Acta Biochimica Polonica, Vol. 65, No 1/2018, 1–7, https://doi.org/10.18388/abp.2017_1588

Review

Purinergic signaling in B cells

Tomasz Przybyła, Monika Sakowicz-Burkiewicz* and Tadeusz Pawełczyk

Department of Molecular Medicine, Medical University of Gdansk, Gdańsk, Poland

Adenosine and adenosine triphosphate are involved in purinergic signaling which plays an important role in control of the immune system. Much data have been obtained regarding impact of purinergic signaling on dendritic cells, macrophages, monocytes and T lymphocytes, however less attention has been paid to purinergic regulation of B cells. This review summarizes present knowledge on ATP- and Ado-dependent signaling in B lymphocytes. Human B cells have been shown to express A1-AR, A2A-AR, A2B-AR and A3-AR and each subtype of P2 receptors. Surface of B cells exhibits two antagonistic ectoenzymatic pathways, one relies on constitutive secretion and resynthesis of ATP, while the second one depends on degradation of adenosine nucleotides to nucleosides and their subsequent degradation. Inactivated B cells remain under the suppressive impact of autocrine and paracrine Ado, whereas activated B lymphocytes increase ATP release and production. ATP protects B cells from Ado-induced suppression and exerts pro-inflammatory effect on the target tissues, and it is also involved in the IgM release. On the other hand, Ado synthesis is necessary for optimal development, implantation and maintenance of the plasmocyte population in bone marrow in the course of the primary immune response. Moreover, Ado plays an important role in immunoglobulin class switching, which is a key mechanism of humoral immune response. Disruption of purinergic signaling leads to severe disorders. Impairment of Ado metabolism is one of the factors responsible for common variable immunodeficiency. There are several lines of evidence that dysfunction of the immune system observed during diabetes may in part depend on disrupted ATP and Ado metabolism in the B cells.

Key words: B cells, purinergic signaling, adenosine, adenosine triphosphate, P1 receptors, P2 receptors

Received: 17 April, 2017; revised: 20 July, 2017; accepted:
16 December, 2017; available on-line: 23 January, 2018

*e-mail: ssak@gumed.edu.pl

Abbreviations: Ado, adenosine; AC, adenylate cyclase; ADA, adenosine deaminase; ADP, adenosine diphosphate; ADK, ecto-adenylate kinase; AMP, adenosine monophosphate; AK, adenosine kinase; ATP, adenosine triphosphate; BCR, B-cell receptors; Breg, regulatory B cells; cAMP, cyclic adenosine monophosphate; CNT, sodium cation dependent concentrative nucleoside transporters; CVID, common variable immunodeficiency; EBV, Epstein Barr virus; ENT, equilibrative nucleoside transporters; ERK1, extracellular signal-regulated kinases; FO B cells, follicular B cells; GC, germinal center; ICS, immunoglobulin class switching; Ino, inosine; CD62L, L-selectin; 5’-NT, CD73, ecto-5’-nucleotidase; NDP, ecto-NDP-kinase; NTPDase1, ectonucleoside triphosphate diphosphohydrolase-1; MHC, major histocompatibility complex; MZ, marginal zone; MMPs, matrix metalloproteinases; MAPK, mitogen activated protein kinase; NTPs, nucleoside triphosphates; PKC, protein kinase C; Tc, cytotoxic T cells; Teff, effector T cells; Treg, regulatory T cells; TCR, T cell receptor; TI, T cell-independent; TLR, toll-like receptor; TF, transcription factor; TFH, follicular helper T cells; Th, helper T cells

Purinergic signaling

Adenosine triphosphate (ATP) and its breakdown products, mainly adenosine diphosphate (ADP) and adenosine (Ado), are ligands for purinergic receptors and function as extracellular messengers. Purinergic signaling plays an important role in many biological processes, including: exocrine and endocrine secretion, immunological responses, aggregation of platelets, vasodilatation and cellular proliferation, differentiation and apoptosis (Burnstock, 2007). Purinergic network consist of two types receptors, namely P1 and P2. P1 receptors selectively bind Ado and subdivide into A1-AR, A2A-AR, A2B-AR and A3-AR subtypes. These receptors belong to the rhodopsin-like family of G protein-coupled receptors and interact with adenylate cyclase (AC). A1-AR and A3-AR negatively affect AC via α subunits of Gi/o protein, and thus inhibiting cyclic adenosine monophosphate (cAMP) synthesis, while A2A-AR and A2B-AR stimulate AC by Gs and promote cAMP accumulation. P2 receptors are activated by ATP and its derivatives, and consist of two types: P2X and P2Y. P2X receptors are ligand-gated ion channels and to date 7 subunits ranging from P2X1 to P2X7 have been identified. .Molecular structure of a single P2X subunit consists of intracellularly located N- and C-termini with consensus binding motifs for protein kinases, two transmembrane-spanning regions (TM1, TM2) responsible for channel gating and lining the ion pore, a large cysteine-rich extracellular loop forming disulfide bridges, a hydrophobic H5 region responsive to channel modulation by cations, and ATP-binding site. The subunits couple into functional homotrimers or heterotrimers, while trimers are capable to form hexamers. Family of P2Y receptors consists of 8 receptor subtypes, namely P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14. Each subunit possesses an extracellular N-terminus and intracellular C-terminus reach in kinase binding motifs. Seven spanning regions lay within the transmembrane domain and are involved in forming of a ligand docking pocket. Intracellular loops and the C-terminus display structural diversity, which determines the degree of coupling with G proteins. Each P2Y receptor connects to a single G protein heterotrimer, typically to Gq/11. P2Y11 is able to interact with Gq/11,as well as with Gs and P2Y12-14 coupled to Gi. Depending on conditions, P2Y form homomultimers or heteromultimers (Burnstock, 2007).

Lymphocytes in the immune system

Human immune system acts through two different types of responses acting in close cooperation. Immediate, innate response comprises myeloid lineage derived cells and lymphocyte natural killer (NK) cells derived from lymphoid progenitors. Constitutive innate response is independent of the count of previous infections and depends only on the type of the inducing factor and remains similar during one’s life time. Extent of the delayed acquired (adaptive) response is increased by recurrent contacts with stimulatory agents due to the phenomenon of immune memory and is carried out by cells of the lymphoid lineage (various types of T and B lymphocytes), as well as the myeloid accessory cells.  For activation, the T lymphocyte requires presentation of a processed antigen by professional antigen presenting cells (dendritic cells, B-cells, or macrophages), together with the major histocompatibility complex (MHC) type I or II (Chaplin, 2010).

Three major types of T lymphocytes are recognized:  Th (helper) CD4+, Tc CD8+ (cytotoxic)  and Treg (regulatory). The major feature of a T lymphocyte is expression on its surface of the αβ T cell antigen receptor (TCR). Naive CD4+ T cells may transform towards Th1, Th2, and Th17 subtypes depending on the type of cytokines released into the microenvironment during activation. Th1 cells express transcription factor (TF) T-bet and produce IL-2, IFN-γ, and lymphotoxin. Th2 cells express TF GATA-3 and release IL-4, IL-5, IL-9, IL-13, and GM-CSF. Th17 cells are marked by TF RORC2 expression and production of IL-6 and IL-17. It is generally approved that Th1 cells promote cellular immune responses, whereas Th2 are involved in humoral mechanisms. Subpopulation of circulating CD4+ T lymphocytes gives rise to regulatory T cells (Tregs), which are involved in modulation of the immune response. This fraction can be divided into natural and induced Tregs. Natural Tregs express surface CD4 and CD25 together with a nuclear forkhead box P3 (Foxp3) TF and secrete immunosuppressive cytokines, like TGFβ and IL-10. Induced Tregs express Foxp3 variably and their major product is IL-10 (Chaplin, 2010).

B cells exhibit expression of the membrane B cell receptor (BCR), which can recognize antigens in their native forms, thus B lymphocytes do not need antigen presentation for activation. Antigen recognition, together with signals from activated Th2 cells, induces B cells to proliferate and generate effector plasma cells and memory B cells (Chaplin, 2010). B cells may also undergo activation in a T cell-independent manner by T cell-independent (TI) antigens (Herlands et al., 2008). TI antigens consist of foreign polysaccharides and unmethylated CpG DNA, and can induce rapid and less specific humoral response when compared to the T cell-dependent activation (Nutt et al., 2015).

The B cells may be subdivided into separate populations depending on their maturity and function. B cell lineages arise directly from common lymphoid 2 progenitor (LCA-2) cells (Tobòn et al., 2013). Bone marrow cells produce signals (cytokines, TFs) which induce differentiation of LCA-2 cells to pre-B cells. Mature B lymphocytes bear only one type of specific BCR, thus the generation of the receptor is a critical step in development of B cells (Tobòn et al., 2013). Immature B cells with already developed BCR migrate from bone marrow to circulation as transitional B cells (Palanichamy et al., 2009). Transitional B cells are characterized by surface expression of CD20, CD38, CD24, BR3 and IgM and nuclear presence of TFs, such as Pax5, EBF, E2A and Oct2 (Melchers, 2015). Current criteria recognize three groups of B cells, i.e. T1, T2 and T3 cells (Palanichamy et al., 2009). Maturation of transitional B cells may occur in the spleen and lymphoidal node follicles, resulting in generation of follicular (FO), marginal zone (MZ), germinal center (GC), and memory B cells (Chung et al., 2002; Melchers, 2015). MZ B cells are non-circulating and reside in the marginal zone of the spleen. They are involved in the early adaptive immune responses since the marginal zones intake high volumes of blood from general circulation (Pillai et al., 2005).  Phenotype of MZ B cells consists of surface IgM, IgD, CD1c, CD24, CD19, CD20, CD21 and nuclear TFs, including Pax5, EBF, E2A and Oct2 (Melchers, 2015). Follicular B cells may circulate in the blood or reside in the primary and secondary follicles of spleen or lymph nodes. These cells take part in production of the bulk of high-affinity antibodies (Chaplin, 2010). They show expression of surface IgM, CD23, CD19, CD20, CD21 and CD22, and nuclear TF Pax5 (Melchers, 2015).

T-cell dependent activation of FO B cells leads to dynamic development of GCs within lymph nodes or spleen. Within GCs, the mature B cells undergo proliferation, differentiation, somatic hypermutation and class switching recombination, which results in generation of B lymphocytes with diverse specificity and antibody affinity (Chaplin, 2010). Antigen selected B cells that leave the GC may further become memory B cells or plasmablasts. GC B cells exhibit surface expression of CD20, CD38 and BR3, together with nuclear TFs BCL6, Pax5 and EBF (Melchers, 2015). Further differentiation of B cells generates short-lived plasmablasts and long-lived plasma cells (Chaplin, 2010). Differentiation of plasmablasts occurs early during infection and may be triggered either by a T cell-dependent or a T cell-independent activation leading to generation of plasma cells (Nutt et al., 2015). Plasma cells are involved in later stages of infection and produce high affinity antibodies against antigens (Nutt et al., 2015). Plasma cells are characterized by surface expression of CD45, CD138, TACI and/or BCMA, CD126, CD184 and CD320. They also express nuclear TFs, like BLIMP1, IRF4 and XBP1 (Melchers, 2015). When the same antigen is introduced next time, the presence of memory cells enables initiation of the secondary immune response which is rapid and more effective (Chaplin, 2010). During the GC response, memory B cells are selected later than those undergoing differentiation to plasma cells. As a result, memory B cells accumulate more immunoglobulin mutations and achieve higher affinity towards a specific antigen. After each round of antigen encounter the pool of differentially responding B cell clones expands and develops a stronger polyclonal response (Victoria et al., 2012). Surface markers of memory B cells are CD20, CD27, CD80, CD84, CD86 and CD148, whereas nuclear TFs include OBF1 and SPI-B (Melchers, 2015).

Regulatory B (Breg) cell is another subtype of B lymphocytes. Bregs suppress function of the pro-inflammatory lymphocytes through the secretion of IL-10, IL-35, and TGF-β (Floudas et al., 2016). Bregs also interact directly with T cells to promote their differentiation towards Tregs. Bregs can arise from most of the B cell types as a result of inflammatory signals and BCR recognition mechanisms. Production of IL-10 is considered an important feature of Bregs. Large fraction of IL-10 producing B (B10) cells is found in the CD19+ CD24 high CD27− CD38 high CD1d high CD5 transitional B cell population. Additionally, B10 cells express markers of activation and memory, CD48 and CD148, respectively (Floudas et al., 2016). Purinergic molecules CD73 and CD39 have been also identified as markers of Bregs. Expression of these molecules allows Bregs to breakdown ATP to Ado and eventually suppress proliferation of T cells (Saze et al., 2013).

In the past two decades much attention has been paid to the role of purinergic signaling in the immune system (Dubyak, 2009; Sakowicz-Burkiewicz & Pawelczyk, 2001; Dubyak, 2012; Rayah et al., 2012; Jacob et al., 2013; Burnstock & Boeynaems, 2014; Di Virgilio & Vuerich, 2015).

Purinergic signaling in the immune cells

Immune cells of different type and lineage express both types of purinergic receptors (Di Virgilio & Vuerich, 2015). The P1 receptors are widely expressed by immune cells of the myeloid and lymphoid origin, and vast numbers of data confirm the important role of A2A-AR and A2B-AR receptors in the control of inflammation (Di Virgilio & Vuerich, 2015). A2A-AR and A2B-AR receptors trigger production of immunosuppressive cAMP (Sakowicz-Burkiewicz et al., 2011; Di Virgilio & Vuerich, 2015). P2X receptor expression has been observed in mononuclear phagocytes, neutrophils, eosinophils, T and B lymphocytes, NK cells and mast cells (Di Virgilio & Vuerich, 2015). P2YRs are widely expressed in the immune cells and have been thoroughly examined in neutrophilic and eosinophilic granulocytes, monocytes, macrophages, dendritic cells, T and B lymphocytes and NK cells (Di Virgilio & Vuerich, 2015). Purinergic receptors are involved in the chemotaxis of inflammatory and dendritic cells, migration of monocytes/macrophages, killing of bacteria by macrophages, secretion of IL-1β, maturation of dendritc cells and their enhanced antigen endocytosis, TCR-mediated activation of T lymphocytes, and suppression of the T cell activity (Burnstock & Boeynaems, 2014; Cekic & Linden, 2016). Relatively less is known about the role of the purinergic signaling in B cells. The main goal of this review is to summarize current knowledge on the impact of purinergic signaling on the B cell function.

Expression of P1 and P2 receptors in B cells

Comparative analysis of A2A-AR expression between populations of lymphocytes revealed that this type of receptor is more abundant on T cells when compared to B cells (Koshiba et al., 1999). It has been also reported that B cells exhibit surface expression of the A3-AR receptor, which becomes overexpressed after activation of B cells (Gessi et al., 2004). In B cells originating from humans and rats, expression of all four types of AR has been detected (Sakowicz et al., 2009; Sakowicz-Burkiewicz et al., 2012). Sluyter and coworkers have shown the presence of P2X1, P2X2, P2X4 and P2X7 on surface of B cells (Sluyter et al., 2001), while Lee and colleagues have detected mRNA for each P2 receptor subtype (Lee et al., 2006). The transcript levels of each P2X and P2Y were similar, except for P2X3 and P2X7, which were significantly lower (Lee et al., 2006). In another study, mRNA of each P2Y subtype was observed, however, P2Y2 and P2Y12 were expressed the most abundantly, while P2Y4 the most weakly (Wang et al., 2004). Examination of P2X1, P2X4 and P2X7 transcripts revealed the dominant expression of P2X4 which was opposite to observations of Lee (Wang et al., 2004; Lee et al., 2006). Blood cells transformed with the Epstein Barr virus (EBV) into lymphoblastic cell lines are considered to be a valuable tool in examination of the immune system. It has been shown that EBV mediated transformation of B cells led to suppression of most of the P2 subtypes and upregulation of P2X7 expression (Lee et al., 2006). Such plasticity in the P2 expression should be considered when choosing an appropriate model for research on purinergic signaling in immune cells.

Distribution of Ado and ATP in B cell environment

Concentration of Ado and ATP in a local environment of B cells is regulated by a network of membrane ectoenzymes (nucleotidases, deaminases, kinases) and transport proteins (Yegutkin et al., 2002; Sakowicz-Burkiewicz et al., 2010). Two antagonistic ectoenzymatic pathways have been observed at the surface of B cells, one relies on constitutive secretion of ATP and continuous resynthesis of high energy phosphates, while the second one depends on degradation of nucleotides to nucleosides and their subsequent inactivation (Yegutkin et al., 2002; Sakowicz-Burkiewicz et al., 2013a).

B cells continuously release ATP, which is subsequently degraded by surface ectoenzymes to Ado (Yegutkin et al., 2002; Sakowicz-Burkiewicz et al., 2010; Sakowicz-Burkiewicz et al., 2013a). Experiments conducted on human naive and memory B cells, as well as on lymphoblastic cell lines, revealed that stimulated B cells release ATP by late endosomal/lisosomal vesicles through Ca2+- and TI-VAMP protein-dependent mechanism (Schena et al., 2013). Ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39) is an ectonucleotidase which hydrolyses nucleoside triphosphates (NTPs) on the surface of B cells and transforms ATP into adenosine diphosphate/monophosphate (ADP/AMP). Subsequently, ecto-5’-nucleotidase (5’-NT, CD73) hydrolyses AMP to Ado (Yegutkin et al., 2002; Sakowicz-Burkiewicz et al., 2010; Sakowicz-Burkiewicz et al., 2013a). Concentration of Ado in the pericellular space of B cells is maintained at a constant level because of uptake through specific transporters and subsequent deamination to inosine (Ino) catalyzed by membrane and intracellular adenosine deaminase (ADA, CD26) (Herrera et al., 2001; Sakowicz-Burkiewicz et al., 2010; Sakowicz-Burkiewicz et al., 2013a). Therefore, pericellular concentration and bioavailability of Ado for P1 receptors is determined by equilibrium between the rates of its synthesis and elimination (Herrera et al., 2001; Sakowicz-Burkiewicz et al., 2010).

Parallel to ATP consuming pathway, an opposite axis exists which promotes the increase in pericellular ATP concentration (Yegutkin et al., 2002; Sakowicz-Burkiewicz et al., 2010; Sakowicz-Burkiewicz et al., 2013a). On the cell surface, besides being hydrolyzed to Ado, AMP may also be transphosphorylated to ADP by an ecto-adenylate kinase (AK1β) (Yegutkin et al., 2002; Sakowicz-Burkiewicz et al., 2013a). ATP provides the main source of phosphates for both enzymes and may derive from the cell’s surroundings or a degradation resistant pool in the membrane domains of B cells (Yegutkin et al., 2002). ATP, resistant to enzymatic degradation by apyrase, is transported from cytosol to the membrane microdomains in lipid rafts probably through plasmatic or exocytotic vesicles (Yegutkin et al., 2006). Eventually, ADP is converted into ATP in a reaction catalyzed by ecto-NDP-kinase (NDP) and this enzyme exhibits a much higher activity when compared to AK1β (Yegutkin et al., 2002; Sakowicz-Burkiewicz et al., 2013a). Aside from ATP, other nucleotide triphosphates have been also shown to provide phosphates for ecto-NDP (Yegutkin et al., 2002; Sakowicz-Burkiewicz et al., 2013a). Antagonistic ectoenzymatic pathways at the B cell membrane are regulated by concentration ratios of nucleoside mono-, di- and triphosphates and by the activities of the enzymes involved. Both factors determine the shift of reaction equilibrium towards degradation of nucleotides or towards their synthesis, resulting in activation of P1 or P2 receptors, respectively.

Processing of purinergic signaling messengers may not be the only function of B cell surface ectoenzymes, as it is possible that they modulate the action of purinergic receptors. The study on function of ADA in T cells conducted by Herrera and co-workers supported such an assumption (Herrera et al., 2001). It has been shown that the membrane ADA is coupled to A2B receptor in T lymphocytes and their interaction enhanced the A2B-AR affinity for its ligand, namely 5’-N-ethylocarboxyamideadenosine, and subsequent production of c-AMP. Moreover, inhibition of the ADA activity did not impair the synergistic effect of ADA-A2B-AR coupling (Herrera et al., 2001). However, similar observations in B cells have not been confirmed yet. Concentration of B cell pericellular Ado may also be controlled in a nonenzymatic manner by the transport of Ado through plasma membrane. Two types of membrane spanning nucleoside transporting channels take part in this process, i.e. equilibrative nucleoside transporters (ENT) and sodium cation dependent concentrative nucleoside transporters (CNT) (Cass et al., 2002; Podgorska et. al., 2005; Molina-Acras et al., 2009). It has been shown that peripheral blood mononuclear cells exhibit expression of CNT2, CNT3, ENT1 and ENT2, however, the Ado uptake is mediated predominantly by ENT1 (Molina-Acras et al., 2003). The expression level of particular nucleoside transporters varies in B cells and depends on their physiological state. Experiments performed on rat B cells indicated that impaired nucleoside transport that takes place in diabetic B lymphocytes results from alterations in the expression of rENT1, rENT2 and rCNT2 transporters, which are independently and differentially regulated by glucose and insulin (Sakowicz et al., 2005). Nonetheless, data focusing particularly on the nucleoside transport in B cells are still limited.

Effector role of Ado and ATP in B cell function

B cells regulate T cell function through purinergic signaling

Recent data suggests that Bregs utilize purinergic signaling to control the function of T cells. Adenine nucleotides and nucleosides activate different signaling pathways, and thus appropriate ratio of Ado and ATP concentrations needs to be maintained in the environment of B cells. Study on impact of ATP and Ado on B and T cells cultured separately and in a co-culture suggested that B cells regulate their own function and function of T cells through purinergic signaling (Saze et al., 2013). Ado derived from enzymatic degradation of ATP suppresses B cell activity in an autocrine fashion through A3-AR, which results in lack of a TCR-dependent response of CD4+, CD8+ T-cells (Saze et al., 2013). On the contrary, the activated B cells become CD39high CD73low and increase production of AMP (Saze et al., 2013). AMP produced by B cells enhances their proliferation in an autocrine manner and at the same time it inhibits proliferation of T cells in a paracrine manner (Saze et al., 2013). Moreover, activated B cells exhibit an increased expression of ADA, which results in further elimination of Ado (Saze et al., 2013). A study on purinergic signaling in Bregs showed that suppression of CD4+ T cell proliferation was exerted under in vivo condition only by subset of CD39high CD73+ CD24+ CD25high and CD38high B cells (Figueiro et al., 2016). This specific fraction of B cells was also capable of producing GM-CSF, TNF-α, IL-6 and moderate levels of IL-10. Such cell phenotype allows vigorous production of 5-AMP and ADO, and subsequent suppression of the T effector cells (Teff). Breg-derived ADO interacts with A2A-AR on Teffs, leading to cAMP accumulation and depletion of their effector function. Moreover, autocrine ADO, AMP, GM-CSF, TNF-α and IL-6 promote expansion of the CD39 high B cells. ADO and AMP promotes proliferation of Bregs through A1A-AR and A2A-AR, whereas GM-CSF and IL-6 have been reported to promote differentiation of B cells to Bregs (Deng et al., 2012; Rosser et al., 2014; Figueiro et al., 2016). Increase in proportion of CD39 high B cells leads to elevation of AMP and ADO levels, and subsequent suppression of activation and proliferation of CD4+ Teff cells (Figueiro et al., 2016). It was proposed that purinergic regulation of Bregs is independent of IL-10 production (Wang et al., 2014). However, recent study demonstrated that IL-10(–/–) B cells showed decreased expression of CD73 and resulted in an impaired Ado production (Kaku et al., 2014).

B cells achieve immunocompetence through purinergic signaling

Resent study demonstrated that Ado synthesis is necessary for optimal development, implantation and maintenance of the plasmocyte population in bone marrow in the course of primary immune response in Mus musculus (Conter et al., 2014). B cells differentiate into plasmocytes in the GCs and during primary immune response the maturing germinal centers become highly enriched in B cells CD73+ and follicular helper T cells (TFH) CD73+ (Conter et al., 2014). TFH cells play a key role in generation and maintenance of germinal centers and in differentiation of plasma cells and memory B cells (Crotty, 2011; Conter et al., 2014). Moreover, it has been shown that activity of CD73 in GCs leads to proper generation of the bone marrow plasmocyte population (Conter et al., 2014). On the other hand, suppressed CD73 expression in B and TFH cells maybe efficiently compensated by other populations of cells (Conter et al., 2014). Researchers hypothesized that arise in CD73 expression in GCs may be the response to hypoxia and an increased level of ATP that naturally occurs in GCs during intense processes of proliferation and apoptosis (Conter et al., 2014). Ado also plays an important role in immunoglobulin class switching (ICS), which is a key mechanism of humoral immune response resulting in differentiation of immunoglobulin isotypes (Schena et al., 2013). It has been observed that stimulation of BCR and toll-like receptor (TLR) in CD39+/CD73+ and CD39+/CD73– subpopulations of human naive and memory B cells resulted in the release of ATP and subsequent Ado formation (Schena et al., 2013). CD73 expression promotes higher frequency of ICS to IgG or IgA in both, the naive and memory B cells (Schena et al., 2013). Additionally, immunophenotype CD73+ is associated with a higher surface expression of CD180, a TLR receptor homolog which regulates B cell sensitivity to TLR9- and CD40L-dependent stimulation (Schena et al., 2013). Moreover, comparing to CD73– B cells, memory CD73+ cells exhibited an increased expression level of Xbp-1, a transcription factor taking part in later stages of B cell development (Schena et al., 2013). Naive and memory CD73+ B cells cultured with either ADA or CD73 inhibitor, upon stimulation showed significant disruption of class switch recombination when compared to untreated cells (Schena et al., 2013). On the other hand, naive and memory CD73– B cells cultivated with Ado, upon stimulation exhibited a significantly higher number of class switched IgG/IgA B cells when compared to CD73- lymphocytes cultured in a medium deprived of Ado (Schena et al., 2013). Release of immunoglobulins, however, may depend on ATP signals. P2X7 activation has been shown to be involved in a T cell-independent production of IgM by B lymphocytes (Sakowicz-Burkiewicz et al., 2013b). Stimulation of B cells with Staphylococcus aureus Cowan strain I (SAC) and IL-2 promotes IgM production and administration of BzATP, a P2X7 agonist, significantly enhanced immunoglobulin secretion (Sakowicz-Burkiewicz et al., 2013b). On the other hand, selective inhibition of P2X7 in stimulated B cells resulted in a total inhibition of IgM release (Sakowicz-Burkiewicz et al., 2013b). However, further investigation needs to be conducted to reveal a specific role of purinergic signaling in the T cell-independent response of B cells.

Ado and ATP control the rolling of lymphocytes during inflammation

Traffic of lymphocytes between blood and tissues is essential for propagation of inflammation and purinergic signaling plays an important role in this process through conditioning kinetics of lymphocytes (Henttinen et al., 2003; Yegutkin et al., 2014). Extravasation of leukocytes to target tissue consists of following steps: temporal adhesion of lymphocytes to vascular endothelium, rolling of cells along endothelium, activation resulting in holding of the cells and subsequent transmigration into tissue (Springer, 1994). Leukocyte-endothelial interactions are regulated via cellular adhesion and purinergic signalization (Panes et al., 1999). Comparison of different endothelial and lymphoid cell lines has shown that endothelial cells exhibit high activity of CD73 and CD39, which results in a phenotype promoting  Ado synthesis, whereas action of lymphoid cells is directed towards pericellular ATP accumulation and Ado elimination (Yegutkin et al., 2002; Henttinen et al., 2003; Yegutkin et al., 2006). B lymphocytes exhibit low or lack of CD39 and CD73 activity, moreover, it has been shown that B cells from the Namalwa leukemic cell line, as well as B lymphocytes isolated from blood of healthy donors, inhibited the CD73 activity of the HUVEC endothelial cell line (Yegutkin et al., 2002; Henttinen et al., 2003; Yegutkin et al., 2006). However, the mechanism governing the inhibition of CD73 activity has not been identified yet (Henttinen et al., 2003). It has been observed that inhibition of endothelial CD73 by B cells was associated with elimination of extracellular Ado owing to ecto-ADA activity (Henttinen et al., 2003). Moreover, it has been shown that the Namalwa lymphocytes used apyrase resistant pericellular pool of ATP as source of γ-phosphates in continuous conversion of AMP to ADP, thus limiting availability of AMP for endothelial CD73 (Henttinen et al., 2003). It can be concluded that a proper course of events constituting inflammatory response, such as leukocyte-endothelial adhesion and subsequent migration of leukocytes to tissues, is determined by B cell dependent shift of local metabolism towards elimination of Ado and accumulation of ATP (Henttinen et al., 2003). Removal of Ado and prevention of its synthesis is controlled at three levels: inhibition of endothelial CD73, catalytic deamination of residual Ado and continuous elimination of substrate for CD73 via conversion of pericellular AMP to ADP (Henttinen et al., 2003). It is assumed that such phenotype allows B cells to avoid suppressive impact of Ado and to sustain micromolar halo of ATP which leads to propagation of ATP signaling in endothelial cells during inflammation (Yegutkin et al., 2002; Henttinen et al., 2003; Yegutkin et al., 2006).

Accumulation of pericellular ATP occurring in B cells activates the P2X7 receptor, resulting in shedding of CD21, CD23 and CD62L from the cell surface (Frémeaux-Bacchi et al., 1998; Gu et al., 1998; Venturi et al., 2003; Sengstake et al., 2006). Disappearance of these molecules modulates immune responses at different levels. Adhesion protein L-selectin (CD62L) is expressed on the surface of B and T cells (Gu et al., 1998). Interaction of CD62L with epitopes on the endothelial venules is the first event in the adhesion cascade leading to integrin interactions and transendothelial migration of leukocytes (Gu et al., 1998). It has been shown that P2X7 stimulation activates the membrane matrix metalloproteinases (MMPs), resulting in the cleavage of CD62L at a proximal extracellular domain and subsequent shedding of soluble CD62L (Gu et al., 1998). Deletion of MMPs cleaved sequence on the proximal membrane domain of CD62L results in continuous migration of activated lymphocytes to peripheral lymph nodes, instead of temporal rolling and movement into thymus, which normally occurs after CD62L shedding (Venturi et al., 2003). CD23 is a membrane spanning protein of type II, acting as a low affinity receptor for IgE (FcϵRII), moreover, it also functions as adhesion molecule involved in transendothelial movement of B cells (Sengstake et al., 2006). CD23 becomes overexpressed in activated B cells and sheds after P2X7 activation, however, it sheds much slower than CD62L (Sengstake et al., 2006). It has been also shown that the level of CD23 decreases in B cells during  migration along the layer of vascular endothelium (Gu et al., 1998; Sengstake et al., 2006). CD21 is a part of membrane coreceptor complex CD19/CD81/Leu-13. The coreceptor complex interacts with BCR to lower the B cell activation threshold (Cherukuri et al., 2001). Activation of P2X7 by ATP results in MMPs mediated proteolytic cleavage of CD21 and its shedding from periphery of B cells (Sengstake et al., 2006). Soluble form of CD21 interacts with complement fragments and forms complexes with CD23 trimers, thereby inhibiting CD23 dependent synthesis of IgE (Frémeaux-Bacchi et al., 1998). ATP is thus a modulator of B cell migration along endothelium, activation and IgE synthesis.

Summary and perspectives

The expression profile of purinergic signaling components, such as purinoreceptors, specific ectoenzymes and nucleoside transporters has been examined extensively in B cells. Function of many of those components has been also revealed. The B lymphocytes remain inactivated under high pericellular concentration of Ado. After activation, B cells increase production of AMP to enhance their own proliferation. Metabolism of activated B cells is shifted towards elimination of Ado and accumulation of pericellular ATP, which enhance the adhesion of lymphocytes to vascular endothelium and subsequent migration to target tissues. P2X7-dependent shedding of surface molecules CD21 and CD62L has been shown to be involved in transendothelial migration of B cells. There is also evidence showing that P2X7 is directly involved in the release of IgM from B cells after TI activation. Although ATP signaling is dominant in activated B lymphocytes, Ado seems to be crucial in achieving immunocompetence by activated cells. It has been observed that maturation of plasmocytes in germinal centers, as well as successful CSR, depends on elevated expression of CD73 in the B cell environment. Disrupted expression of P1 receptors and enzymes metabolizing Ado and ATP has been observed in a course of CVID and diabetes (Table 1). Action of A1-AR, A2A-AR, A2B-AR, A3-AR and P2X7 receptors in B cells has been so far investigated the most extensively, however, insight into function of other P2 receptors is necessary.

Acknowledgements

This work was supported by the Medical University of Gdansk research grant: ST-52-02-0052/07.

References

Burnstock G (2004) Introduction: P2 receptors. Curr Top Med Chem 4: 793–803

Burnstock G (2007) Purine and pyrimidine receptors. Cell Mol Life Sci 64: 1471–1483. http://doi.org/10.1007/s00018-007-6497-0

Burnstock G, Boeynaems JM (2014) Purinergic signalling and immune cells. Purinergic Signal 10: 529–564. http://doi.org/10.1007/s11302-014-9427-2

Cass CE, Young JD, Baldwin SA, Cabrita MA, Graham KA, Griffiths M, Jennings LL, Mackey JR, Ng AM, Ritzel MW, Vickers MF, Yao SY (2002) Nucleoside transporters of mammalian cells. In Membrane Transporters as Drug Targets. Gordon L. Amidon, Wolfgang Sadée eds, pp 313–352. Springer

Cekic C, Linden J (2016) Purinergic regulation of the immune system. Nat Rev Immunol 16: 177–192. http://doi.org/10.1038/nri.2016.4

Chaplin DD (2010) Overview of the immune response. J Allergy Clin Immunol 125: S3–S23. https://doi.org/10.1016/j.jaci.2009.12.980

Cherukuri A, Cheng PC, Pierce SK (2001) The role of the CD19/CD21 complex in B cell processing and presentation of complement-tagged antigens. J Immunol 167: 163–172. doi:org/10.4049/jimmunol.167.1.163

Chung JB, Sater RA, Fields ML, Erikson J, Monroe JG (2002) CD23 defines two distinct subsets of immature B cells which differ in their responses to T cell help signals. Internat Immunol 14: 157–166.  https://doi.org/10.1093/intimm/14.2.157

Conter LJ, Song E, Shlomchik MJ, Tomayko MM (2014) CD73 expression is dynamically regulated in the germinal center and bone marrow plasma cells are diminished in its absence. PloS ONE 9: e92009. http://doi.org/10.1371/journal.pone.0092009

Crotty S (2011) Follicular helper CD4 T cells (Tfh). Ann Rev Immunol 29: 621–663. http://doi.org/10.1146/annurev-immunol-031210-101400

Deng J, Galipeau J (2012) Reprogramming of B cells into regulatory cells with engineered fusokines. Infect Disord Drug Targets 12: 248–254 http://dx.doi.org/10.2174/187152612800564392

Di Virgilio F, Vuerich M (2015) Purinergic signaling in the immune system. Auton Neurosci 191: 117–123. http://doi.org/10.1016/j.autneu.2015.04.011

Dubyak GR (2009) Both sides now: multiple interactions of ATP with pannexin-1 hemichannels. Focus on “A permeant regulating its permeation pore: inhibition of pannexin 1 channels by ATP”. Am J Physiol Cell Physiol 296: C235–C241. http://doi.org/10.1152/ajpcell.00639.2008

Dubyak GR (2012) P2X7 receptor regulation of no-classical secretion from immune effector cells. Cell Microbiol 14: 1697–1706. http://doi.org/10.1111/cmi.12001

Figueiró F, Muller L, Funk S, Jackson EK, Battastini AMO, Whiteside TL (2016) Phenotypic and functional characteristics of CD39high human regulatory B cells (Breg). Oncoimmunology 5: e1082703-e1082703-10doi:10.1080/2162402X.2015.1082703.

Floudas A, Amu S, Fallon PG (2016) New insights into IL-10 dependent and IL-10 independent mechanisms of regulatory B cell immune suppression. J Clin Immunol 36: 25–33. https://doi.org/10.1007/s10875-016-0263-8

Frémeaux-Bacchi V, Fischer E, Lecoanet-Henchoz S, Mani JC, Bonnefoy JY, Kazatchkine MD (1998) Soluble CD21 (sCD21) forms biologically active complexes with CD23: sCD21 is present in normal plasma as a complex with trimeric CD23 and inhibits soluble CD23-induced IgE synthesis by B cells. Int Immunol 10: 1459–1466

Gessi S, Varani K, Merighi S, Cattabriga E, Avitabile A, Gavioli R, Fortini C, Leung E, Mac Lennan S, Borea PA (2004) Expression of A3 adenosine receptors in human lymphocytes: up-regulation in T cell activation. Mol Pharmacol 65: 711–719. http://doi.org/10.1124/mol.65.3.711

Gu B, Bendall LJ, Wiley JS (1998) Adenosine triphosphate–induced shedding of CD23 and L-selectin (CD62L) from lymphocytes is mediated by the same receptor but different metalloproteases. Blood 92: 946–951

Henttinen T, Jalkanen S, Yegutkin GG (2003) Adherent leukocytes prevent adenosine formation and impair endothelial barrier function by Ecto-5′-nucleotidase/CD73-dependent mechanism. J Biol Chem 278: 24888–24895. http://doi.org/10.1074/jbc.M300779200

Herlands RA, Christensen SR, Sweet RA, Hershberg U, Shlomchik MJ (2008) T cell-independent and toll-like receptor-dependent antigen-driven activation of autoreactive B cells. Immunity 29: 249–260. http://doi.org/10.1016/j.immuni.2008.06.009

Herrera C, Casado V, Ciruela F, Schofield P, Mallol J, Lluis C, Franco R (2001) Adenosine A2B receptors behave as an alternative anchoring protein for cell surface adenosine deaminase in lymphocytes and cultured cells. Mol Pharmacol 59: 127–134. http://doi.org/10.1124/mol.59.1.127

Jacob F, Novo CP, Bachert C, Van Crombruggen K (2013) Purinergic signaling in inflammatory cells: P2 receptor expression, functional effects, and modulation of inflammatory responses. Purinergic Signal 9: 285–306 http://doi.org/10.1007/s11302-013-9357-4

Kaku H, Cheng KF, Al-Abed Y, Rothstein TL (2014) A novel mechanism of B-cell mediated immune suppression through CD73-expression and adenosine production. J Immunol 193: 5904–5913. doi:10.4049/jimmunol.1400336.

Kocbuch K, Sakowicz-Burkiewicz M, Grden M, Szutowicz A, Pawel-czyk T (2009) Effect of insulin and glucose on adenosine metabolizing enzymes in human B lymphocytes. Acta Biochim Pol 56: 439–446

Koshiba M, Rosin DL, Hayashi N, Linden J, Sitkovsky MV (1999) Patterns of A2A extracellular adenosine receptor expression in different functional subsets of human peripheral T cells. Flow cytometry studies with anti-A2A receptor monoclonal antibodies. Mol Pharmacol 55: 614–624

Lee DH, Park KS, Kong ID, Kim JW, Han BG (2006) Expression of P2 receptors in human B cells and Epstein-Barr virus-transformed lymphoblastoid cell lines. BMC Immunol 7: 22–32. http://doi.org/10.1186/1471-2172-7-22

Melchers F (2015) Checkpoints that control B cell development. J Clin Invest 125: 2203–2210. doi:10.1172/JCI78083

Molina-Arcas M, Bellosillo B, Casado FJ, Montserrat E, Gil J, Colomer D, Pastor-Anglada M (2003) Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia. Blood 101: 2328–2334. http://doi.org/10.1182/blood-2002-07-2236

Molina-Arcas M, Casado FJ, Pastor-Anglada M (2009) Nucleoside transporter proteins. Curr Vasc Pharmacol 7: 426–434

Nutt SL, Hodgkin PD, Tarlinton DM, Corcoran LM (2015) The generation of antibody-secreting plasma cells. Nat Rev Immunol 15: 160–171. http://doi.org/10.1038/nri3795

Palanichamy A, Barnard J, Zheng B, Owen T, Quach T, Wei C, Looney JR, Sanz J, Anolik JH (2009) Novel human transitional B cell populations revealed by B cell depletion therapy. J Immunol 182: 5982–5993. doi:10.4049/jimmunol.0801859

Panés J, Perry M, Granger DN (1999) Leukocyte-endothelial cell adhesion: avenues for therapeutic intervention. Brit J Pharmacol 126: 537–550. http://doi.org/10.1038/sj.bjp.0702328

Pillai S, Cariappa A, Moran T (2005) Marginal zone B cells. Annu Rev Immunol 23: 161–196. doi:10.1146/annurev.immunol.23.021704.115728.

Podgorska M, Kocbuch K, Pawelczyk T (2005) Recent advances in studies on biochemical and structural properties of equilibrative nucleoside transporters. Acta Biochim Pol 52: 749–758

Rayah A, Kanellopoulos JM, Di Virgilio F (2012) P2 receptors and immunity. Microbes Infect 14: 1254–1262. http://doi.org/10.1016/j.micinf.2012.07.006

Rosser EC, Oleinika K, Tonon S, Doyle R, Bosma A, Carter NA, Harris KA, Jones SA, Klein N, Mauri C (2014) Regulatory B cells are induced by gut microbiota-driven interleukin-1beta and interleukin-6 production. Nat Med 20: 1334–1339. http://dx.doi.org/10.1038/nm.3680

Sakowicz M, Szutowicz A, Pawelczyk T (2005) Differential effect of insulin and elevated glucose level on adenosine transport in rat B lymphocytes. Int Immunol 17: 145–154. http://doi.org/10.1093/intimm/dxh195

Sakowicz-Burkiewicz M, Kocbuch K, Grden M, Szutowicz A, Pawelczyk T (2006) Diabetes-induced decrease of adenosine kinase expression impairs the proliferation potential of diabetic rat T lymphocytes. Immunology 118: 402–412

Sakowicz-Burkiewicz M, Kocbuch K, Grden M, Szutowicz A, Pawelczyk T (2009) Protein kinase C mediated high glucose effect on adenosine receptors expression in rat B lymphocytes. J Physiol Pharmacol 60: 145–153

Sakowicz-Burkiewicz M, Kocbuch K, Grden M, Szutowicz A, Pawel-czyk T (2010) Adenosine 5’-triphosphate is the predominant source of peripheral adenosine in human B lymphoblasts. J Physiol Pharmacol 61: 491–499

Sakowicz-Burkiewicz M, Pawelczyk T (2011) Recent advances in understanding the relationship between adenosine metabolism and the function of T and B lymphocytes in diabetes. J Physiol Pharmacol 62: 505–512

Sakowicz-Burkiewicz M, Kocbuch K, Grden M, Maciejewska I, Szutowicz A, Pawelczyk T (2012) Impact of adenosine receptors on immunoglobin production by human peripheral blood B lymphocytes. J Physiol Pharmacol 63: 661–668.

Sakowicz-BurkiewiczM,Grden M, Maciejewska I, Szutowicz A, Pawelczyk T (2013a) High glucose impairs ATP formationon the surface of human peripheral blood B lymphocytes. Int J Biochem Cell Biol 45: 1246–1254. https://doi.org/10.1016/j.biocel.2013.03.008

Sakowicz-Burkiewicz M, Kocbuch K, Grden M, Maciejewska I, Szutowicz A, Pawelczyk T (2013b) High glucose concentration impairs ATP outflow and immunoglobulin production by human peripheral B lymphocytes: involvement of P2X7 receptor. Immunobiology 218: 591–601. http://doi.org/10.1016/j.imbio.2012.07.010

Saze Z, Schuler PJ, Hong CS, Cheng D, Jackson EK, Whiteside TL (2013) Adenosine production by human B cells and B cell–mediated suppression of activated T cells. Blood 122: 9–18. http://doi.org/10.1182/blood-2013-02-482406

Schena F, Volpi S, Faliti CE, Penco F, Santi S, Proietti M, Schenk U, Damonte G, Salis A,Bellotti M, Fais F, Tenca C, Gattorno M, Eibel H, Rizzi M, Warnatz K, Idzko M, Ayata CK, Rakhmanov M, Galli T, Martini A, Canossa M, Grassi F, Traggiai E (2013) Dependence of immunoglobulin class switch recombination in B cells on vesicular release of ATP and CD73 ectonucleotidase activity. Cell Rep 3: 1824–1831. http://doi.org/10.1016/j.celrep.2013.05.022

Sengstake S, Boneberg EM, Illges H (2006) CD21 and CD62L shedding are both inducible via P2X7Rs. Int Immunol 18: 1171–1178. http://doi.org/10.1093/intimm/dxl051

Sluyter R, Barden JA, Wiley JS (2001) Detection of P2X purinergic receptors on human B lymphocytes. Cell Tissue Res 304: 231–236

Springer TA (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76: 301–314. http://doi.org/ 10.1016/0092-8674(94)90337-9

Tobón GJ, Izquierdo JH, Cañas CA (2013) B Lymphocytes: development, tolerance, and their role in autoimmunity – focus on systemic Lupus erythematosus. Autoimmune Diseases 2013: 827254. doi: 10.1155/2013/827254

Venturi GM, Tu L, Kadono T, Khan AI, Fujimoto Y, Oshel P, Bock CB, Miller AS, Albrecht RM, Kubes P, Steeber DA, Tedder TF (2003) Leukocyte migration is regulated by L-selectinendoproteolyticrelease. Immunity 19: 713–724. https://doi.org/10.1016/S1074-7613(03)00295-4

Victora GD, Nussenzweig MC (2012) Germinal Centers. Ann Rev Immunol 30: 429–457. doi: 10.1146/annurev-immunol-020711-075032

Wang L, Jacobsen SEW, Bengtsson A, Erlinge D (2004) P2 receptor mRNA expression profiles in human lymphocytes, monocytes and CD34+ stem and progenitor cells. BMC Immunology 5: 16–21. http://doi.org/10.1186/1471-2172-5-16

Wang Y, Han X (2014) B Cells with regulatory function in human diseases. Autoimmune Dis Ther Approaches 1: 107–120

Warrier AC, Rao NY, Kulpati DS, Mishra TK, Kabi BC (1995) Evaluation of adenosine deaminase activity and lipid peroxidation levels in diabetes mellitus. Indian J Clin Biochem 10: 9–13. http://doi.org/10.1007/BF02873661

Wu G, Marliss EB (1991) Deficiency of purine nucleoside phosphorylase activity in thymocytes from the immunodeficient diabetic BB rat. Clin Experiment Immunol 86: 260–265. http://doi.org/10.1111/j.1365-2249.1991.tb05807.x

Yegutkin GG, Henttinen T, Samburski SS, Spychala J, Jalkanen S (2002) The evidence for two opposite, ATP-generating and ATP-consuming, extracellular pathways on endothelial and lymphoid cells. Biochem J 367: 121–128. http://doi.org/10.1042/BJ20020439

Yegutkin GG, Mikhailov A, Samburski SS, Jalkanen S (2006) The detection of micromolarpericellular ATP pool on lymphocyte surface by using lymphoid ecto-adenylate kinase as intrinsic ATP sensor. Mol Biol Cell 17: 3378–3385. http://doi.org/10.1091/mbc.E05-10-0993

Yegutkin GG, Auvinen K, Karikoski M, Rantakari P, Gerke H, Elima K, Maksimow M, Quintero IB, Vihko P, Salmi M, Jalkanen S (2014) Consequences of the lack of CD73 and prostatic acid phosphatase in the lymphoid organs. Mediators Inflamm 2014: 485743. http://doi.org/10.1155/2014/485743