Preparations of intravenous immunoglobulins diminish the number and proinflammatory response of CD14+CD16++ monocytes in common variable immunodeficiency (CVID) patients
Introduction
The cells of the monocyte lineage derive from myelomonocytic stem cells in bone marrow. They mature to monocytes, enter blood, circulate and then migrate into the tissues. In the different tissues these cells (now termed macrophages) differentiate into phenotypically and functionally distinct cell types, like alveolar and peritoneal macrophages, osteoclasts, microglia cells, etc. While the heterogeneity of tissue macrophages is well established, monocyte heterogeneity, before introduction of monoclonal antibodies (mAbs) and flow cytometry, has been initially defined by the presence or absence of the high affinity Fc-receptor [1], which later was classified as CD64. Using mAbs against the CD14 and CD16 (Fc gamma IIIA receptor [FcγRIIIA]) molecules, human monocytes were then subdivided into two subpopulations: CD14++CD16 (classical) and CD14+CD16++ (non-classical or “proinflammatory”) cells [2]. The CD14+CD16++ subset represents approximately 10% of total blood monocytes, i.e. about 50 cells/μl. In comparison to the main subpopulation of classical monocytes, this subset is characterized by an enhanced expression of human leukocyte antigens (HLA) class II, CD11a, CD11c, CD18, Toll-like receptors (TLR)-2, and increased ability to produce proinflammatory cytokines (tumor necrosis factor [TNF], interleukin [IL]-12), whereas classical monocytes produce higher amounts of IL-10 and moderate levels of TNF [3], [4], [5], [6], [7]. The CD14+CD16++ monocytes may be also identified in another way as CD14+HLA-DR++ cells [5] and they develop into tissue macrophages or preferentially into dendritic cells [8], [9].
The numbers of CD14+CD16++ monocytes are dramatically increased in patients with sepsis, but are also elevated in HIV-1 infection, asthma, sarcoidosis, peridontitis, atopic eczema, pancreatitis, atherosclerosis, hemodialysis, alveolar proteinosis, rheumatoid arthritis, hemophagocytic lymphohistiocytosis and Kawasaki disease [3], [10]. On the other hand, CD14+CD16++ monocytes are depleted by treatment with glucocorticoids [11], [12]. These findings suggest that the non-classical CD14+CD16++ monocytes are of great importance in inflammatory diseases being the main source of TNF among leukocytes, and that they may be an important target for anti-inflammatory therapy.
Some of the inflammatory diseases that are characterized by an increased number of CD14+CD16++ monocytes, like hemophagocytic syndrome [13], [14] and Kawasaki disease [15], [16], are treated with high doses of intravenous immunoglobulins (IVIG) [17], [18]. We hypothesized that IVIG treatment may act on the CD14+CD16++ monocytes. Therefore, the present study on the in vivo and in vitro effects of IVIG on numbers and function of monocyte subsets was undertaken. The CVID patients were investigated, as they receive regular immunoglobulin substitution. Here we show that IVIG at even supplementary doses can strongly reduce numbers of the non-classical CD14+CD16++ monocytes and also their proinflammatory cytokine production via triggering of FcγRIIB (CD32B).
Section snippets
Patients
We studied adult (n = 18, average age – 36.2 ± 13.7 years, average age at diagnosis – 18.2 ± 19.8 years, M/F = 8/10) patients with common variable deficiency (CVID) [19] and, as a control, 17 age-matched healthy adults (average age – 34.1 ± 6.7 years, M/F = 10/7). All patients were under supervision of the outpatient units of the Department of Clinical Immunology and the Department of Internal Medicine and Gerontology of the Jagiellonian University Medical College. During the study period they presented no
Identification of monocyte subpopulations
Monocyte subpopulations were determined in a 4-color flow cytometry single platform assay using anti-CD45, -HLA-DR, -CD14, -CD16 mAbs and TruCOUNTTM tubes for determination of the absolute number of monocytes. As it is shown in Fig. 1A, a threshold was set on CD45 staining in order to exclude non-leukocyte events and in the CD45/side scatter plot a gate (G1) was set around monocytes extending into the upper region of the lymphocyte population. Events in this gate were shown in a CD14/HLA-DR
Discussion
In this study a new labeling approach to enumerate CD14++CD16 and CD14+CD16++ monocyte subsets in the blood was used [22]. Apart from the adequate identification of these subpopulations this procedure allows for efficient exclusion of NK cells from analysis. The 4-color flow cytometry and a single platform assay were used to determine the absolute numbers of monocyte subsets in peripheral blood. An additional washing step before staining of the whole blood samples was introduced, because in
Acknowledgments
We wish to thank Ms. Mariola Ożóg for skillful technical assistance. We acknowledge with thanks the gift of anti-CD32B mAbs by MacroGenics Inc. This work was supported by the National Committee for Scientific Research grant No. 3220/PO1/2007/32. M. Siedlar was supported by the Alexander von Humboldt Foundation. L. Ziegler-Heitbrock was supported by grant Zi288 from Deutsche Forschungsgemeinschaft.
References (43)
- et al.
Identification and characterization of novel monocyte subpopulation in human peripheral blood
Blood
(1989) - et al.
Antitumor response of CD14+/CD16+ monocyte subpopulation
Exp. Hematol.
(2004) - et al.
The novel subset of CD14/CD16 blood monocytes is expanded in sepsis patients
Blood
(1993) - et al.
Diagnostic criteria for primary immunodeficiencies
Clin. Immunol.
(1999) - et al.
Natural killer (NK) activity in human responders and nonresponders to stimulation by anti-CD16 antibodies
Cell. Immunol.
(1995) - et al.
Concurrent presence of agonistic and antagonistic anti-CD95 autoantibodies in intravenous Ig preparations
J. Allergy Clin. Immunol.
(2003) - et al.
SHIP recruitment attenuates Fc gamma RIIB-induced B cell apoptosis
Immunity
(1999) - et al.
Differential cytokine expression in human blood monocyte subpopulations: a polymerase chain reaction analysis
Blood
(1996) - et al.
Kawasaki disease: aetiopathogenesis and therapeutic utility of intravenous immunoglobulin
Autoimmun. Rev.
(2010) - et al.
Isolation and functional characteristics of FcR+ and FcR human monocyte subsets
J. Immunol.
(1984)