugated anti-human CD64 (FcγRIa, 10.1), and PE-conjugated anti-human CD11b (ICRF44). For immunostaining of mice neutrophils, primary antibodies used were: FITC-conjugated anti-mouse Ly-6G (Gr-1, clone RB6-8C5), FITC-conjugated anti-mouse FcγRIII/FcγRII, Alexa-fluor 647-conjugated anti-mouse FcγRI, and PE-conjugated anti-mouse CD11b were used. Isotype primary antibody controls were used to assess nonspecific staining. Staining was done for 30 minutes at 4°C for all antibodies. All antibodies were obtained from BD Pharmingen. Cell acquisition (counting) was performed on a FACSort flow cytometer using the CellQuest software (BD Biosciences).

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,1919 - Rajarathnam K, Sykes BD, Kay CM, Dewald B, Geiser T, Baggiolini M, et al. Neutrophil activation by monomeric interleukin-8. Science. 1994;264(5155):90-2. doi: 10.1126/science.8140420 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;2020 - Tomay F, Wells K, Duong L, Tsu JW, Dye DE, Radley-Crabb HG, et al. Aged neutrophils accumulate in lymphoid tissues from healthy elderly mice and infiltrate T- and B-cell zones. Immunol Cell Biol. 2018;96(8):831-40. doi: 10.1111/imcb.12046 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 evaluated FcγRIIa, FcγRIIIb, and FcγRIa expression on neutrophils by flow cytometry following 18 hours of exposure with LPS (100 ng/ml), CXCL8 (10 ng/ml), LPS + CXCL8, or medium alone (Figure 2A-C). Exposure to LPS alone significantly increased the expression of all FcγRs receptors in both groups (Figure 2A-C); however, neutrophils from elderly individuals had lower values than the young group, except for FcγRIIIb (Figure 2B). CXCL8 increased the percentage of FcγRIIa+ neutrophils in samples from the young group when compared with the elderly group (Figure 2A). Moreover, exposure to CXCL8 increases the frequency of FcγRIa+ neutrophils in samples from both groups (Figure 2C). After exposure to CXCL8, no significant difference was observed in the percentage of FcγRIIIb+ neutrophils in samples from elderly and young groups (Figure 2B). Culture with LPS plus CXCL8 increased the frequency of FcγRIa+ neutrophils in samples from elderly group (Figure 2C). After culture, the percentage of FcγRIIIb+ neutrophils in samples from the young group was always lower when compared with ex vivo values (data not shown). Despite the lower frequency of CD11b+ neutrophils in samples from the elderly group, upon in vitro stimulation, its expression was increased. Exposure to LPS or CXCL8 did not increase the percentage of CD11b+ neutrophils in samples from young individuals (Figure 2D). Together, the data demonstrate a statistically significant difference in FcγRIIa and FcγRIa expression between the groups, but not between FcγRIIIb expressions in samples from either young or elderly individuals (Figure 2).

Figure 2
Young human samples present higher FcγR+ neutrophils than older adults’ samples after in vitro activation.

Different expression pattern of FcγRIII, FcγRII and FcγRI on activated neutrophils from aged mice compared to young matched controls

FcγRIII, FcγRII, FcγRI, and CD11b expressions were evaluated on bone marrow-derived neutrophils from aged and young mice and analyzed in resting conditions (Figure 3) or after in vitro activation with cytokines or LPS (Figure 4). Data obtained clearly showed similar expressions of IgG receptors on neutrophils in a steady state (Figure 3A-B). LPS and TNF promoted increased expression of FcγRIII/FcγRII on neutrophils from young and aged mice (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).

Figure 3
Aged mice have higher frequency of CD11b+ neutrophils.

Figure 4
Young murine samples have higher FcγRI+ and CD11+ neutrophils than aged mice after in vitro activation.

Discussion

The association of age-related disorders with inflammatory pathogenesis2121 - Martinis M, Franceschi C, Monti D, Ginaldi L. Inflamm-ageing and lifelong antigenic load as major determinants of ageing rate and longevity. FEBS Lett. 2005;579(10):2035-9. doi: 10.1016/j.febslet.2005.02.055,2222 - Licastro F, Candore G, Lio D, Porcellini E, Colonna-Romano G, Franceschi C, et al. Innate immunity and inflammation in ageing: a key for understanding age-related diseases. Immun Ageing. 2005;2:8. doi: 10.1186/1742-4933-2-8 shows the great impact of immunosenescence in older adults’ quality of life. Previous work from our laboratory demonstrated that elderly individuals exhibit differences in the phenotype of circulating or tissue-infiltrating neutrophils, associated with oral dysbiosis and development of stomatitis.1414 - Gasparoto TH, Vieira NA, Porto VC, Campanelli AP, Lara VS. Differences between salivary and blood neutrophils from elderly and young denture wearers. J Oral Rehabil. 2011;38(1):41-51. doi: 10.1111/j.1365-2842.2010.02126.x,1515 - Gasparoto TH, Sipert CR, Oliveira CE, Porto VC, Santos CF, Campanelli AP, et al. Salivary immunity in elderly individuals presented with Candida-related denture stomatitis. Gerodontology. 2012;29(2):e331-9. doi: 10.1111/j.1741-2358.2011.00476.x,1717 - Gasparoto TH, Oliveira CE, Vieira NA, Porto VC, Cunha FQ, Garlet GP, et al. Activation pattern of neutrophils from blood of elderly individuals with Candida-related denture stomatitis. Eur J Clin Microbiol Infect Dis. 2012;31(6):1271-7. doi: 10.1007/s10096-011-1439-z. Type I FcγRs play essential roles in immune responses to pathogens and are expressed by several innate immune cells, including neutrophils. However, the impact of immunosenescence in FcγRs remains poorly understood. In our article, we demonstrated that, in humans, aging decreases the frequency of FcγRIIb+ neutrophils and could negatively modulate FcγRIIa and FcγRIIIb expression by neutrophils upon stimulation with cytokine and LPS.

The results obtained show decreased levels of CXCL8 in the serum from the older adults when compared with young individuals. Considering that basal levels of CXCL8 are found in the blood serum from healthy individuals, maintaining neutrophils in the homeostasis and, probably, controlling their phenotype,2323 - Martich GD, Danner RL, Ceska M, Suffredini AF. Detection of interleukin 8 and tumor necrosis factor in normal humans after intravenous endotoxin: the effect of antiinflammatory agents. J Exp Med. 1991;173(4):1021-4. doi: 10.1084/jem.173.4.1021 we speculated if CXCL8, the most neutrophil activator factor, would be at different levels on the blood and affect FcγRs expression on neutrophils. Elderly individuals displayed significantly less FcγRIIIb+ neutrophils when compared with young subjects, which is in accordance with a previous study.2424 - Butcher SK, Chahal H, Nayak L, Sinclair A, Henriquez N v, Sapey E, et al. Senescence in innate immune responses: reduced neutrophil phagocytic capacity and CD16 expression in elderly humans. J Leukoc Biol. 2001;70(6):881-6. doi: 10.1189/jlb.70.6.881.
https://doi.org/10.1189/jlb.70.6.881...
Furthermore, CXCL8 was not sufficient to increase FcγRIIIb+ neutrophils, suggesting that this chemokine is not important to FcγRIIIb expression on human neutrophils.2525 - Loos T, Opdenakker G, van Damme J, Proost P. Citrullination of CXCL8 increases this chemokine’s ability to mobilize neutrophils into the blood circulation. Haematologica. 2009;94(10):1346-53. doi: 10.3324/haematol.2009.006973

Indeed, our results showed that the stimulation with CXCL8 did not induce FcγRIIa expression on neutrophils from elderly subjects. The percentage of the second most abundant FcγRs, the FcγRIIa, was more prominent on freshly isolated neutrophils from elderly individuals, suggesting the existence of a compensatory mechanism of FcγRIIIb/FcγRIIa expression during immunosenescence. We also demonstrated that, although LPS increased FcγRIIa expression on neutrophils from the elderly group, FcγRIIa induction upon other stimulation is lower when compared with neutrophils obtained from young individuals, suggesting that aged neutrophils probably require a more robust signaling activation. Neutrophil activation by FcγR usually requires the co-engagement of FcγRIIIb and FcγRIIa,2626 - Vossebeld PJ, Homburg CH, Roos D, Verhoeven AJ. The anti-Fc gamma RIII mAb 3G8 induces neutrophil activation via a cooperative actin of Fc gamma RIIIb and Fc gamma RIIa. Int J Biochem Cell Biol. 1997;29(3):465-73. doi: 10.1016/s1357-2725(96)00160-4 and upon efficient cross-linking, neutrophils produce inflammatory mediators and reactive oxygen species, and trigger neutrophil extracellular trap (NET) formation,77 - Tsuboi N, Asano K, Lauterbach M, Mayadas TN. Human neutrophil Fcgamma receptors initiate and play specialized nonredundant roles in antibody-mediated inflammatory diseases. Immunity. 2008;28(6):833-46. doi: 10.1016/j.immuni.2008.04.013,2727 - Unkeless JC, Shen Z, Lin CW, DeBeus E. Function of human Fc gamma RIIA and Fc gamma RIIIB. Semin Immunol. 1995;7(1):37-44. doi: 10.1016/1044-5323(95)90006-3 so the lower frequency of FcγRIIa+ neutrophils detected in samples from older adults after in vitro stimulation might imply in lower neutrophil activation.2828 - Marois L, Vaillancourt M, Marois S, Proulx S, Paré G, Rollet-Labelle E, et al. The ubiquitin ligase c-Cbl down-regulates FcgammaRIIa activation in human neutrophils. J Immunol. 2009;182(4):2374-84. doi: 10.4049/jimmunol.0801420 Concerning mice neutrophils, our results showed no significant differences in the expression of FcγRIII/FcγRII in freshly isolated neutrophils from young and aged mice. LPS and TNF induced an increase of FcγRIII/FcγRII+ neutrophils in both groups.

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,2929 - Hoffmann JJ. Neutrophil CD64: a diagnostic marker for infection and sepsis. Clin Chem Lab Med. 2009;47(8):903-16. doi: 10.1515/CCLM.2009.224. it can be upregulated after activation.3030 - Nuutila J. The novel applications of the quantitative analysis of neutrophil cell surface FcgammaRI (CD64) to the diagnosis of infectious and inflammatory diseases. Curr Opin Infect Dis. 2010;23(3):268-74. doi: 10.1097/QCO.0b013e32833939b0,3131 - Qureshi SS, Lewis SM, Gant VA, Treacher D, Davis BH, Brown KA. Increased distribution and expression of CD64 on blood polymorphonuclear cells from patients with the systemic inflammatory response syndrome (SIRS). Clin Exp Immunol. 2001;125(2):258-65. doi: 10.1046/j.1365-2249.2001.01596.xIn vitro, LPS markedly increased the frequency of FcγRIa+ neutrophils. However, the induction of FcγRIa was even higher in young individuals when compared with older individuals. FcγRIa upregulation allows neutrophils to efficiently trigger antibody-dependent cytotoxicity (ADCC).3232 - Repp R, Valerius T, Sendler A, Gramatzki M, Iro H, Kalden JR, et al. Neutrophils express the high affinity receptor for IgG (Fc gamma RI, CD64) after in vivo application of recombinant human granulocyte colony-stimulating factor. Blood. 1991;78(4):885-9. doi: 10.1182/blood.V78.4.885.885 Thus, as suggested for FcγRIIIb, neutrophils from elderly individuals might need stronger signal receptor assembling and signaling pathways to trigger FcγRIa expression or even other inflammatory-induced receptors.3333 - Wessels I, Jansen J, Rink L, Uciechowski P. Immunosenescence of polymorphonuclear neutrophils. ScientificWorldJournal. 2010;10:145-60. doi: 10.1100/tsw.2010.14

CD11b+ neutrophils were more abundant in the blood from young subjects, where aged mice had a higher frequency of CD11b+ neutrophils than young ones. However, the groups that displayed higher numbers of neutrophils expressing CD11b did not increase its expression effectively upon in vitro stimulation. Studies evaluating the effects of immunosenescence on CD11b expression are scarce and conflicting. It has been reported that CD11b can be both unaltered and increased with age. CD11b is an integrin that forms complexes with CD18 and regulates adhesion of neutrophils to the endothelium, a fundamental step for leukocyte migration towards inflamed/infected tissue3434 - Phillipson M, Heit B, Colarusso P, Liu L, Ballantyne CM, Kubes P. Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade. J Exp Med. 2006;203(12):2569-75. doi: 10.1084/jem.20060925 and its induction is a sign of neutrophil activation.3535 - Golay J, Valgardsdottir R, Musaraj G, Giupponi D, Spinelli O, Introna M. Human neutrophils express low levels of FcγRIIIA, which plays a role in PMN activation. Blood. 2019;133(13):1395-405. doi: 10.1182/blood-2018-07-864538 In addition, CD11b is stored in secretory, gelatinase, and specific granules3636 - Kabutomori O, Iwatani Y, Koh T, Fushimi R, Amino N. Decrease in the density of IgG-Fc receptor III (CD16) on “toxic” neutrophils. Acta haematologica. 1993;89(3):163-4. doi: 10.1159/000204512 and can be rapidly upregulated on stimulated cells by stimulation with bacterial products, including LPS and fMLP.3737 - Vlkova M, Chovancova Z, Nechvatalova J, Connelly AN, Davis MD, Slanina P, et al. Neutrophil and Granulocytic Myeloid-Derived Suppressor Cell-Mediated T Cell suppression significantly contributes to immune dysregulation in common variable immunodeficiency disorders. J Immunol. 2019;202(1):93-104. doi: 10.4049/jimmunol.1800102 Since LPS is recognized by different receptors on neutrophils, especially TLR4, which is reduced on neutrophils from older adults; this fact might be influencing LPS-activation of neutrophils.1515 - Gasparoto TH, Sipert CR, Oliveira CE, Porto VC, Santos CF, Campanelli AP, et al. Salivary immunity in elderly individuals presented with Candida-related denture stomatitis. Gerodontology. 2012;29(2):e331-9. doi: 10.1111/j.1741-2358.2011.00476.x Although we did not pursue the investigation of the molecular mechanisms underlying the downmodulation of CD11b on human neutrophils, the observation that elderly individuals displayed a lower quantity of systemic CXCL8 might be associated with the decreased frequency of CD11b+ neutrophils, since CXCL8 upregulates CD11b expression.2525 - Loos T, Opdenakker G, van Damme J, Proost P. Citrullination of CXCL8 increases this chemokine’s ability to mobilize neutrophils into the blood circulation. Haematologica. 2009;94(10):1346-53. doi: 10.3324/haematol.2009.006973 Another possible explanation for the downregulation of CD11b might be related to TNF and CCL3, which decayed prematurely in aged mice during innate granuloma formation3838 - Chiu BC, Stolberg VR, Freeman CM, Chensue SW. Mononuclear phagocyte-derived interleukin-10 suppresses the innate pulmonary granuloma cytokine response in aged mice. Am J Pathol. 2007;171(3):829-37. doi: 10.2353/ajpath.2007.061122 and were demonstrated to induce the expression of CD11b on human neutrophils.3939 - Montecucco F, Steffens S, Burger F, da Costa A, Bianchi G, Bertolotto M, et al. Tumor necrosis factor-alpha (TNF-alpha) induces integrin CD11b/CD18 (Mac-1) up-regulation and migration to the CC chemokine CCL3 (MIP-1alpha) on human neutrophils through defined signalling pathways. Cell Signal. 2008;20(3):557-68. doi: 10.1016/j.cellsig.2007.11.008 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.4040 - Butcher S, Chahel H, Lord JM. Review article: ageing and the neutrophil: no appetite for killing? Immunology. 2000;100:411–6. 10.1046/j.1365-2567.2000.00079.x 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.3737 - Vlkova M, Chovancova Z, Nechvatalova J, Connelly AN, Davis MD, Slanina P, et al. Neutrophil and Granulocytic Myeloid-Derived Suppressor Cell-Mediated T Cell suppression significantly contributes to immune dysregulation in common variable immunodeficiency disorders. J Immunol. 2019;202(1):93-104. doi: 10.4049/jimmunol.1800102 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.

Figure 1
CXCL8 levels and expression of FcγRIIIb and CD11b on neutrophils from healthy elderly individuals are decreased.

References

  • 1
    - Faurschou M, Borregaard N. Neutrophil granules and secretory vesicles in inflammation. Microbes Infect. 2003;5(14):1317-27. doi: 10.1016/j.micinf.2003.09.008
  • 2
    - Segal AW. How neutrophils kill microbes. Annu Rev Immunol. 2005;23:197-223. doi: 10.1146/annurev.immunol.23.021704.115653
  • 3
    - Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 2011;11(8):519-31. doi: 10.1038/nri3024
  • 4
    - Futosi K, Fodor S, Mocsai A. Neutrophil cell surface receptors and their intracellular signal transduction pathways. Int Immunopharmacol. 2013;17(3):638-50. doi: 10.1016/j.intimp.2013.06.034
  • 5
    - Bauer JW, Baechler EC, Petri M, Batliwalla FM, Crawford D, Ortmann WA, et al. Elevated serum levels of interferon-regulated chemokines are biomarkers for active human systemic lupus erythematosus. PLoS Med. 2006;3(12):e491. doi: 10.1371/journal.pmed.0030491
  • 6
    - Kruger P, Saffarzadeh M, Weber AN, Rieber N, Radsak M, von Bernuth H, et al. Neutrophils: between host defence, immune modulation, and tissue injury. PLoS Pathog. 2015;11(3):e1004651. doi: 10.1371/journal.ppat.1004651
  • 7
    - Tsuboi N, Asano K, Lauterbach M, Mayadas TN. Human neutrophil Fcgamma receptors initiate and play specialized nonredundant roles in antibody-mediated inflammatory diseases. Immunity. 2008;28(6):833-46. doi: 10.1016/j.immuni.2008.04.013
  • 8
    - Pincetic A, Bournazos S, DiLillo DJ, Maamary J, Wang TT, Dahan R, et al. Type I and type II Fc receptors regulate innate and adaptive immunity. Nat Immunol. 2014;15(8):707-16. doi: 10.1038/ni.2939
  • 9
    - Nimmerjahn F, Ravetch JV. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol. 2008;8(1):34-47. doi: 10.1038/nri2206
  • 10
    - Anania JC, Chenoweth AM, Wines BD, Hogarth PM. The Human FcγRII (CD32) Family of Leukocyte FcR in Health and Disease. Front Immunol. 2019;10:464. doi: 10.3389/fimmu.2019.00464
  • 11
    - Kerntke C, Nimmerjahn F, Biburger M. There is (scientific) strength in numbers: a comprehensive quantitation of fc gamma receptor numbers on human and murine peripheral blood leukocytes. Front Immunol. 2020;11:118. doi: 10.3389/fimmu.2020.00118
  • 12
    - Gruver AL, Hudson LL, Sempowski GD. Immunosenescence of ageing. J Pathol. 2007;211(2):144-56. doi: 10.1002/path.2104
  • 13
    - Goronzy JJ, Weyand CM. Understanding immunosenescence to improve responses to vaccines. Nat Immunol. 2013;14(5):428-36. doi: 10.1038/ni.2588
  • 14
    - Gasparoto TH, Vieira NA, Porto VC, Campanelli AP, Lara VS. Differences between salivary and blood neutrophils from elderly and young denture wearers. J Oral Rehabil. 2011;38(1):41-51. doi: 10.1111/j.1365-2842.2010.02126.x
  • 15
    - Gasparoto TH, Sipert CR, Oliveira CE, Porto VC, Santos CF, Campanelli AP, et al. Salivary immunity in elderly individuals presented with Candida-related denture stomatitis. Gerodontology. 2012;29(2):e331-9. doi: 10.1111/j.1741-2358.2011.00476.x
  • 16
    - Wenisch C, Patruta S, Daxbock F, Krause R, Horl W. Effect of age on human neutrophil function. J Leukoc Biol. 2000;67(1):40-5. doi: 10.1002/jlb.67.1.40
  • 17
    - Gasparoto TH, Oliveira CE, Vieira NA, Porto VC, Cunha FQ, Garlet GP, et al. Activation pattern of neutrophils from blood of elderly individuals with Candida-related denture stomatitis. Eur J Clin Microbiol Infect Dis. 2012;31(6):1271-7. doi: 10.1007/s10096-011-1439-z.
  • 18
    - Chiarella P, Reffo V, Bruzzo J, Bustuoabad OD, Ruggiero RA. Therapeutic anti-tumor vaccines: from tumor inhibition to enhancement. Clin Med Oncol. 2008;2:237-45. doi: 10.4137/cmo.s538
  • 19
    - Rajarathnam K, Sykes BD, Kay CM, Dewald B, Geiser T, Baggiolini M, et al. Neutrophil activation by monomeric interleukin-8. Science. 1994;264(5155):90-2. doi: 10.1126/science.8140420
  • 20
    - Tomay F, Wells K, Duong L, Tsu JW, Dye DE, Radley-Crabb HG, et al. Aged neutrophils accumulate in lymphoid tissues from healthy elderly mice and infiltrate T- and B-cell zones. Immunol Cell Biol. 2018;96(8):831-40. doi: 10.1111/imcb.12046
  • 21
    - Martinis M, Franceschi C, Monti D, Ginaldi L. Inflamm-ageing and lifelong antigenic load as major determinants of ageing rate and longevity. FEBS Lett. 2005;579(10):2035-9. doi: 10.1016/j.febslet.2005.02.055
  • 22
    - Licastro F, Candore G, Lio D, Porcellini E, Colonna-Romano G, Franceschi C, et al. Innate immunity and inflammation in ageing: a key for understanding age-related diseases. Immun Ageing. 2005;2:8. doi: 10.1186/1742-4933-2-8
  • 23
    - Martich GD, Danner RL, Ceska M, Suffredini AF. Detection of interleukin 8 and tumor necrosis factor in normal humans after intravenous endotoxin: the effect of antiinflammatory agents. J Exp Med. 1991;173(4):1021-4. doi: 10.1084/jem.173.4.1021
  • 24
    - Butcher SK, Chahal H, Nayak L, Sinclair A, Henriquez N v, Sapey E, et al. Senescence in innate immune responses: reduced neutrophil phagocytic capacity and CD16 expression in elderly humans. J Leukoc Biol. 2001;70(6):881-6. doi: 10.1189/jlb.70.6.881.
    » https://doi.org/10.1189/jlb.70.6.881
  • 25
    - Loos T, Opdenakker G, van Damme J, Proost P. Citrullination of CXCL8 increases this chemokine’s ability to mobilize neutrophils into the blood circulation. Haematologica. 2009;94(10):1346-53. doi: 10.3324/haematol.2009.006973
  • 26
    - Vossebeld PJ, Homburg CH, Roos D, Verhoeven AJ. The anti-Fc gamma RIII mAb 3G8 induces neutrophil activation via a cooperative actin of Fc gamma RIIIb and Fc gamma RIIa. Int J Biochem Cell Biol. 1997;29(3):465-73. doi: 10.1016/s1357-2725(96)00160-4
  • 27
    - Unkeless JC, Shen Z, Lin CW, DeBeus E. Function of human Fc gamma RIIA and Fc gamma RIIIB. Semin Immunol. 1995;7(1):37-44. doi: 10.1016/1044-5323(95)90006-3
  • 28
    - Marois L, Vaillancourt M, Marois S, Proulx S, Paré G, Rollet-Labelle E, et al. The ubiquitin ligase c-Cbl down-regulates FcgammaRIIa activation in human neutrophils. J Immunol. 2009;182(4):2374-84. doi: 10.4049/jimmunol.0801420
  • 29
    - Hoffmann JJ. Neutrophil CD64: a diagnostic marker for infection and sepsis. Clin Chem Lab Med. 2009;47(8):903-16. doi: 10.1515/CCLM.2009.224.
  • 30
    - Nuutila J. The novel applications of the quantitative analysis of neutrophil cell surface FcgammaRI (CD64) to the diagnosis of infectious and inflammatory diseases. Curr Opin Infect Dis. 2010;23(3):268-74. doi: 10.1097/QCO.0b013e32833939b0
  • 31
    - Qureshi SS, Lewis SM, Gant VA, Treacher D, Davis BH, Brown KA. Increased distribution and expression of CD64 on blood polymorphonuclear cells from patients with the systemic inflammatory response syndrome (SIRS). Clin Exp Immunol. 2001;125(2):258-65. doi: 10.1046/j.1365-2249.2001.01596.x
  • 32
    - Repp R, Valerius T, Sendler A, Gramatzki M, Iro H, Kalden JR, et al. Neutrophils express the high affinity receptor for IgG (Fc gamma RI, CD64) after in vivo application of recombinant human granulocyte colony-stimulating factor. Blood. 1991;78(4):885-9. doi: 10.1182/blood.V78.4.885.885
  • 33
    - Wessels I, Jansen J, Rink L, Uciechowski P. Immunosenescence of polymorphonuclear neutrophils. ScientificWorldJournal. 2010;10:145-60. doi: 10.1100/tsw.2010.14
  • 34
    - Phillipson M, Heit B, Colarusso P, Liu L, Ballantyne CM, Kubes P. Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade. J Exp Med. 2006;203(12):2569-75. doi: 10.1084/jem.20060925
  • 35
    - Golay J, Valgardsdottir R, Musaraj G, Giupponi D, Spinelli O, Introna M. Human neutrophils express low levels of FcγRIIIA, which plays a role in PMN activation. Blood. 2019;133(13):1395-405. doi: 10.1182/blood-2018-07-864538
  • 36
    - Kabutomori O, Iwatani Y, Koh T, Fushimi R, Amino N. Decrease in the density of IgG-Fc receptor III (CD16) on “toxic” neutrophils. Acta haematologica. 1993;89(3):163-4. doi: 10.1159/000204512
  • 37
    - Vlkova M, Chovancova Z, Nechvatalova J, Connelly AN, Davis MD, Slanina P, et al. Neutrophil and Granulocytic Myeloid-Derived Suppressor Cell-Mediated T Cell suppression significantly contributes to immune dysregulation in common variable immunodeficiency disorders. J Immunol. 2019;202(1):93-104. doi: 10.4049/jimmunol.1800102
  • 38
    - Chiu BC, Stolberg VR, Freeman CM, Chensue SW. Mononuclear phagocyte-derived interleukin-10 suppresses the innate pulmonary granuloma cytokine response in aged mice. Am J Pathol. 2007;171(3):829-37. doi: 10.2353/ajpath.2007.061122
  • 39
    - Montecucco F, Steffens S, Burger F, da Costa A, Bianchi G, Bertolotto M, et al. Tumor necrosis factor-alpha (TNF-alpha) induces integrin CD11b/CD18 (Mac-1) up-regulation and migration to the CC chemokine CCL3 (MIP-1alpha) on human neutrophils through defined signalling pathways. Cell Signal. 2008;20(3):557-68. doi: 10.1016/j.cellsig.2007.11.008
  • 40
    - Butcher S, Chahel H, Lord JM. Review article: ageing and the neutrophil: no appetite for killing? Immunology. 2000;100:411–6. 10.1046/j.1365-2567.2000.00079.x
  • Funding sources:This study was supported by grants # 10/19317-6 and # 2009/14127-7, São Paulo Research Foundation (FAPESP) and by CNPq (MCTI/Universal # 441673/2014-7). This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Publication Dates

  • Publication in this collection
    31 Mar 2021
  • Date of issue
    2021

History

  • Received
    23 Sept 2020
  • Reviewed
    14 Dec 2020
  • Accepted
    15 Dec 2020
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