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The Journal of Immunology, 1999, 163: 3185-3193.
Copyright © 1999 by The American Association of Immunologists

The Human B Cell Response to IL-13 Is Dependent on Cellular Phenotype as Well as Mode of Activation1

Dwayne Ford*, Catherine Sheehan*, Christopher Girasole*, Rory Priester*, Nicola Kouttab*, John Tigges*, Thomas C. King{dagger}, Andrea Luciani*, John W. Morgan* and Abby L. Maizel2,*

* Department of Pathology, Roger Williams Medical Center, Boston University School of Medicine, Boston, MA 02118; and {dagger} Lifespan Health Care System, Brown University School of Medicine, Providence, RI 02908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Normal mature quiescent human B lymphocytes, isolated as a function of buoyant density, require activation for up-regulation of IL-13R constituents. Cell activation through a combination of surface Ig and CD40 receptor ligation leads to the most substantial message production for IL-13R{alpha}1. Functional consequences of this receptor variation, in initially quiescent cells, includes demonstrable effects on cellular proliferation in response to ligand exposure. Variations in the method of surface activation, with particular emphasis on the CD40 receptor, reveals that immobilized CD40 ligand may be sufficient, in and of itself, to up-regulate IL-13R{alpha}1, which may bear significance for B-lymphocyte bystander proliferation. Regulation of the IL-13R{alpha}1 protein and message also differs as a function of cellular phenotype. Although values are greater in memory than naive B cells, as they are initially isolated from extirpated tonsils, variations in the magnitude of message and protein, as a function of surface stimulation, are more substantial in the naive subset. The magnitude of variation in message production in naive cells is associated with a more vigorous proliferative response to IL-13 than seen in memory lymphocytes. The cellular response to IL-13, as a function of activation and phenotype, is the converse of that demonstrated for IL-2. Evaluation of proliferation, receptor message, ligand binding protein production, and the response to putatively synergistic cytokines reveals that IL-2 is the predominant lymphokine utilized by memory cells. This is in contradistinction to IL-13, which along with IL-4, are the predominant moieties for naive lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The IL-13R is composed of combinations of a 140-kDa protein, identifiable as the IL-4R{alpha} (1, 2), a 65- to 70-kDa protein termed IL-13R{alpha}1, and a 55- to 60-kDa receptor termed IL-13R{alpha}2. The ligand binding protein termed IL-13R{alpha}1 refers to that entity encoded by the cDNA isolated by Aman et al. (3), by Miloux et al. (4) and by Gauchat et al. (5), originally based on the murine cDNA isolated by Hilton et al. (6). The protein encoded by the cDNA isolated by Caput et al. (7), which is related to the murine orthologue cloned by Donaldson et al. (8) and the murine IL-13 binding protein identified by Zhang et al. (9), is herein referred to as IL-13R{alpha}2. Studies of a relatively broad subdivision of cell types predicts and/or demonstrates that one may find cell lineages that possess the three proteins in specific combinations (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). A cellular subset has been shown to include IL-4R{alpha}, IL-13R{alpha}1, and IL-13R{alpha}2, another subset contains IL-4R{alpha} in association with IL-13R{alpha}1, and a third subset may possesses IL-13R{alpha}1 in combination with IL-13R{alpha}2 in amounts that may divert from 1:1 stochiometry. The potential of a heterotrimeric IL-13R constituting the IL-4R{alpha}-chain, IL-13R{alpha}1, and the common ({gamma}c)3 protein has recently been suggested (12). A direct role for {gamma}c in the IL-13 binding complex has been difficult to demonstrate in cells of lymphoid derivation (14, 19, 24, 25, 26) and other multiple lineages (13, 20, 21, 27, 28, 29), although exceptions to these observations have been made (12, 30, 31). The ability of {gamma}c to alter IL-13R function, by as yet unknown mechanisms, has been hypothesized from relatively complex transfection studies (7, 32).

In the majority of instances examined to date, the receptor constituents appear to be constitutively expressed (18). Recent work has demonstrated that in contradistinction to cells of epithelial, neuroectodermal, or fibroblastic lineage, mature lymphoid cells require activation for upregulation of the IL-13R constituents (14, 19). The demonstration of regulatory requirements for receptor expression has been shown directly by examination of message and protein production (14, 19) and indirectly by examination of the functional consequences of receptor binding (33). IL-13R{alpha}1, as it exists on mature human B cells, first binds to IL-13 with subsequent sequestration of IL-4R{alpha} to form a functional receptor. This entity is found in relatively low numbers on the cell surface, binds protein with intermediate affinity, and is strictly regulated as a function of cell surface activation (19). A higher affinity IL-13 ligand binding protein, IL-13R{alpha}2, demonstrated to be absent from mature, quiescent lymphoid cells, has been suggested to function as a decoy receptor (13) in the manner reported for the IL-1RII receptor (34). It has yet to be determined whether this form of IL-13 ligand binding protein invariably transmits a cognate signal. This is particularly germane considering the scant cytoplasmic region of this protein (7) and the recent report that murine IL-13R{alpha}2, which shows 59% overall amino acid identity with human IL-13R{alpha}2, contains no box 1 or box 2 signaling motifs (8).

Control of IL-13 function may be further refined by the presence or absence of soluble receptors and/or ligand binding proteins (9, 14). In cells of lymphoid lineage, activation signals delivered through the Ag and the CD40 receptors are important for the modulation of IL-13R number responsiveness. The signal modulated through CD40 (35) appears to be required for maximal IL-13 efficacy (19, 36, 37). This dependency has been shown for IL-13R{alpha}1 but is, at present, unknown for IL-13R{alpha}2. This latter receptor constituent is absent from the majority of mature lymphoid populations examined thus far, although a subpopulation of germinal center cells has been shown to possess small amounts of message for this protein. In addition, a plasmacytoid cell line has been shown to possess IL-13R{alpha}2 (38). Whether germinal center cells undergoing plasmacytoid differentiation up-regulate this component is presently unknown. The need for cellular activation to up-regulate the components of the functional IL-13R on most mature lymphoid cells has been documented, although multiple questions remain. The current studies have further evaluated the control of receptor message and the cellular response. It was determined that IL-13 responsiveness is not only dependent upon state of cellular activation, but also varies as a function of whether the cellular population has undergone conversion from naive to memory phenotype. In addition, the predominant lymphokine controlling the proliferation of naive and memory cells varies as a function of individual receptor component production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cytokines and reagents

Recombinant human cytokines, the anti-CD40 mouse mAb, and reagents for routine phenotyping were as previously described, (19). Anti-human {kappa} and anti-human {lambda} Abs were covalently coupled to activated Immunobead Matrix from Irvine Scientific (Santa Ana, CA) and used at 10 µg/ml beads. Anti-human CD40 Abs were also covalently linked to activated Immunobead Matrix and utilized as indicated in the text. Soluble anti-CD40 and bound anti-CD40 Abs were utilized independently. Additional reagents for phenotypic analysis, not previously described (19), were anti-CD122PE, obtained from Becton Dickinson (San Jose, CA), FITC-conjugated anti-CD25 from Immunotech (Westbrook, ME), and anti-CD132PE purchased from PharMingen (San Diego, CA). Reagents for magnetic separation were as follows: anti-CD38 Ab was obtained from Becton Dickinson and PharMingen, anti-IgD Ab was obtained from Dako (Carpinteria, CA) and Immunotech, and anti-IgA Ab was obtained from Immunotech and PharMingen. Anti-IgG Ab was from Sigma (St. Louis, MO) and PharMingen. Rat anti-mouse IgG1 Magnetic Microbeads, CS+, and VS+ separation columns were acquired from Miltenyi Biotec (Auburn, CA) and used with the VarioMACS (Miltenyi Biotec) magnetic separator. Reagents for immunoprecipitation were as follows: anti-{gamma}c (TUGh4) was purchased from PharMingen; anti-IL-4R{alpha}-N-17 (aa 27–43) and C-20 (aa 801–820), anti-IL-2Rß C-20 (aa 532–551) and S-20 (aa 510–529) rabbit polyclonal Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-IL-13R{alpha}1 C-19 (aa 379–397) and N-15 (aa 62–76) were produced by Zymed (San Francisco, CA) and affinity purified in our laboratory. Reagents for Western blotting analysis were as follows: anti-{gamma}c C-20 (aa 342–361) and anti-IL-4R{alpha} C-20 (aa 801–820) were purchased from Santa Cruz Biotechnology. Anti-IL-13R{alpha}1 C-19 (aa 379–397) was produced by Zymed. Primer pairs utilized for competitive PCR may be found in Table IGo.


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  		Table I. Primer pair sequences for competitive PCR assays

 
Isolation of tonsillar B lymphocytes

Tonsillar human B lymphocytes were prepared essentially as described (2, 19, 39). Cells prepared in this manner were routinely >=98% B lymphocytes as determined by immunofluorescence with anti-CD3, -CD19, -CD20, -CD45, and -CD14 Abs. Tonsillar B lymphocytes were further fractionated as a function of buoyant density (40). The density of cells prepared in this manner may be categorized as high (>1.094 g/ml Percoll solution), moderate (>1.089 g/ml), or low (>1.082 g/ml). The phenotype of these cells has been previously described (19).

Cell surface phenotyping

Phenotypic analysis of cell surface Ags was performed as previously described with the fluorescent intensity of the cells analyzed on a Becton Dickinson FACSCalibur flow cytometer (41).

Isolation and enrichment of naive and memory cells

Naive and memory human B lymphocytes were isolated as described by Liu et al. (42). Naive B lymphocytes were enriched from either high density or a combination of high and moderate density cells by negative selection following removal of CD38bright, IgG+, and IgA+ cells. Memory B lymphocytes were enriched from a combination of moderate and low density cells by negative selection following removal of CD38bright and IgD+ cells. Cells were labeled with Abs, washed twice, and then incubated with rat anti-mouse IgG1 microbeads (Miltenyi Biotec) at 4°C for 15 min. The resulting magnetically labeled cells were washed twice in supplemented PBS and then magnetically fractionated in Miltenyi CS+ or VS+ columns. Only the negatively separated fractions with the appropriate phenotype, as previously described, were used in experiments.

Measurement of proliferation

Cell proliferation, as measured by [3H]thymidine incorporation, was performed as previously described (39).

Immunochemical identification of receptor components

Freshly isolated quiescent high density B lymphocytes or magnetically separated memory and naive B lymphocytes were used for 0-h controls. Subsequently, 24-h activated cells were harvested by centrifugation and frozen at -20°C until extraction and immunoprecipitation. Extraction of cell pellets was performed for 30 min on ice in lysis buffer containing freshly added protease inhibitors (50 mM HEPES (pH 7.5), 0.5% Brij 97, 50 mM NaCl, 50 mM NaF, 5 mM EDTA, 1 mM Na3VO4, 2 mM PMSF, 10 mM aprotinin, 10 µg/ml leupeptin, 10 µg/ml benzamidine, 10 µg/ml trypsin-chymotrypsin inhibitor, and 10 µg/ml pepstatin) and centrifuged for 15 min at 14,000 x g at 4°C. Supernatants were then precleared for 1 h using 40 µl protein G-Sepharose beads in 20 mM sodium phosphate buffer (pH 7.0) at 4°C in an end-over-end mixer. The resulting supernatant was then immunoprecipitated for 1.5–2 h with either 10 µg anti-{gamma}c (TUGh4), or 5 µg each of anti-IL-4R{alpha} N-17 (aa 27–43) and C-20 (aa 801–820), or 5 µg each of anti-IL-2Rß C-20 (aa 532–551) and S-20 (aa 510–529), or 5 µg each of anti-IL-13R{alpha} C-19 (aa 379–397) and N-15 (aa 62–76) at 4°C in an end-over-end mixer. Ab/protein complexes were precipitated for an additional hour using 50 µl of a 1:2 slurry of protein G-Sepharose beads in 20 mM sodium phosphate buffer (pH 7.0), washed three times with ice-cold lysis buffer, boiled in Laemmli buffer, and resolved by 7.5% SDS-PAGE. The resolved proteins were transferred to nitrocellulose membranes and blocked in Superblock buffer (Pierce, Rockford, IL) overnight at 4°C on an orbital shaker. The blocked membranes were washed once for 15 min and twice for 5 min with Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 (TBS-T). Unconjugated primary Abs were diluted in Superblock to 1 or 1.5 µg/ml and used to blot the membranes for 1 h at room temperature. The membranes were subsequently washed as above and incubated with 1:40,000–80,000 dilution of HRP-conjugated donkey anti-rabbit Ig Abs (Amersham Pharmacia Biotec, Piscataway, NJ) for 30 min. The membranes were then washed again as above, exposed to enhanced chemiluminescence (ECL) reagents for 1 min and developed for between 2 and 30 min using ECL film. Band intensity when quantitated was presented as relative arbitrary units.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-13 proliferative response is dependent upon CD40 stimulation

Experiments initially evaluated the proliferative response of quiescent, mature lymphocytes as a function of variations in the method of activation. In the first protocol evaluated herein, data are presented concerning the method of presentation of the CD40 signal to quiescent, mature B cells. As is evident in Fig. 1GoA, CD40 stimulation, provided by means of soluble anti-CD40 Ab alone, was ineffective in providing stimulation capable of inducing responsiveness to IL-13. Yet stimulation through the CD40 receptor, by means of this soluble Ab, was not an inert event. Examination of the response seen in Fig. 1GoB demonstrates that activation through the Ig receptor is a relatively weak direct stimulant for IL-13 and IL-2 responsiveness as opposed to its effect on IL-4-mediated proliferation. In Fig. 1GoC, the synergy between anti-Ig and anti-CD40 becomes clearly evident, and is most pronounced for IL-13 and weak for IL-2 (compare Fig. 1Go, B and C), thereby confirming the notion that anti-CD40 provides an essential activation modality with particular importance for responsiveness to IL-13. In addition to examining the efficacy of IL-13- and IL-4-induced proliferation, control data are included in Fig. 1Go comparing the response of the above lymphokines to IL-2, a cytokine capable of promoting B cell proliferation in specific cellular subsets (43). Although the magnitude of the response to IL-4 is most substantial in the quiescent mature subset of B cells, the efficacy of IL-2 is substantially reduced in magnitude compared to other cytokines (see Fig. 1Go, B and C).



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  		FIGURE 1. Effect of IL-2, IL-4, and IL-13 on cell proliferation of mature, quiescent B lymphocytes. High-density (1.094 µg/ml) tonsillar B-lymphocytes (1 x 105/200 µl) were cultured in microtiter wells with either soluble anti-CD40 Ab at 5 µg/ml (A), insoluble anti-{kappa}/{lambda} Abs at 10 µg/ml of beads (B), or anti-CD40 Abs at 5 µg/ml and insoluble anti-{kappa}/{lambda} at 10 µg/ml of beads (C). Cells were exposed to IL-2 ({blacksquare}), IL-4 (•), and IL-13 ({blacktriangleup}). A and B are plotted as increasing concentrations of cytokines vs [3H]thymidine incorporation. C is plotted as increasing concentrations of insoluble anti-{kappa}/{lambda} Abs with a fixed amount of soluble anti-CD40 Abs (5 µg/ml) and cytokines (10 µg/ml). All cells were cultured for 72 h and then pulsed with 1.0 µCi [3H]thymidine for an additional 16–18 h. The data are those of a representative experiment with triplicate wells per experimental determination. Multiple experiments (n = 5) indicated the same trend.

 
Method of activation through the CD40 receptor impacts B cell proliferation

The efficacy of CD40 stimulation provided through separate modalities was next examined. Anti-CD40 Ab, covalently linked to an insoluble matrix support, was unable to provide a signal sufficient, in and of itself, to enable IL-13 reactivity (data not shown). Activation using the above-described insolubilized Ab was similar to that seen with the soluble reagent in Fig. 1GoA. In contradistinction to the physically immobilized Ab reagent, study of cellular reactivity to CD40L, presented in an insoluble form by CDw32-L cells transfected with such ligand (44), revealed a relatively marked response to IL-13 alone (Fig. 2Go). The results demonstrating the ability of CD40 ligand to be sufficient, in and of itself, to activate cells to be responsive to IL-13 was also confirmed when message production was determined. Table IIGo presents data evaluating the amount of message, as determined by competitive RT-PCR, for each of the proteins related to construction of both the IL-4 and the IL-13 receptors on mature B lymphocytes. It is demonstrated that statistically significant variations in IL-13R{alpha}1 are the most pronounced as a function of either combined immunoglobulin ligation and soluble CD40 stimulation (p < 0.001) or insoluble CD40 ligand (CD40L) itself (p < 0.05). The degree of the variation in the message encoding {gamma}c, the common receptor protein utilized by IL-2, -4, -7, and -15, is less substantial as a function of activation although combined immunoglobulin and CD40 stimulation shows a statistically significant increase (p < 0.05). In addition, the total amount of {gamma}c message itself, which is larger in magnitude (p < 0.005) than the other moieties evaluated, correlates well with its utilization by multiple receptor entities. The message for IL-4R{alpha}, statistically reduced compared to the other moieties (p < 0.005), appears to be limiting in value, consistent with a prior report on endothelial cells (45). Of interest are the data depicted in Fig. 2Go where both IL-4 and IL-13 respond well to insoluble CD40L stimulation alone. It is apparent that the amount of message for the IL-4R{alpha} component is sufficient to accommodate the marked increases in IL-13R{alpha}1, utilized for both IL-13 or IL-4 receptor function, in addition to up-regulation of {gamma}c that may be utilized for IL-4 responsiveness. Fig. 3Go depicts assessment of the protein constituents themselves as determined by Western blot analysis. Comparison of the message amounts for IL-13R{alpha}1 seen in Table IIGo and the relative protein amounts seen in Fig. 3Go supports the contention that activation-induced changes, as measured by the two separate modalities, are, in the main, correlative. One issue of note is that the magnitude of message variation for IL-13R{alpha}1 as a function of CD40L exposure, 467%, exceeds the relative variation seen in actual protein amount, 142%. The converse situation is demonstrated for {gamma}c where message varies by 50%, compared to unstimulated cells where protein varies by 371%. It should be emphasized that the two methods of evaluation differ in quantitative accuracy. The competitive PCR is a relatively strict quantitative assay, the results from which yield the actual number of amplicons available. This is in contradistinction to the Western blot analysis that, as presently performed, reveals relative determinations. The depiction of change in Fig. 3Go is a demonstration of relative change for a specific ligand binding protein. It is not quantitative in respect to comparing different individual protein amounts of separate receptor units except in a qualitative manner. Furthermore, the relationship between protein amount and message production may not be strictly linear. One must account for variations in both messenger half-life and protein turnover.



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  		FIGURE 2. Effect of CD40L, in comparison to anti-CD40 Ab, on IL-4 and IL-13-induced proliferation. High-density (1.094 µg/ml) tonsillar B lymphocytes (1 x 105/200 µl) were cultured in microtiter wells in the presence of 5 x 103 mitomycin C-treated CD40L-transfected L cells in the presence of increasing concentrations of IL-4 ({blacksquare}) or IL-13 (•). Mitomycin C-treated CD40L-transfected L cells alone ({blacktriangleup}) in the presence of either IL-4 or IL-13 is plotted as a negative control. An additional negative control, demonstrating the lack of effect of nontransfected L cells, has been clearly shown in an earlier publication (56 ). All cells were cultured for 72 h and then pulsed with 1.0 µCi [3H]thymidine for an additional 16–18 h. The data are those of a representative experiment (n = 3), with triplicate wells per experimental determination, with multiple experiments (n = 3) indicating the same finding.

 

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  		Table II. mRNA quantitation for the components of the IL-4/IL-13 receptor in human B lymphocytes

 


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  		FIGURE 3. Comparisons of protein expression, by Western blot analysis, in quiescent, high density mature B lymphocytes. High-density (1.094 µg/ml) mature tonsillar B lymphocytes (~1 x 108 cells) were activated for 24 h in the presence of either insoluble {kappa}/{lambda} Abs (10 µg/ml beads), anti-CD40 Abs (5 µg/ml), or a 1:20 ratio of CD40L-transfected L cells to B lymphocytes. Nonactivated cells were harvested subsequent to Percoll density separation. All cells were harvested by centrifugation, and proteins were solubilized in extraction buffer, precleared and immunoprecipitated with appropriate Abs as indicated in Materials and Methods. IP proteins were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose membranes, and blotted with 1–1.5 µg/ml primary and 1:40,000–80,000 dilution of secondary Abs. Molecular weight markers were used to indicate approximate size. Band intensity was quantitated by Scan Analysis software (Biosoft, MA) and presented as relative arbitrary units.

 
IL-13 proliferation as a function of B cell phenotype

The above-described results relate to a population of relatively quiescent B lymphocytes isolated as a function of buoyant density, a reflection of activation state, as opposed to differentiated phenotype. Although cells isolated in this manner are quiescent, the population contains lymphocytes in varying stages of maturation. Consequently, IL-13 responsiveness of phenotypically fractioned quiescent naive and memory B lymphocytes was next evaluated. Cells were isolated such that the naive cells are enriched for lymphocytes that are CD38-, CD44+, IgD+, IgG-, and IgA-, whereas the memory cells are CD38-, CD44+, IgD-, and IgG+. As seen in Fig. 4Go, phenotype is evidently a determining factor in the cellular responsiveness to ligand exposure. The response of lymphocytes to IL-4 (data not shown) when activated through the Ig and CD40 receptors, although reduced by 25% in memory as opposed to naive cells, remains essentially similar when comparing the two phenotypes. Yet the responses to IL-13 and IL-2 show statistically significant variations in ligand response as a function of phenotype. IL-2, as previously mentioned and shown herein (refer Fig. 1Go), has been demonstrated to function as a proliferative ligand for defined subsets (43). The evaluation of IL-2 in the present context, along with IL-13, was to provide a meaningful comparative cytokine not directly utilizing any shared receptor components necessary for induction of IL-13 proliferation. The data derived from six independent replicate comparisons of naive and memory cells were evaluated statistically (Student’s t tests) in terms of the cytokine response of B lymphocytes stimulated through the Ig and CD40 receptors. Comparing the optimal proliferative response of the six independent tonsillar preparations, naive cells proliferated ~2.6-fold more vigorously to IL-13 than memory cells (p < 0.001), whereas the response of B lymphocytes to IL-2 revealed that memory cells proliferated ~1.9-fold more vigorously than naive cells (p < 0.001). These mean determinations are consistent with the individual experiment presented in Fig. 4GoA (IL-13) and Fig. 4GoB (IL-2).



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  		FIGURE 4. Differential effects of IL-13 and IL-2 on memory versus naive B lymphocytes. Magnetically separated naive (circles) and memory (squares) tonsillar B lymphocytes (1 x 105/200 µl) were cultured in microtiter wells with insoluble anti-{kappa}/{lambda} Abs alone at 10 µg/ml of beads (open symbols) or in combination with anti-CD40 Abs at 5 µg/ml (filled symbols) in the presence of increasing concentrations of IL-13 or IL-2. All cells were cultured for 72 h and then pulsed with 1.0 µCi [3H]thymidine for an additional 16–18 h. The data are those of a representative experiment with triplicate wells per experimental determination, with multiple experiments (n > 5) indicating the same finding.

 
Differential effects of cytokines on memory and naive cells in the presence of additional activation signals

To further examine the phenotypic variation in IL-2 and IL-13 proliferative responsiveness, the effects mediated by an additional surface activation signal were next evaluated. Previous experimentation had demonstrated the capacity of IL-10 to augment responses of B cells to IL-2 exposure (46). Cells of either naive or memory derivation were incubated in the presence of activating signals through the Ig and CD40 receptors in the presence or absence of IL-10 and in the presence or absence of IL-13 or IL-2. In terms of proliferation, the response to IL-13 of both memory and naive cells was essentially additive in the presence of IL-10 in that the total proliferative effect reflected the addition of that response indicated by either cytokine alone (data not shown). The proliferative response of IL-2 diverged from this pattern. As noted previously (see Fig. 4GoB), naive lymphocytes respond relatively ineffectively to IL-2. As seen in Fig. 5Go, IL-10 marginally synergizes with the cytokine in the naive subset, yet shows a more pronounced, statistically significant (p < 0.001) effect in the memory group where IL-2 was noted to preferentially induce a vigorous proliferative response.



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  		FIGURE 5. Effect of IL-10 on IL-2 responsiveness in memory and naive B lymphocytes. Magnetically separated naive and memory tonsillar B lymphocytes (1 x 105/200 µl) were cultured in microtiter wells with either soluble anti-CD40 Ab at 5 µg/ml, insoluble anti-{kappa}/{lambda} Abs at 10 µg/ml of beads, or anti-CD40 Abs at 5 µg/ml and insoluble anti-{kappa}/{lambda} at 10 µg/ml of beads, in the presence of IL-10 ({square}), IL-2 (), or IL-2 and IL-10 ({blacksquare}). All cells were cultured for 72 h and then pulsed with 1.0 µCi [3H]thymidine for an additional 16–18 h. The data are those of a representative experiment of multiple experiments (n = 3) with triplicate wells per experimental determination.

 
Determination of message and protein for receptor constituents in memory and naive cells

To evaluate the receptor dynamics associated with those proliferation effects seen in the memory and naive subpopulations, message production was evaluated for the ligand binding proteins of interest. Table IIIGo presents message quantitation, by competitive RT-PCR, to evaluate whether differences in proliferative response patterns relate to variations in the individual components. From the information presented, it is first evident that basal message amounts for the unstimulated cellular components examined are greater in memory than naive cells (p < 0.05 for {gamma}c, IL-13R{alpha}1, and IL-2R{alpha}). Yet, for {gamma}c, IL-13R{alpha}1, and IL-4R{alpha}, the extent and magnitude of message variation is reduced in the memory cells as a function of external stimulation. Determination of amplicon numbers associated with anti-{kappa}/{lambda} plus anti-CD40 stimulation, those activation conditions associated with maximal proliferation in the presence of a single lymphokine, reveal that the IL-13R{alpha}1 values are consistent with the statistically significant differences in memory and naive cells, i.e., message for IL-13R{alpha}1 is higher in naive cells than memory cells (p < 0.05). The situation for the components of the IL-2R are the converse of that seen with IL-13. Of significant import is the observation that message for IL-2R{alpha}, ß (p < 0.01 across all stimulation conditions) and {gamma}c (p < 0.05 for unstimulated memory vs naive) are all increased under specific conditions of external activation in the memory cells. Particular attention to IL-2R{alpha}, IL-2Rß, and {gamma}c are essential in memory cells, considering the combined effect of these ligand binding chains on IL-2R affinity (47). This is exemplified by the specific evaluation of IL-2R{alpha} and IL-2Rß, which clearly show statistically significant differences (p < 0.01 for IL-2R{alpha} and p < 0.05 for IL-2Rß) concerning IL-10 synergy when comparing memory and naive cells.


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  		Table III. mRNA quantitation of the components of the IL-4/IL-13 and IL-2 receptors in naive and memory B lymphocytes

 
To continue to examine the receptor components in the memory and naive cells, Western blot analysis was performed on IL-13R{alpha}1 and IL-2Rß, the two components with the most substantial impact on their respective receptors. The variation of the immunoprecipitated proteins, shown in Fig. 6Go, is consistent with the proliferative response seen in Fig. 4Go. Naive cells exhibit a marked variability in expression of IL-13R{alpha}1, with maximal protein detected after dual surface ligation of the Ig and CD40 receptors (Fig. 6GoA, lane 3). The marked variation in protein amount is consistent with the message determinations. Memory cells (see Fig. 6GoB) demonstrated relatively constant IL-13R{alpha}1 protein without the intensive variations seen in naive cells. It should be mentioned that the Western blot analyses were performed on immunoprecipitated material derived from variable numbers of naive as opposed to memory cells to maximize protein visualization. Quantitative comparisons are therefore adequately accurate within a phenotype, yet not between phenotypes, where only broad qualitative differences may be determined. Given this caveat, examination of Fig. 6GoB demonstrates the detection of IL-2Rß in memory cells and its apparent absence in naive cells (see Fig. 6GoA). The effect of IL-10 on IL-2Rß in memory cells is consistent with the proliferative responses documented herein, which correlates well with the observed synergy between IL-10 and IL-2 in memory B lymphocytes. Flow cytometric analysis was next performed to evaluate all three IL-2R components. Table IVGo presents mean fluorescent intensity of the measured fluorescence for the entire populations scanned, which is a direct reflection of cell surface protein density. One may note that the results virtually mimic those seen from the message analysis data with the important notion that IL-2 protein for all constituents is increased in memory cells, consistent with the proliferative predominance of IL-2 on memory cells.



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  		FIGURE 6. Differential protein expression of IL-13R{alpha}1 and IL-2Rß evaluated by Western blot analysis in naive and memory B lymphocytes. Naive B lymphocytes (A) and memory B lymphocytes (B) (~8 x 107 cells) were activated in the presence of either insoluble {kappa}/{lambda} Abs (10 µg/ml beads) with or without anti-CD40 Abs (5 µg/ml) or with insoluble {kappa}/{lambda} Abs (10 µg/ml beads) plus anti-CD40 Abs (5 µg/ml) and IL-10 (20 ng/ml) for 24 h. Nonactivated cells were harvested subsequent to magnetic separation. All cells were harvested by centrifugation and proteins were solubilized in extraction buffer, precleared, and immunoprecipitated with appropriate Abs as indicated in Materials and Methods. IP proteins were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose membranes, and blotted with 1–1.5 µg/ml primary and 1:40,000–80,000 dilution of secondary Abs. Molecular weight markers were used to indicate approximate size.

 

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  		Table IV. Flow cytometric evaluation of IL-2R components in naive and memory B lymphocytes

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results presented herein have established an additional level of control for the cytokine IL-13. Cellular transmission of signals associated with IL-13 ligand binding are dependent not only upon mode of cellular activation but also upon cellular phenotype. The level of message for IL-13R{alpha}1, the ligand binding unit of the functional receptor on human B lymphocytes, markedly varies in response to surface stimulation in those lymphocytes that are quiescent at the time of external perturbation. The degree of variation is particularly marked in those unactivated naive B lymphocytes that are characterized by their CD38-/CD44+ status and their specific Ig isotype expression. Mature quiescent lymphocytes, from which these naive cells are isolated, respond with a log fold increase in IL-13R{alpha}1 message expression following Ig and CD40 ligation. The marked expansion in the message for IL-13R{alpha}1 is part of a relatively broad increase in message production, yet IL-13R{alpha}1 appears to be up-regulated to a more substantial extent. From a transcriptional perspective, IL-4R{alpha}, although responding with particular sensitivity to Ig cross-linking, appears to be limiting in amount as has been seen with endothelial cells (48). The message for {gamma}c, which varies over a more limited range, remains in relatively high concentration apparently ensuring its availability for the multiple ligand binding chains to which it participates in functional receptor formation (49). In the present context this relates to IL-4 and IL-2 responsiveness only in that numerous reports to date, including our own, have confirmed the absence of {gamma}c in most receptor configurations associated with IL-13 binding (see Introduction). Of note in the present studies is the variation in receptor message, protein, and subsequent cell proliferation associated with alterations in the method of stimulation of the CD40 receptor. It seems intuitive that utilization of the actual receptor ligand, i.e., CD40L, presented in a physiological manner, would eventuate in the most pronounced stimulation. This was in fact confirmed herein, yet the result does bring forth specific biologic issues of interest. One may initially question whether the effects of CD40L on its receptor, in and of itself, may underlie bystander proliferative effects noted in B-lymphocyte cell systems (44). It may be envisioned that B cells interacting with CD40L, in the absence of specific Ig cross-linking, would be a relatively rare event. The fate of such stimulated cells would seem to parallel those B lymphocytes, following multiple rounds of somatic mutation, that instead of binding Ag with increasing affinity, bind Ag with limited affinity and are thereupon eliminated from the Ag-specific pool. Those mature B cells binding with high affinity are destined to vigorously proliferate, with a fraction of these cells eventually entering the memory cell compartment (50).

The memory cells evaluated in the present study are interesting from multiple perspectives. These cells, as initially isolated, possess receptor message and protein amounts invariably higher than that found in naive cells. Alteration in surface phenotype, in terms of a more rapid up-regulation of specific Ags, has previously been demonstrated for memory B cells (42). In the present context, although memory cells possess elevated message and protein for multiple ligand binding chains, particular attention is directed to the IL-2R {alpha}- and ß-chains. Prior experimentation has revealed that the high affinity IL-2R is comprised of a three-chain structure composed of the IL-2R{alpha}, a 55-kDa protein that inherently binds ligand with low affinity, and the IL-2Rß, a 75-kDa protein that along with {gamma}c completes the high affinity binding unit. The memory lymphocytes evaluated herein not only possess higher unstimulated values for these components, when compared to naive cells, but these B cells also up-regulate the receptor elements to a greater extent, especially in the presence of IL-10. The differential in protein amount is underscored by Western blot analysis examining IL-2Rß protein expression. This element is undetectable in naive B lymphocytes, even when starting amounts of cells utilized for immunoprecipitation are in excess (i.e., ~100 x 106 cells per assay point), whereas it is readily apparent in memory cells. The higher initial values, and the increases associated with anti-Ig, anti-CD40, and IL-10 exposure are biologically associated with functional consequences such as increased cell proliferation. This proliferation occurs with a receptor that binds ligand in the picomolar range and results in proliferation essentially 2-fold higher than seen in naive cells. The predominance of IL-2 on memory cells is the converse of IL-13, which shows predominant proliferative effects on naive B cells. IL-13 message and protein, although increased in unstimulated memory cells, demonstrates a reduced response to external surface ligation such that essentially one-half the amount of message for IL-13R{alpha}1 is available to the memory cell. This is functionally reflected in a diminished memory cell response to IL-13. The proliferative response to IL-4 remains somewhat more intact (only reduced ~25%). The maintenance of IL-4 responsiveness may be due to its capability of transmitting a signal through a receptor composed of IL-4R{alpha} in association with either {gamma}c or IL-13R{alpha}1. Of importance to the above discussion is the caveat that in the present studies both message and protein were evaluated in a static manner, such that determinations were done at defined time intervals without associated evaluations of turnover. In addition, although the relationship between receptor message, protein amount, and eventual proliferative response has been clearly documented herein, specific examples can be found where direct linearity does not apply. In these conditions, multifactorial mechanisms, including message stability and protein turnover must be considered. Furthermore, the situation may be more complex when receptor configuration can be composed of three separate elements (e.g., IL-4 and IL-2). The apparent inverse relationship, demonstrated herein between IL-4/IL-13 vs IL-2 in memory and naive cells, is similar to the contrasting effects of IL-4 and IL-2 previously reported for specific assay conditions used to examine the proliferative responses in normal and malignant B cells (37, 51, 52, 53, 54, 55). In several prior studies, it was demonstrated that IL-4 inhibited specific incubation-dependent B cell responses to IL-2, whereas the effect of IL-4 on other B cell trophic cytokines was not apparent. The relationship between inhibitory cytokine exposure and receptor acquisition for the other lymphokines apparently accounted for some of the effects noted (53). Interestingly, this phenomenon would further exacerbate the contrasting effects noted in the present study. Further examination of the influence of each cytokine on quantitative message and protein expression should help resolve these interesting similar effects.


   Acknowledgments
 
We thank Linda Mulzer for excellent preparation of the manuscript and Deborah Morgan for technical assistance. We also thank Ajay Gulati for initial experiments utilizing magnetic separation.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant DK49649 and by funds from the Roger Williams Medical Center Department of Pathology Research Foundation. Back

2 Address correspondence and reprint requests to Dr. Abby L. Maizel, Department of Pathology, Roger Williams Medical Center, 825 Chalkstone Avenue, Providence, RI 02908. E-mail address: Back

3 Abbreviations used in this paper: {gamma}c, common {gamma}; CD40L, CD40 ligand. Back

Received for publication March 26, 1999. Accepted for publication July 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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