to b or not to b? glucocorticoid impact on b lymphocyte fate and function

To B or Not to B? Glucocorticoid Impact on BLymphocyte Fate and Function

William J. Kovacs

Division of Endocrinology, Diabetes, and Metabolism, The Pennsylvania State University, College ofMedicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033

Lymphocytes have been used to study the action of glu-cocorticoids for more than a half-century. Although it

had been recognized that adrenal steroids administered invivo could cause atrophy of the thymus and lymph nodesof the rat (1), Dr Allan Munck and his colleagues were thefirst to show that a specific biochemical change (reductionin glucose use) could be elicited by glucocorticoids actingon thymocytes and that these effects seem to be mediatedthrough hormone interaction with highly specific intra-cellular binding sites (2–5). These receptors were also rec-ognized to mediate the apoptotic response of lymphoidcells of some species, and murine lymphoma cell lines thatexhibited resistance to these lytic effects of glucocorticoidswere used as a tool to explore the structure-function re-lationships of the glucocorticoid receptor (GR) (6–9).

The power of this approach drove many important dis-coveries in the field of steroid hormone action. In what hasseemed at times like a parallel universe, glucocorticoidswere being applied in the clinical setting for the control ofboth normal and abnormal immune system function longbefore we knew anything about how they worked (10).These hormonal compounds were used to treat patientswith immune system neoplasms and autoimmune, aller-gic, or inflammatory diseases as well as those undergoingorgan transplantation, even though the mechanisms bywhich these various clinical effects were mediated re-mained largely obscure. We have now begun to recognizethat the glucocorticoid actions on the immune systemlikely extend beyond apoptotic responses and suppressionof cytokine gene expression in thymus-derived lympho-cytes (11). This recognition has led to the expectation thatother specific cellular and molecular targets of glucocor-ticoids can be identified in the immune system and, hope-

fully, exploited for therapeutic application in the clinicalsetting.

In this issue of Endocrinology, Gruver-Yates et al (12)present an exploration of the expression of the GR acrossthe developmental pathway of (bone marrow derived) B-lymphocytes, the antibody-producing cells of the humoralimmune system. The authors use flow cytometric tech-niques to meticulously characterize GR expression in im-mature B cell subsets extracted from bone marrow andfrom peripheral lymphoid organs, and they demonstratefunctionality of these receptors by assessing the apoptoticresponses of these cells to glucocorticoids administered invitro and in vivo. Their results are of particular interest inthat they identify B-lymphocytes at all stages of develop-ment as potential targets of glucocorticoid action.

Although endocrinologists seem to have heretoforelargely ignored the B lymphocyte as a glucocorticoid tar-get, immunologists (for at least a time) showed consider-able interest in the idea. In vivo experiments in humansreceiving supraphysiological doses of exogenous gluco-corticoids [100 or 400 mg of hydrocortisone (13) or 60 mgof prednisone (14)] resulted in transient reductions in thenumber of circulating lymphocytes after 4–6 hours. Theseeffects reverted to normal by 24 hours. The most pro-nounced reductions occurred in T cells, but circulating Bcell numbers were also reduced by nearly 50% (14). Theability of the remaining circulating lymphocytes to re-spond to phytohemagglutinin (a T cell mitogen) was un-altered, but responses to pokeweed mitogen (a B cell mi-togen) were reduced by the highest dose of hydrocortisone(13). Because previous studies had demonstrated resis-tance of human lymphoid cells to glucocorticoid-mediatedlysis (15), the investigators interpreted their findings in

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2014 by the Endocrine SocietyReceived November 25, 2013. Accepted December 5, 2013.

For article see page 463

Abbreviation: GR, glucocorticoid receptor.

N E W S A N D V I E W S

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vivo as consistent with glucocorticoid-driven redistribu-tion of lymphocytes from the circulation to peripherallymphoid organs.

Early studies of the direct effects of glucocorticoids onhuman B cells in vitro revealed that glucocorticoids couldalso stimulate some B cell functions. The mitogen activa-tion of B cells (in the presence of T cells) could be sup-pressed by hydrocortisone, but only at millimolar concen-trations, whereas physiological concentrations ofglucocorticoids or the typical therapeutic-range doses ex-erted marked stimulatory effects on antibody production(16, 17).

As techniques advanced for the isolation of pure pop-ulations of B cells, and with increased understanding of thenatural signals required for B cell activation, the effects ofglucocorticoids in vitro were reexamined. In experimentsusing purified B cells incubated in the presence of IL-4, theaddition of glucocorticoids has been found to result inaugmentation of IgE synthesis and secretion, an effect thatappeared to depend on a glucocorticoid-mediated induc-tion of expression of CD 40 ligand (an important costimu-latory pathway) on the B cell surface (18–20). These ex-periments suggested that glucocorticoid effects onhumoral immunity might be exerted not only by negative(apoptotic) processes but also by stimulatory effects insome specific physiological contexts. Although Gruver-Yates et al (12) use the apoptotic response to probe func-tionality of GR in their various B cell subsets, they arecareful to point out that GR action might not always andeverywhere elicit apoptotic responses in the B cell com-partment. In reductio ad absurdum, if glucocorticoidswere simply B cell killers, an organism might be able tohave B lymphocytes or glucocorticoids but not both. Toillustrate, we ran a few experiments using human B lym-phocytes isolated by CD19 magnetic beads (as used byGruver-Yates et al) and cultured them in RPMI 1640 with10% charcoal-stripped (steroid free) fetal calf serum for 6days. As one might expect, few cells can be demonstratedto be alive at that point under basal conditions (Figure 1A)but with the simple addition of dexamethasone at 1 �Mconcentration, a measurable fraction of the cells survivesthe ordeal (Figure 1B), consistent with the older findingsthat glucocorticoids could be supportive of human B cellsurvival and development.

So if glucocorticoids are capable of signaling at manydifferent steps along a continuum of B cell development inthe central compartment and during B cell activation in theperiphery and if they can exert both apoptotic and sup-portive effects, what could be the significance? First, weknow that the early B cell precursors frequently expressautoreactive antibody specificities (21) and that these au-toreactive clones are selected out by a number of processes

including clonal deletion (an apoptotic process). How glu-cocorticoid action impacts this or other tolerance-induc-ing processes will be of considerable interest. As B cellscontinue along their developmental pathway, they en-counter additional checkpoints at which autoreactiveclones may be tolerized by distinct processes of clonal de-letion, anergy induction, and receptor editing (22–24).Some of these checkpoints have been found to be modu-lated by sex steroids (25–27); glucocorticoid effects areunexplored. Second, when a mature B cell in the peripheryencounters antigen in the context of T cell help (cytokinestimulation and CD40 engagement), the processes of so-matic hypermutation and immunoglobulin heavy-chaingene class switch recombination are initiated (28, 29). So-

Figure 1. Glucocorticoid treatment can enhance human peripheralblood B cell viability in vitro. Human B cells were isolated fromperipheral blood using CD19 magnetic beads (Miltenyi Biotech) andcultured in RPMI 1640 without phenol red and with 10% charcoal-stripped fetal calf serum for 6 days. Parallel cultures haddexamethasone added at a concentration of 1 �M. Cells wereanalyzed by flow cytometry with live/dead stains (Life Technologies).Brightly stained cells are dead; cells that exclude the fluorescent dyeare viable. Shown is one representative of three replicate experimentswith different human donors.

340 Kovacs Glucocorticoid Impact on B Lymphocytes Endocrinology, February 2014, 155(2):339–342


matic hypermutation in the variable regions of an immu-noglobulin gene expands the repertoire of antibody spec-ificity (and again raises the possibility of the emergence ofauto reactive clones), whereas heavy-chain gene classswitch recombination changes the functional class of im-munoglobulin produced from IgM to IgG or IgE. Both ofthese processes are initiated by the DNA-modifying en-zyme, activation-induced cytidine deaminase (AICDA), agene now known to be regulated by estrogens and pro-gestins (30–32). We recently observed evidence that glu-cocorticoids could also modulate the expression ofAICDA mRNA, suggesting a potential impact of adrenalsteroids on these late steps of B cell maturation in theperiphery (33).

The experiments reported here by Gruver-Yates et al(12) will, I hope, rekindle some interest from endocrinol-ogists on the impact of hormonal signals on the humoralimmune system. Their detailed survey of GR expressionin the B cell developmental pathway suggests multiplesites for glucocorticoid action with potential for impor-tant physiologic impact on the humoral immune sys-tem’s responses to pathogens and vaccines as well as theprocesses leading to emergence of humoral autoimmunephenomena.

Acknowledgments

Thanks go to N. J. Olsen for critical reading of the manuscript.

Address all correspondence and requests for reprints to: Wil-liam J. Kovacs, MD, The Pennsylvania State University, Collegeof Medicine, Division of Endocrinology, Diabetes, and Metab-olism, Mail Code H044, 500 University Drive, Hershey, PA17033–0850. E-mail: [email protected].

This work was supported by a novel research grant from theLupus Research Institute (New York, New York).

Disclosure Summary: The authors have nothing to disclose.

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to b or not to b? glucocorticoid impact on b lymphocyte fate and function

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