The CD44 variant isoforms CD44v6 and CD44v7 are expressed by distinct leukocyte subpopulations and exert non-overlapping functional activities

Simone Seiter1,2, Dirk-Steffen Schmidt2 and Margot Zöller2,3

1 Department of Dermatology, University of the Saarland, 66424 Hamburg/Saar, Germany
2 Department of Tumor Progression and Immune Defense, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
3 Department of Applied Genetics, University of Karlsruhe, 76049 Karlsruhe, Germany

Correspondence to: M. Zöller, Department of Tumor Progression and Immune Defense, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have described recently that anti-CD44s, anti-CD44v6 and anti-CD44v7 interfere with delayed-type hypersensitivity (DTH) reactions. Yet, TNBS-induced colitis can be cured only by anti-CD44v7. To clarify the mechanisms underlying the divergent functional activities of CD44v6 and CD44v7 we explored their contribution to lymphocyte activation in vivo and in vitro. CD44v6 and CD44v7 are distinctly expressed on subpopulations of activated lymphocytes. Expression of CD44v6 is mainly restricted to T cell blasts. CD44v7 has been detected on CD4+ cells, B cells and monocytes. Mitogenic and antigenic stimulation of lymphocytes in vitro was impaired in the presence of anti-CD44v6 and anti-CD44v7. Accordingly, anti-CD44v6 and anti-CD44v7 mitigated the DTH reaction in 2,4-dinitro-1-fluorobenzene-sensitized and challenged mice. However, the seemingly similar effects of CD44v6- and CD44v7-specific antibodies resulted from different activities. Anti-CD44v6 treatment led to a down-regulation of IL-2 and IFN-{gamma} production predominantly by CD8+ cells. In anti-CD44v7-treated mice expression of IL-12 was decreased. Elevated levels of IL-10 accompanied this reduction. The latter resulted from an anti-CD44v7-mediated blockade of interactions between CD4+ cells and monocytes as well as an active triggering of B cells. Thus, anti-CD44v6 and anti-CD44v7 interfere with lymphocyte activation at very specific points. CD44v6 functions predominantly at the T cell level. CD44v7 influences production of proinflammatory cytokines by B cells as well as an interaction between CD4+ cells and antigen-presenting cells. As CD44 isoforms do not differ in their intracytoplasmatic tail, the distinct activities must result from expression on different leukocyte subsets and interactions with distinct ligands.

Keywords: CD44 isoforms, co-stimulatory function, cytokines, delayed-type hypersensitivity


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD44 comprises a set of transmembrane glycoproteins, whose members differ by glycosylation (1), and by insertion of up to 10 variant exons between exon 5 and exon 6 of the CD44 standard isoform (CD44s) (2). Considering functions of CD44 on hematopoietic cells, CD44s has been described as a lymphocyte homing receptor (35). In addition, the molecule is involved in lymphocyte maturation (611), traffic (1215) and activation (1623). Knowledge of functional activity of CD44 variant isoforms v6 and v7 is still scarce. Lymphocyte activation has been shown to be accompanied by up-regulation of CD44v6 (2427) and delayed-type hypersensitivity (DTH) reactions can be mitigated by anti-CD44v6, but also by anti-CD44v7 (28,29). On the other hand, a severe pancolitis can be prevented and cured only by anti-CD44v7, while anti-CD44v6 exerts no effect at all (30). Interestingly, only peripheral blood lymphocytes of patients with a systemic autoimmune disease display increased levels of CD44v7, whereas elevated levels of CD44v6 were noted in peripheral blood lymphocytes of patients with infectious, allergic as well as autoimmune diseases (31).

The finding that anti-CD44v6 and anti-CD44v7 mitigated a Th1-induced DTH reaction, but only anti-CD44v7 interfered with a Th1-mediated gut-associated chronic inflammatory process was unexpected and prompted us to explore whether functional activities of these CD44 isoforms differ principally. We approached the question by antibody blocking studies in vitro and in vivo. Functional activity was evaluated with respect to co-stimulatory activity, modulation of activation markers and cytokine secretion. Although both CD44v6 and CD44v7 are involved in the process of lymphocyte activation, our results demonstrate that these CD44 isoforms display distinct modes of action. CD44v6 functions as a co-stimulatory molecule predominantly on T cells. CD44v7 rather appears to be a ligand for co-stimulatory molecules.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice and treatment
BALB/c mice were obtained from Wiga (Sulzfeld, Germany). They were kept under specific pathogen-free conditions and were fed sterilized food and tap water ad libitum. Animals were used for experiments at the age of 8 weeks. Contact allergy was induced by painting 20 µl of 0.5% 2,4-dintro-1-fluorobenzene (DNFB) in 4:1 acetone:olive oil on the rear footpads of mice for a successive 2 days. Four days later, mice were challenged by painting 10 µl of 0.2% DNFB to each side of both ears (32). Concomitantly with painting, mice received either rat IgG or anti-CD44s or anti-CD44v6 or anti-CD44v7 (200 µg/100 µl PBS), i.v. Ear thickness was measured 0, 24, 48 and 72 h after challenge.

mAb
The following mAb were used: anti-CD44s (IM-7, rIgG2b), anti-CD44v6 (11A6, rIgG2a; Schmidt et al., manuscript in preparation), LN7.2 (anti-CD44v7, mIgG2a) (30, Wittig et al., submitted), anti-IFN-{gamma} (R4-6A2, anti-rIgG1), anti-µ (331.12, rIgG2b), anti-CD3 (145-2C11, hIgG), anti-CD4 (YTA3.2.1, rIgG2b), anti-CD8 (YTS169.4.1, rIgG2b), anti-Mac-1 (YBM6.1.1, rIgG2a), anti-IL-2R (7D4, rIgM) and anti-ICAM-1 (YN1/1.7.4, rIgG2a). Culture supernatants were purified by passage over Protein G–Sepharose and mAb were used in vitro at a concentration of 10 µg/ml. Anti-CD40, anti-CD40L, anti-B7-1, anti-B7-2, anti-CD28, anti-CTLA-4, and pairs of anti-IL-2, anti-IL-4, anti-IL-10, anti-IL-12, anti-IFN-{gamma} and anti-tumor necrosis factor (TNF)-{alpha} were obtained commercially (PharMingen, Hamburg, Germany). For flow cytometry, FITC- and phycoerythrin (PE)-labeled isotype-specific secondary antibodies were used. For FACS analysis 3–5x105 cells were stained according to routine procedures. When assaying cytokine-producing cells, lymphocytes were fixed and permeabilized in advance. As far as they had been stimulated in vitro, 5 µM monensin was added to the culture medium during the last 8 h of culture. Fluorescence was determined with an Epics XL (Coulter, Krefeld, Germany) or a FACSCalibur (Becton Dickinson, Heidelberg, Germany).

Lymphocyte preparation and activation
Ears, draining lymph nodes (drLN) and the spleen were excised, and peritoneal exudate cells (PEC) were washed out. Ears were split and put with the inner face down in 1% trypsin for 3 times 20 min at 37°C. LN and spleen were meshed through fine gauze. Subpopulations of lymphocytes were enriched by panning on anti-mouse IgM-coated plates (33). The non-adherent population was defined as T cell enriched and the adherent population as B cell enriched. To separate CD4+ and CD8+ cells, the T cell-enriched population was incubated with anti-CD4 or anti-CD8. After washing, cells were seeded on anti-rat IgG-coated plates. The adherent cells were collected after 2 h of incubation.

Mitogenic and antigenic stimulation was performed with 7.5µg/ml concanavalin A (Con A), 25 µg/ml lipopolysaccharide (LPS), 100 µg/ml DNP-ovalbumin (OVA) or with DNFB haptenized syngeneic lymphocytes. Cells were cultured for 1–3 days. Thereafter, surface markers and cytokine production were determined. Alternatively, 10 µCi/ml [3H]thymidine was added for the last 16 h of culture. Cells were harvested with an automatic harvester and thymidine incorporation was determined in a ß-counter. [3H]Thymidine incorporation is presented as proliferation index, whereby [3H]thymidine incorporation in the presence of control (mouse and/or rat) IgG has arbitrarily been taken as 1.0

ELISAspot assay (34)
Plates were coated with anti-IL-2, anti-IL-4, anti-IL-10, anti-IFN-{gamma} and anti-TNF-{alpha} in bicarbonate buffer, pH 8.9. Plates were washed and blocked with 100 µg/ml BSA. Cells were added to the coated plates. Plates were incubated for 18–24 h at 37°C, cells were lysed, plates were washed 3 times and 50 µl biotinylated antibody was added for overnight at 4°C. After four washings, the enzyme streptavidin–alkaline phosphatase was added in PBS–gelatin–Tween 20. Plates were incubated for 45 min at 37°C. Thereafter they were washed 5 times before adding the substrate in 1% agar. Spots were counted after incubation at 37°C for 4 h.

Statistics
Significance of differences were calculated by the two-tailed Student's t-test.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression profile of CD44v6 and CD44v7 on lymph node cells
CD44v6 and CD44v7 are not expressed on resting LNC, but become transiently up-regulated during lymphocyte activation, although with different time kinetics. CD44v6 is predominantly detected on lymphoblasts; expression of CD44v7 is strongest prior to blast transformation. In comparison to CD44s, the intensity of CD44v6 and CD44v7 expression is weak (28). As revealed by fluorescence analysis of subpopulations of activated leukocytes (Table 1Go) and double fluorescence analysis (Fig. 1Go), CD44v6 is mainly expressed on T cell blasts, whereas CD44v7 is expressed by CD4+ cells, B cells and monocytes.


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Table 1. Subpopulations of in vivo activated drLNC expressing CD44v6 and CD44v7
 


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Fig. 1. Expression of CD44v6 and CD44v7 on leukocyte subpopulations. (A) drLNC of BALB/c mice were collected 5 days after sensitization with DNFB. LNC were stained with biotinylated anti-CD44v6 and anti-CD44v7, and counterstained with streptavidin–PE. A single parameter overlay is shown. White area, negative control; grey area, anti-CD44v7; black area, anti-CD44v6. (B) LNC were cultured in the presence of Con A (7.5 µg/ml), and SC as well as PEC in the presence of LPS (25 µg/ml). LNC were stained with anti-CD4 or anti-CD8, SC with anti-µ and PEC with anti-CD11b as first antibody and FITC-labeled anti-rat IgG as second antibody (x-axis). All probes were stained with either biotinylated anti-CD44v6 or anti-CD44v7 and were counterstained with streptavidin–PE (y-axis). The negative control has been treated with rat IgG as control for the first antibody, FITC-labeled anti-rat IgG and streptavidin–PE. The population of large cells (LNC and SC) and of large and more granulated cells (PEC) has been gated. Anti-CD44v6 preferentially stained T cells and a fraction of monocytes; anti-CD44v7 stained part of CD4+ cells, B cells and monocytes.

 
Blocking of lymphocyte activation in vitro by anti-CD44v6 and anti-CD44v7
Because CD44v6 and CD44v7 are hardly expressed on resting lymphocytes, it could be assumed that these molecules are functionally active during lymphocyte stimulation. Therefore, we first evaluated whether anti-CD44v6 and anti-CD44v7 interfere with lymphocyte proliferation (Fig. 2Go). Addition of anti-CD44 to the culture medium, indeed, resulted in a blockade of the proliferative response of spleen cells (SC) towards the nominal antigen DNP-OVA. Although the mitogenic response of SC to Con A and LPS was hardly influenced by the anti-CD44v antibodies, anti-CD44s as well as anti-CD44v6 and anti-CD44v7 inhibited the response of B cell-depleted SC towards Con A. Anti-CD44s and anti-CD44v7, but not anti-CD44v6, also inhibited the LPS response of T cell depleted SC.



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Fig. 2. Blockade of mitogenic and antigenic responses by anti-CD44. Unseparated SC and SC depleted either of T cells or of B cells (2.5x105 / well) were stimulated in vitro by Con A (7.5 µg/ml), LPS (25 µg/ml) or DNP-OVA (100 µg/ml). Culture medium contained in addition either control (mouse plus rat) IgG or anti-CD44s or anti-CD44v6 or anti-CD44v7 (10 µg/ml). Cells were cultured for 72 h adding [3H]thymidine during the last 16 h of culture. Proliferation indices as compared to Con A or LPS or DNP-OVA plus control IgG (defined as 1.0) are shown. Values represent the mean + SD of five experiments. Significance of differences indicated by asterisks: *P < 0.1, **P < 0.01, ***P < 0.001.

 
The anti-CD44 treatment also exerted some effect on the expression of co-stimulatory molecules or their ligands (Table 2Go), e.g. expression of the IL-2R, of CD40 and of B7-1 and B7-2 was reduced in anti-CD44s-treated cultures; anti-CD44v6 treatment led to a reduction in IL-2R expression; in the presence of anti-CD44v7 expression of CD40L was significantly lower than in control cultures.


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Table 2. Expression of lymphocyte activation markers in the presence of anti-CD44v6 and anti-CD44v7
 
These findings provided a first hint that anti-CD44v6 might display functional activity at the T cell level, while anti-CD44s and anti-CD44v7 might function both at the T cell and at the B cell level.

Anti-CD44v6 and anti-CD44v7 mitigate a DNFB-mediated DTH reaction
BALB/c mice were sensitized and challenged by painting with DNFB in an oil:acetone mix. Mice concomitantly received i.v. injections of anti-CD44 antibodies. As has been reported, treatment with anti-CD44s strongly reduced edema formation and retarded the infiltration of leukocytes (13,35). Anti-CD44v6 and anti-CD44v7 had less effect on edema formation (28) (Fig. 3AGo). However, a significantly reduced number of leukocytes was recovered from the injured ear (Fig. 3BGo) and expansion of lymphocytes in the drLN was slightly impaired (Fig. 3CGo). Furthermore, after sensitization and challenge with DNFB, control mice frequently appeared rather exhausted with weight loss, fever and fatigue. Occasionally local Schwartzmann reactions were noted (data not shown) and beyond 3 days after the challenge drLN frequently were emaciated. Because wasting symptoms were only seen in animals not receiving anti-CD44, it is obvious that the antibodies efficiently interfered with pathological over-reactivity of the immune system.




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Fig. 3. Influence of anti-CD44v6 and anti-CD44v7 on lymphocyte expansion and migration in a DNFB induced DTH reaction. (A) Ears were fixed in formaldehyde 2 days after DNFB challenge. Hematoxylin & eosin-stained 5 µm paraffin sections of ears from mice receiving control (mouse plus rat) IgG (a), anti-CD44s (b), anti-CD44v6 (c) and anti-CD44v7 (d) are shown. (B) Leukocytes infiltrating the ear and (C) drLNC and were collected 1–3 days after challenge with DNFB. The mean recovery of cells ± SD of five mice per group is shown. Significance of differences indicated by asterisks: *P < 0.1, **P < 0.01, ***P < 0.001.

 
Furthermore, the lymphocyte subset distribution of the infiltrate differed (Table 3Go). In anti-CD44s-treated mice, the proportion of monocytes was reduced. Anti-CD44v6 treatment led to a strong reduction of CD8+ cells. In anti-CD44v7-treated mice the infiltrate contained fewer CD4+ cells.


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Table 3. Leukocyte subset distribution in the infiltrate of DNFB-sensitized and challenged mice
 
The changes in the numbers of infiltrated cells and in the relative frequencies of leukocyte subsets in the infiltrate could either reflect a selective inhibition of the activation of leukocyte subsets by CD44 isoform-specific antibodies or be due to an effect of anti-CD44 antibodies on leukocyte migration and/or recruitment. Anti-CD44s is known to intervene with leukocyte activation as well as migration (15,19). Since anti-CD44v6 and anti-CD44v7 do not influence adhesion to hyaluronan (data not shown), we hypothesized that these antibodies may preferentially interfere with leukocyte activation.

Impact of anti-CD44 on cytokine production
To support the hypothesis that anti-CD44v6 and anti-CD44v7 selectively inhibit the activation of leukocyte subsets, cytokine expression was evaluated in unseparated leukocyte populations as well as in defined subpopulations and in mixtures of subpopulations. Cytokine (IL-2, IFN-{gamma}, IL-12 and TNF-{alpha}) expression was determined by flow cytometry, cytokine secretion by ELISAspot and cytokine production by ELISA.

In the first setting, LNC from untreated donors were stimulated in vitro by DNP-OVA in the presence of CD44-specific antibodies and the percentage of cytokine-expressing cells was evaluated in comparison to cultures containing control IgG (Fig. 4AGo). The strongest reduction in cytokine expression was seen with anti-CD44s, which inhibited the expression of all four cytokines. Anti-CD44v6, instead, interfered exclusively with IL-2 and IFN-{gamma} expression. Anti-CD44v7 efficiently modulated TNF-{alpha}, IL-12 and IFN-{gamma} expression, but had no effect on IL-2. The finding that anti-CD44v7 more efficiently hampered expression of the proinflammatory cytokines IL-12 and TNF-{alpha} points towards CD44v7 being functionally active at the antigen-presenting cell rather than the T cell level.



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Fig. 4. Influence of anti-CD44v6 and anti-CD44v7 on cytokine expression and secretion of in vitro and in vivo stimulated lymph node cells. (A) LNC from untreated mice were stimulated in vitro with DNP-OVA in the presence of control (mouse plus rat) IgG, anti-CD44s, anti-CD44v6 and anti-CD44v7 (10 µg/ml). Cells were stimulated for 3 days adding monensin (5 µM/ml) during the last 8 h of culture. (B) Mice were sensitized by painting the footpads with DNFB at day 0 and 1. At day 0 and day 3 mice received an i.v. injection of 200 µg/200 µl control IgG, anti-CD44s, anti-CD44v6 and anti-CD44v7 respectively. drLN were collected after 5 days. (C and E) drLNC and (D) leukocytes infiltrating the ear were isolated 2 days after a challenge with DNFB (7 days after priming). Mice received three i.v. injections of antibodies, as described above. (A–D) The percentage of IL-2, IFN-{gamma}, IL-12 and TNF-{alpha} expressing cells was determined by flow cytometry, and (E) the frequency of actively secreting cells by ELISAspot. The mean percentage of stained cells/number of spots + SD of three in vitro experiments (A) and of four (B) and five (C–E) in vivo experiments are shown. Significance of differences indicated by asterisks: *P < 0.1, **P < 0.01, ***P < 0.001.

 
The interpretation was strengthened by the analysis of cytokine expression in drLNC after sensitization with DNFB under concomitant treatment with CD44-specific antibodies (Fig. 4BGo), and in the state of hyper-reactivity in DNFB sensitized and challenged mice (Fig. 4C–EGo). In the in vivo situation, too, anti-CD44s and anti-CD44v6 exerted a strong effect on IFN-{gamma} expression. Anti-CD44v7 again modulated preferentially IL-12 expression and, possibly as a consequence thereof, IFN-{gamma} expression. TNF-{alpha} expression was reduced in sensitized mice, yet was unaltered in the state of hyper-reactivity. Treatment with anti-CD44v6 and anti-CD44v7 exerted similar effects on leukocytes infiltrating the ear as described for the drLNC. Instead, the effect of anti-CD44s on cytokine expression by infiltrated cells was less impressive (Fig. 4DGo). As shown for drLNC, cytokine secretion did not differ significantly from the pattern of cytokine expression (Fig. 4EGo). This also accounted for cytokine production as determined by ELISA (data not shown).

Thus, anti-CD44s, anti-CD44v6 and anti-CD44v7 efficiently interfered with cytokine expression, production and secretion, but the three antibodies differed with respect to the predominantly blocked cytokine. Furthermore, in a pathological state of hyper-reactivity, the effect of anti-CD44s was stronger in drLNC than locally, whereas in anti-CD44v6- and anti-CD44v7-treated mice similar effects were observed on infiltrated cells and drLNC. We interpreted the findings in the sense that (i) CD44s, but not CD44v6 and CD44v7, influenced leukocyte migration, and (ii) anti-CD44s, anti-CD44v6 and anti-CD44v7 interfered with different leukocyte subsets.

Anti-CD44v6, but not anti-CD44v7, directly blocks cytokine expression by T cells.
To answer the question whether anti-CD44v6 and anti-CD44v7 directly block the activation of defined leukocyte subsets or interfere with a cell–cell interaction, drLNC from DNFB sensitized BALB/c mice, which had received either control IgG or anti-CD44v6 or anti-CD44v7, were separated into populations enriched for CD4+ cells, CD8+ cells, B cells and monocytes. The separated populations were maintained in culture for 24 h in the presence of anti-CD44v6 or anti-CD44v7 and tested for cytokine expression (Fig. 5AGo). Treatment with anti-CD44v6 influenced IL-2 and IFN-{gamma} expression by CD4+ and CD8+ cells. Besides, TNF-{alpha} expression by monocytes was reduced. The cytokine expression profile was different in the presence of anti-CD44v7. The antibody had no influence on cytokine expression by CD8+ cells. However, cytokine expression by CD4+ cells, B cells and monocytes was altered in the presence of anti-CD44v7. Notably, the influence of anti-CD44v7 on cytokine expression by these leukocyte subpopulations was not uniform: expression of IFN-{gamma} by CD4+ cells and monocytes was decreased; the percentage of monocytes producing TNF-{alpha} also was decreased and fewer B cells expressed IL-12. On the other hand, the number of monocytes expressing IL-12 was slightly increased. As evaluated by ELISAspot and ELISA, cytokine expression corresponded to cytokine secretion (Fig. 5BGo) and cytokine production (data not shown). Thus, anti-CD44v6 and anti-CD44v7 clearly influenced distinct leukocyte subpopulations.



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Fig. 5. Cytokine expression by subpopulations of leukocytes. Mice were sensitized with DNFB and received two i.v. injections of 200 µg/200 µl rat IgG or anti-CD44v6 or anti-CD44v7. drLNC as well as PEC were collected after 5 days. CD4+ cells, CD8+ cells, B cells and monocytes were enriched as described in Methods. Cells were cultured for 24 h. Cytokine expression was evaluated by flow cytometry (A) and cytokine secretion by ELISAspot (B). The mean percentage of stained cells + SD of three experiments is shown. Significance of differences indicated by asterisks: *P < 0.1, **P < 0.01, ***P < 0.001.

 
To evaluate whether the antibodies also exerted an influence on the interaction between different subpopulations, CD8+ cells were co-cultured with CD4+ cells and CD4+ cells as well as B cells were co-cultured with monocytes. Cytokine expression by the distinct subpopulations was evaluated by double fluorescence staining. The results are shown in Table 4Go and examples are presented in Fig. 6Go. When CD8+ cells were cultured together with CD4+ cells in the presence of anti-CD44v6 a reduced percentage of CD8+ cells produced IL-2 and IFN-{gamma}. The percentage of reduction by anti-CD44v6 was similar in cultures containing only CD8+ cells or CD8+ cells plus CD4+ cells. The same observation did hold true for cytokine production by CD4+ cells, i.e. anti-CD44v6 exerted a similar effect on cytokine expression by CD4+ cells cultured separately or together with CD8+ cells (Table 4AGo). In the next setting CD4+ cells were cultured together with monocytes. In the presence of anti-CD44v7, CD4+ cells produced TNF-{alpha} and IL-12 at a reduced level. This was not observed when CD4+ cells were cultured separately (Table 4BGo and Fig. 6Go). When B cells were cultured with monocytes in the presence of anti-CD44v7, TNF-{alpha} production was reduced and IL-12 production was more strongly reduced than in cultures containing only B cells (Table 4BGo). With respect to the decreased expression of IL-12 by CD4+ cells and B cells which have been cultured with monocytes in the presence of anti-CD44v7 it should be remembered (Fig. 5Go) that expression of IL-12 in monocytes was up-regulated by anti-CD44v7.


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Table 4. Anti-CD44v7 blocks an interaction between monocytes and CD4+ cells/B cells
 


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Fig. 6. Influence of anti-CD44v6 and anti-CD44v7 on cytokine expression by leukocyte subpopulations and mixtures of leukocyte subpopulations. drLNC and PEC were collected from DNFB-sensitized mice, which had received two i.v. injections of 200 µg/200 µl rat IgG or anti-CD44v6 or anti-CD44v7. Subpopulations were enriched as described in Methods and were separately or together maintained in culture for 24 h. Thereafter the non-adherent cells were collected. Cytokine expression was evaluated by double fluorescence analysis. Cells were first stained with biotinylated cytokine-specific antibodies and were counterstained with streptavidin–PE (y-axis). Thereafter cells were stained with anti-CD8-FITC, anti-CD4-FITC or anti-IgM–FITC (x-axis). The negative controls have been treated with rat IgG as control for the first antibody, streptavidin–PE and FITC-labeled anti-rat IgG. (A) Influence of anti-CD44v6 on IL-2 and IFN-{gamma} expression by CD8+ cells cultured separately or together with CD4+ cells. (B) and (C) Influence of anti-CD44v7 on IL-12 and TNF-{alpha} expression by CD4+ cells (B) and by B cells (C) cultured separately or together with monocytes.

 
These findings gave an independent confirmation of our hypothesis that the distinct effects of anti-CD44v6 and anti-CD44v7 are due to the involvement of different leukocyte subpopulations. Besides, the partly opposing effects of anti-CD44v7 on enriched populations of CD4+ cells, B cells and monocytes versus mixtures of these populations supported the idea that anti-CD44v7 may affect not only the CD44v7 receptor bearing cell but, in addition and by preventing an interaction with the so far undefined ligand, the ligand bearing population.

Anti-CD44v7 apparently interferes with signaling from sensitized monocytes towards B cells and Th cells
If the latter hypothesis holds true and CD44v7 functions as a receptor on monocytes for a ligand expressed by CD4+ cells and B cells, CD4+ cells and B cells should respond differently to monocytes from DNFB- versus monocytes from DNFB plus anti-CD44v7-treated mice. To answer the question, B cells or CD4+ cells were cultured with monocytes from untreated, DNFB-treated and DNFB plus anti-CD44v7-treated mice collecting only the non-adherent cells for the determination of cytokine expression (Fig. 7Go).



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Fig. 7. Influence of anti-CD44v7 on interactions between monocytes, B cells and Th cells. LNC and PEC were collected from unsensitized, DNFB-sensitized and DNFB-sensitized plus anti-CD44v7-treated mice. Mice of the first two groups received mouse IgG as a control to the anti-CD44v7 antibody. CD4+ cells, B cells and monocytes were enriched as described in Methods. Subpopulations of cells were co-cultured for 24 h. Thereafter only the non-adherent cells were collected and cytokine expression was evaluated by flow cytometry. The contamination with monocytes (CD11b+) as revealed by flow cytometry was in the range of 3–5%. (A) IL-12- and TNF-{alpha}-expressing B cells in co-cultures with monocytes. (B) IL-10-expressing B cells and CD4+ cells in co-cultures with monocytes The mean percentage of cytokine-expressing cells in five independently performed experiments is shown. SD were in the range of 4–10%. Corresponding results were obtained in an additional experiment where cytokine expression in B cells and CD4+ cells was determined by double fluorescence staining (data not shown).

 
When B cells were cultured with monocytes from untreated or DNFB-treated mice, TNF-{alpha} and IL-12 expression was unaltered or increased. Instead, when monocytes were derived from DNFB plus anti-CD44v7-treated mice, less B cells expressed IL-12 and TNF-{alpha}. This was independent of the activation state of the B cells, i.e. it was seen with B cells from untreated and DNFB-sensitized mice. The effect was also independent on whether or not the B cells had already been in contact with anti-CD44v7 (Fig. 7AGo). From this experiment it becomes apparent that anti-CD44v7 blocked an interaction between monocytes and B cells which normally stimulates IL-12 and TNF-{alpha} production by the B cells. Since a purified population of B cells from DNFB and anti-CD44v7-treated mice produced high levels of IL-12 and TNF-{alpha}, it must be the monocyte which is directly affected by anti-CD44v7. Yet, the effect becomes apparent at the (ligand bearing) B cell.

Since IL-10 and IL-12 have been described to be of mutual counteracting influence, we finally evaluated IL-10 expression in co-cultures of B cells or CD4+ cells with monocytes (Fig. 7BGo). IL-10 expression in B cells was strongly reduced in the presence of monocytes from DNFB-treated as compared to monocytes from untreated mice. In the presence of monocytes from DNFB plus anti-CD44v7-treated mice, IL-10 expression by B cells was restored. Also, a higher percentage of CD4+ cells expressed IL-10 when cultured with monocytes from DNFB plus anti-CD44v7-treated mice. The phenomenon, again, was independent of whether or not the B cell and the CD4+ cell had been in contact with anti-CD44v7. Thus, the experiment confirms that CD4+ cells and B cells are released from suppression to produce IL-10 by blocking CD44v7 on monocytes. Finally, it should be noted that the percentage of IL-10-expressing B cells as well as CD4+ cells was increased in DNFB plus anti-CD44v7-treated mice (Fig. 7A and BGo). Whether the up-regulation of IL-10 expression by CD4+ cells and by B cells is a consequence of the reduced expression of IL-12 and TNF-{alpha} or is an independent phenomenon will be discussed.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Several other groups and ourselves have described the involvement of CD44v6 in lymphocyte activation (2427). In view of this evidence we were surprised to notice that anti-CD44v6 treatment was ineffective in a TNBS-induced colitis, while a complete cure could be achieved with anti-CD44v7 (30). The divergent effects of anti-CD44v6 and anti-CD44v7 were not restricted to the mucosa-associated immune system, e.g. anti-CD44v6 and anti-CD44v7, like anti-CD44s, also mitigated a Th1-mediated DTH reaction. Yet, anti-CD44v6 exerted only a minor effect on a FITC-induced, supposedly Th2-mediated DTH reaction (28). To attain further hints as to the cause of the distinct effects of anti-CD44v6 and anti-CD44v7 we evaluated the influence of these two antibodies on leukocyte activation and cytokine expression in a DNFB-induced DTH reaction. Our data support the idea that the distinct activities of CD44v6 and CD44v7 are due to different expression patterns. In addition, blocking of CD44v7 does not only alter functional activity of the CD44v7 receptor bearing cell, but also has bearing on cells expressing a so far undefined CD44v7 ligand.

Though the main objective of our study was a definition of functional differences between CD44v6 and CD44v7, we briefly want to comment on the effects mediated by anti-CD44s. There are numerous reports claiming that CD44s can function as a co-stimulatory receptor (19,21,37) as well as a ligand for a co-stimulatory molecule (38) in lymphocyte activation. In addition, there are reports on the involvement of CD44s in DTH reactions (13,28,29,35,39), i.e. blockade of a CD44–hyaluronic acid interaction by anti-CD44s inhibits the migration of monocytes into and within the site of inflammation (39). Furthermore, it has been described before in an elegant study by Grendele et al. (15) that CD44s plays an important role in leukocyte extravasation. The relative reduction of monocytes seen in the infiltrate of anti-CD44s-treated mice is in line with these studies. In addition, the most strongly reduced edema formation in anti-CD44s-treated mice may be a consequence of the impaired activation of monocytes and a reduction in monocyte-derived mediators. Finally, we want to comment on the fact that the number of leukocytes in the injured organ has been reduced, but cytokine expression by the infiltrated cells has been unimpaired. This finding, too, supports the interpretation that anti-CD44s interferes directly with the process of leukocyte emigration, i.e. the few emigrating cells are functionally unaltered. Notably, the phenomenon has not been seen in anti-CD44v6 and anti-CD44v7-treated mice, where lymphocytes in the draining node and in the infiltrate displayed concordant activity profiles. Thus, these CD44 isoforms may not contribute to leukocyte emigration.

CD44v6, originally described as a marker of metastasizing tumor cells in the rat (40), has soon after its discovery been associated with lymphocyte activation (41) and has been supposed to be involved in clonal expansion of T cells during antigenic stimulation (24). The finding that expression of CD44v6 is up-regulated in human autoimmune diseases like glomerulonephritis, diabetes, rheumatoid arthritis and chronic inflammatory bowel disease (26,4244) is well in line with an involvement of CD44v6 in lymphocyte activation.

CD44v6 is predominantly expressed on T cells. Blockade of CD44v6 inhibits T cell proliferation, but has no influence on B cell proliferation. Anti-CD44v6 has no major influence on the expression of additional co-stimulatory molecules and their ligands like B7-1/B7-2–CD28/CTLA-4 or CD40–CD40 ligand. In vitro as well as in vivo, production and secretion of IL-2 and IFN-{gamma} are impaired in the presence of anti-CD44v6, while expression of the proinflammatory cytokine IL-12 is unimpaired. The reduced production of TNF-{alpha} by monocytes could be a consequence of the reduced amount of IFN-{gamma}, which is known to stimulate TNF-{alpha} production by monocytes. In addition, a reduced number of CD8+ cells has been recovered from the infiltrate in DNFB-painted mice. These findings are in line with the interpretation that CD44v6 is predominantly involved in the activation of cytolytic T cells. So far, we do not know the signals triggered by CD44v6 ligation. Irrespective of this open question, the data demonstrate that the functional principle of CD44v6 differs from that of CD44s. Whereas CD44s–CD44s ligand interactions can initiate signaling in both the CD44s and the CD44s ligand-bearing cells (19,21,37,38), occupancy of CD44v6 by the antibody apparently influenced only the CD44v6-expressing T cell. Interestingly, a recent report described that priming of CD8+ cells in DTH reactions occurs independently of priming of CD4+ cells (45). The finding that anti-CD44v6 interfered predominantly with the CD8+ population could well explain that anti-CD44v6 mitigated a DNFB-induced, but not a TH2-mediated FITC-induced DTH reaction (28) and that it failed to modulate a Th1-mediated TNBS induced colitis (30).

Expression of CD44v7 differs from expression of CD44v6, i.e. CD44v7 is expressed on small subpopulations of activated CD4+ cells, B cells and monocytes. Similar to anti-CD44v6, anti-CD44v7 mitigated a Th1-mediated DTH reaction. When lymphocytes were stimulated in the presence of anti-CD44v7, expression of CD40 ligand and occasionally (data not shown) of CD40 was reduced. Anti-CD44v7-induced changes in cytokine expression were rather complex in that a strong decrease in the proinflammatory cytokine IL-12 and a less impressive reduction of IFN-{gamma} was observed, while expression of IL-2 was unaltered. The effect was seen in the drLN as well as in infiltrated cells, the infiltrate containing a significantly reduced number of CD4+ cells. An analysis of cytokine expression by leukocyte subpopulations revealed that subpopulations responded differently to anti-CD44v7. Two observations need particular attention.

First, DNFB treatment led to a strong up-regulation of IL-12 expression by monocytes. B cells cultured with monocytes from DNFB-treated mice also produced increased levels of TNF-{alpha} and IL-12. Anti-CD44v7 did not prevent up-regulation of IL-12 expression by monocytes. However, B cells which had been cultured together with monocytes in the presence of anti-CD44v7 produced IL-12 and TNF-{alpha} at a reduced rate. This implies that anti-CD44v7 blocked monocytes to deliver a signal towards B cells which initiates TNF-{alpha} and IL-12 production.

Second, after DNFB plus anti-CD44v7 treatment, B cells and CD4+ cells expressed increased levels of IL-10. When B cells and CD4+ cells had been cultured with monocytes from DNFB-treated mice, they produced strongly reduced levels of IL-10. When monocytes were treated with anti-CD44v7, IL-10 production by CD4+ cells and B cells was restored or even up-regulated. It has been described recently that a population of CD4+ cell, which is naturally suppressed by IL-12, starts to produce IL-10 upon release of suppression (46). This IL-10 producing CD4+ cells efficiently down-modulate overshooting Th1 reaction (46). Similarly, we noted that in co-cultures of monocytes with CD4+ cells the latter produced increasing amounts of IL-10 in the presence of anti-CD44v7. The presence of anti-CD44v7 also restored IL-10 production by B cells co-cultured with monocytes. There are three possible interpretations of these findings. (i) Anti-CD44v7 blocks a monocyte–B cell interaction. Our data are in line with this interpretation, i.e. that the mitigating effect of anti-CD44v7 in the DTH reaction is based on the down-modulation of TNF-{alpha} and IL-12 expression by B cells, and as a consequence the down-modulation of IFN-{gamma} by CD4+ cells. It should, however, be noted that the level of IL-12 expression did not fall short of the level in untreated mice. (ii) Alternatively, monocytes actively block IL-10 expression by CD4+ cells and anti-CD44v7 interferes with this blockade. The observation that an increased percentage of CD4+ cells cultured with monocytes from DNFB plus anti-CD44v7-treated mice expressed IL-10 would be in line with this interpretation. (iii) Anti-CD44v7 provides a direct trigger for B cells to express IL-10. Increased IL-10 expression by B cells treated with anti-CD44v7 has, indeed, been observed in the DNFB-induced DTH and the TNBS-induced colitis model (30). Furthermore, it has been shown that CD44 variant isoforms are up-regulated on activated dendritic cells and that antibodies against CD44 induce aggregation, up-regulation of accessory molecules and cytokine secretion in activated DC (47). Taking our three hypotheses, we favor the idea that anti-CD44v7 exerts a dualistic function, i.e. provides a trigger for B cells and releases CD4+ cells from suppression by monocytes. Further experiments are required to elucidate whether CD44v7, indeed, fulfills distinct functions at the level of monocytes versus CD4+ cells and B cells. It may be mentioned in this context that such distinct effects of an adhesion molecule would be nothing peculiar for CD44v7, but are well documented for integrins known to mediate both signaling in and signaling out (48). Definition of the ligand and of CD44v7-initiated signaling may help to answer the open question.

In summary, although anti-CD44v6 as well as anti-CD44v7 inhibited lymphocyte activation in vitro and mitigated a Th1-mediated DTH reaction, the two antibodies distinctly hampered leukocyte activation. Whereas anti-CD44v6 blocked a co-stimulatory molecule (CD44v6) on T cells, anti-CD44v7 either interfered with signal transduction in the ligand as well as the receptor bearing cell or it exerted, depending on the CD44v7+ leukocyte subpopulation, agonistic versus antagonistic activities. So far, our data do not allow differentiating between these alternatives. Yet, both models provide an explanation how splice variants in the extracellular part of a given cell surface molecule can be involved in distinct signaling pathways.


    Acknowledgments
 
We thank Dr U. Günthert, Basel Institute for Immunology, Basel, Switzerland, for the CD44v7 specific hybridoma. This work was supported by the Deutsche Forschungsgemeinschaft, grant no. Zo40-5/3.


    Abbreviations
 
CD44s CD44 standard isoform
CD44v CD44 variant isoform
Con A Concanavalin A
DNFB 2,4-dinitro-1-fluorobenzene
drLNC draining lymph node cells
DTH delayed-type hypersensitivity
LPS lipopolysaccharide
OVA ovalbumin
PE phycoerythrin
PEC peritoneal exudate cell
SC spleen cell
TNF tumor necrosis factor

    Notes
 
Transmitting editor: C. Martinez-A

Received 23 April 1999, accepted 29 September 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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