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Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and lymphotoxin, or type 2 CD4 T cells (Th2), which
produce IL-4, IL-5, IL-9, IL-13, and other cytokines. Th1
differentiation hinges on the presence of IL-12 during initial priming,
whereas Th2 differentiation requires the presence of IL-4 (1, 2). The
source of this early IL-4 is unclear, but eosinophils (3), basophils
(4), memory T cells (5), NK1.1+ CD4 T cells (6), and 
T cells (7) have been implicated. Recently, it has been shown that
addition of IL-6 to naive T cells during initial priming in vitro
promotes the production of low levels of IL-4, which in an autocrine
fashion drives Th2 differentiation (8). IL-6 is produced by a wide variety of cells including macrophages, T lymphocytes, endothelial cells, hepatocytes, and fibroblasts (9, 10). This cytokine plays a role in the terminal differentiation of B cells (11); promotes proliferation of endothelial cells (12), T cells (9), and plasmablastic cells (13); stimulates the production of acute phase proteins by hepatocytes (14); is a differentiation factor for CTL (15); and can act in an anti-inflammatory capacity (16). The finding that IL-6 can promote the production of IL-4 by ThP cells in vitro adds another facet to the diverse functional repertoire of IL-6. With the development of mice with a genetically disrupted IL-6, a greater understanding of the role of IL-6 during Th differentiation in vivo is possible.
The eggs of the parasitic helminth Schistosoma mansoni are potent inducers of a Th2 responses (17). We have used schistosome eggs to study the induction and development of Th2 responses, and in a model in which eggs were injected into the peritoneal cavity we have observed the very early production of IL-4 by eosinophils (3). This cell type is recruited to the peritoneal cavity in an IL-5-dependent, T cell-independent fashion (3, 18). We have postulated that the early eosinophil-derived IL-4 plays a role in pushing the responding Th population along the Th2 differentiation pathway. However, IL-5-/- mice, in which early IL-4 production in response to i.p.-injected schistosome eggs is absent, continue to be able to mount a robust Th2 response.4 These studies indicate that the IL-5/eosinophil-dependent egg stimulated early IL-4 is not essential for Th2 response induction.
To better understand how schistosome eggs initiate the development of a type 2 response in the host, we have investigated the possibility that IL-6, produced in response to schistosome eggs, can promote the initial production of IL-4 by ThP cells that, in an autocrine fashion, drives differentiation into Th2 cells. In this report we show that IL-6 is rapidly produced in response to schistosome eggs, but that the egg-induced early production of IL-4 by CD4 T cells persists in the absence of IL-6 and that the development of a type 2 T cell response is not abolished in IL-6-/- mice. Although IL-6 does not appear to be required during in vivo T cell differentiation, it plays an important function in in vitro T and B cell proliferation and in B cell responsiveness following immunization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to eight-week old, male or female, wild type (WT) hybrid (C57BL/6 x SV129) (#101045) and (C57BL/6 x SV129) IL-6-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME). S. mansoni (Puerto Rican strain NMRI) eggs were isolated from the livers of infected mice, washed extensively, resuspended at 50,000 eggs/ml in low endotoxin PBS (Sigma, St. Louis, MO), and stored at -70°C until used as previously described (19, 20). Mice were injected with 50 µl of egg suspension or with an equal volume of PBS per hind footpad.
Abs, Ags, and reagents
FITC- and PE-labeled anti-CD8, PE-labeled CD4, FITC- and PE-labeled anti-B220, and biotin-labeled anti-IL-2R p55 Abs were purchased from PharMingen (San Diego, CA). Streptavidin-PE was purchased from Jackson ImmunoReseach (West Grove, PA). Rat anti-IL-4R IgG1 mAb M1 was generously donated by Immunex (Seattle, WA) and used at 2.5 µg/ml. Plate-bound anti-CD3 mAb (PharMingen) was used at 1 µg/well.
rIL-6 (Intergen, Purchase, NY) was used at 1–10 ng/ml in in vitro cultures. Soluble egg Ag (SEA) was prepared as previously described (19, 20) and used at 50 µg/ml. LPS was purchased from Sigma and used at 5 µg/ml. CFSE (5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester) was purchased from Molecular Probes (Eugene, OR) and used at 2.5 µM.
Lymph node (LN) cell preparation and proliferation
Popliteal LN were harvested from egg- or PBS-injected mice, and single cell suspensions prepared using sterile 70-µm nylon sieves (Falcon, Franklin Lakes, NJ) as previously described (21). LN cells were resuspended at 5 x 106/ml in complete T cell medium containing DMEM (Life Technologies, Gaithersburg, MD), 10% FCS (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies), 10 mM HEPES (Life Technologies), L-glutamine (Life Technologies), and 5 x 10-5 M 2-ME (Sigma). Cells (106/well) were cultured in 96-well flat-bottom plates (Falcon) at 37°C/5% CO2. Culture supernatants were harvested at 24 and 72 h for cytokine analysis. M1 anti-IL-4R Ab was added to LN cultures (2.5 µg/ml) from mice injected 1 day previously to enable the measurement of IL-4 protein (3).
For [3H]thymidine proliferation assays, LN cells were plated at 2.5 x 104/well in round-bottomed 96-well plates (Falcon). After 4 days at 37°C/5% CO2, the cells were pulsed with [3H]thymidine (1 µCi; Amersham, Arlington Heights, IL) for 18 h before harvesting with a 96-well plate harvester (Tomtec, Orange, CT). [3H]Thymidine incorporation was measured using a liquid scintillation counter (Wallac, Gaithersburg, MD).
For CFSE proliferation assays, LN cells were labeled by incubation with 2.5 µM CFSE in PBS on ice for 8 min. Labeled and unlabeled cells were plated at 6.4 x 105 cells/ml in round-bottomed 96-well plates and incubated for 72 h at 37°C/5% CO2. Cells were then stained for expression of surface markers as described below (see Flow cytometry).
CD4 T cells were purified from total LN cell suspensions using mouse CD4 Dynabeads (Dynal, Oslo, Norway) following the manufacturer’s instructions. Briefly, cells were incubated with mouse CD4 Dynabeads at a ratio of 8 beads:1 target cell at 4°C for 1 h. CD4 T cells were isolated with a magnet, and the Dynabeads were removed using Detachabead. After washing the CD4 T cells were resuspended in complete T cell medium and cultured in 96-well round-bottomed plates at 8 x 104/well at 37°C/5% CO2. Supernatants were removed after 72 h. Purity of the CD4 populations was assessed by flow cytometry as described below.
RT-PCR
Popliteal LN and injected footpads were harvested directly into RNAzol (Tel Test, Friendswood, TX) and snap frozen. Total LN mRNA was isolated and cDNA made using SuperScript II reverse transcriptase (Life Technologies) as previously described (22). Hypoxanthine phosphoribosyltransferase (HPRT) transcripts were amplified using competitive PCR as previously described (23) and used to normalize cDNA levels for cytokine analysis. HPRT transcripts were amplified using 37 cycles and cytokine transcripts were amplified using 41 cycles. Primers used for HPRT, IL-4, and IL-6 amplification were as described previously (23). PCR products were run on a 2.5% agarose gel, stained using ethidium bromide, and analyzed using the Eagle Eye program (Stratagene, La Jolla, CA).
Flow cytometry
Cells were stained on ice for 20 min with either FITC-, PE-, or biotin-labeled Abs. After washing twice with 1% FCS (Sigma) and 0.08% NaN3 (Sigma) in PBS, directly labeled cells were resuspended in fixative (1% formaldehyde (Sigma) in PBS). Biotin-labeled cells were incubated with PE-streptavidin for 20 min on ice, washed, and then resuspended in fixative. Cells were analyzed using a FACS Caliber flow cytometer (Becton Dickson, Franklin Lakes, NJ) with the Cell Quest program (Becton Dickson).
Cytokine assays
Sandwich ELISAs were used to measure IL-4, IL-5, IL-10, and
IFN- in culture supernatants as previously described (24, 25, 26). For
the IL-6 ELISA, rat anti-IL-6 mAb 20F3 (kind gift from F.
Finkelman, University of Cincinnati College of Medicine, Cincinnati,
OH), biotin-labeled rat anti-IL-6 mAb 32C11 (PharMingen), and rIL-6
(Intergen) were used following the PharMingen protocol. Rat
anti-IL-2 mAb JES6-1A12 and biotin-labeled rat anti-IL-2 mAb
JES6-5H4 (PharMingen) and a rIL-2 standard (kind gift from Dr. W.
C. Van Voorhis, University of Washington, Seattle) were used for the
IL-2 ELISA following the PharMingen protocol.
Isotype specific ELISAs
Plasma was collected from blood drawn from mice by heart puncture at the time of LN removal and stored at -20°C. Immulon 2 plates (Dynex, Chantilly, VA) were coated with 0.5 µg SEA/well in 0.1 M sodium carbonate buffer overnight at 4°C. After washing with 0.05% Tween in PBS, the wells were blocked with 10% FCS in PBS for 1 h at 37°C. Plasma samples, diluted in 10% FCS in PBS, were added to the wells and incubated for 2 h at 37°C. After washing, peroxidase-labeled Abs specific for mouse IgG1 and IgG2a (Southern Biotechnology Associates, Birmingham, AL) or total mouse Ig (Amersham) Abs were added. After 1 h at 37°C the plates were washed and developed using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Kirkegarde & Perry Laboratories, Gaithersburg, MD). Absorbance was read at 405 nm.
Statistical analysis
Data were analyzed using the Student’s t test.
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Results |
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To determine whether IL-6 could be playing a role in the
differentiation of egg-specific ThP cells into Th2 cells, the
production of IL-6 was first analyzed in response to the injection of
schistosome eggs into the hind footpads of WT B6/129 mice. Within
24 h of egg injection, IL-6 transcripts were up-regulated at the
site of Ag deposition and in the draining popliteal LN (Fig. 1
A). Cultured LN cells from
egg-injected mice, but not those from PBS-injected animals, produced
low levels of IL-6 constitutively (Fig. 1B). Levels of IL-6
were increased over 20-fold by stimulation with either anti-CD3 or
LPS (Fig. 1C). Even under these strong polyclonal activation
conditions, cells from egg-injected mice made significantly more IL-6
than did those from PBS-injected mice. Interestingly, re-exposure of LN
cells from egg-injected mice to SEA in vitro also led to an increase
(
2-fold) in IL-6 production (Fig. 1B). These results
indicate that eggs induce IL-6 production at a time and site consistent
with a role for this cytokine in promoting the differentiation of
egg-specific ThP cells into Th2 cells.
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To examine the role of IL-6 in promoting early IL-4 production by
CD4 T cells, the early (24 h) responses to egg injection of
IL-6-/- mice were compared with those of WT mice. LN
cells from both egg-injected WT and IL-6-/- mice produced
IL-4 within 24 h of stimulation with anti-CD3 in vitro (Fig. 2A). LN cells from mice
injected with PBS also produced IL-4 within 24 h but the levels of
IL-4 produced were consistently lower than those seen following egg
injection (Fig. 2A). Because no significant differences were
observed in the levels of IL-4 produced by WT and IL-6-/-
LN cells from egg-injected animals after 24 h of in vitro culture,
the initial production of IL-4 in total LN cultures does not depend
upon the presence of IL-6.
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B). Egg-injection primed the CD4 T cells to produce
substantially higher levels of IL-4, although WT CD4 T cells
consistently produced more IL-4 than did the IL-6-/- CD4
T cells (Fig. 2B). The reduced levels of IL-4 measured in
the cultures of IL-6-/- CD4 T cells suggest that IL-6 may
play a role in the priming of CD4 T cells to produce IL-4 although it
is not the sole signal required in the presence of a strong Th2
stimulus (i.e., schistosome eggs). IL-6-/- mice develop an egg-specific Th2 response following egg injection
Although the absence of IL-6 during CD4 T cell priming did not
abolish IL-4 production, the reduced levels produced by the
IL-6-/- CD4 T cells could potentially affect the overall
development of a Th2 response. Therefore, the role of IL-6 in the
development of a Th2 response was investigated by isolating LN cells
7–8 days after egg injection. RT-PCR of RNA isolated from LN from
IL-6-/- and WT mice revealed that the levels of IL-4
transcripts were equivalent in the two mouse strains (Fig. 3A). Only low levels of IL-4
transcripts were detected in the LN of PBS-injected mice at this time
(Fig. 3A). In vitro stimulation of the LN cells with Ag
supported this finding as no significant difference was detected in the
levels of IL-4 in 72 h supernatants of the LN cultures from
IL-6-/- and WT mice (Fig. 3B). In addition,
both WT and IL-6-/- mice developed a Th2 response to egg
Ag as demonstrated by the production of IL-4, IL-5, and IL-10 (Fig. 3, B–D) and by negligible levels of IFN- (Fig. 3F). IL-2 was also measured in 24-h culture supernatants
from both mouse strains (Fig. 3E). Notably, IL-10 and IL-2
levels were lower in the supernatants of cells from egg-injected
IL-6-/- mice than in those from WT mice (Fig. 3, D and E). No cytokines were found in the
supernatants of LN cells from PBS-injected mice stimulated with SEA
(data not shown). Purified CD4 cells from WT and IL-6-/-
egg-injected mice displayed a similar cytokine profile after
stimulation with anti-CD3 (Fig. 4,
A–D), although the disparity in levels of
cytokines produced between IL-6-/- and WT mice was less
apparent than for the whole LN cell cultures. These results strongly
suggest that the absence of IL-6 does not prevent the development of a
Th2 cytokine response to schistosome eggs.
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Because the Ab isotypes are also indicative of Th response
development and IL-6 is known to play an important role in B cell
differentiation and Ab production (11, 27), the Ab response to egg Ag
was studied 7 days after egg injection. SEA-specific Ab in plasma were
detected using ELISAs. As shown in Fig. 5, plasma from WT mice contains
SEA-specific Ab in which both IgG1 and IgG2a isotypes can be detected,
albeit at low levels. Total Ig and IgG2a were considerably decreased in
the IL-6-/- mice, with the most striking difference being
the total absence of IgG1. These results demonstrate an impairment in
the production of both Th1 (IgG2a) and Th2 (IgG1)-associated Ab
isotypes. Histological examination of the LN taken 7 days after egg
injection revealed an impaired germinal center formation in the
IL-6-/- mice compared with the WT mice (data not shown)
as was expected given previous reports (27). Therefore, despite the
similar Th2 response development in the IL-6-/- mice
compared with WT mice, Ab production of both Th1 and Th2-associated
isotypes and germinal center formation is impaired, supporting the
finding that in the absence of IL-6, B cell development and
differentiation is affected.
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Despite the lack of effect of the absence of IL-6 on Th2 cytokine
production, its marked effect on B cell responses prompted the
investigation into other known functions of IL-6 which could modify
response development after egg injection. In addition to its role in T
and B cell differentiation, IL-6 has been shown to promote
proliferation of spleen cells in vitro (28). The role of IL-6 in
proliferation was clearly demonstrated by visual assessment of the
culture wells after 48 h. WT LN cells from mice injected with eggs
7 days previously had more pronounced blast and cluster formation when
stimulated with anti-CD3 (Fig. 6A) than did cells from
IL-6-/- mice (Fig. 6B). Comparable results
were seen on day 1 after egg injection (data not shown). Furthermore,
the proliferation of the IL-6-/- LN cells as measured by
[3H]thymidine incorporation was also severely reduced
compared with WT (Fig. 6C). Similar results were found when
proliferation was measured by flow cytometry of cells labeled with CFSE
(Fig. 7). CFSE is a fluorescent
cytoplasmic tag which becomes evenly distributed between daughter cells
after mitosis and thus, reduction in cellular fluorescence correlates
with proliferation. Addition of rIL-6 to cultures stimulated with
anti-CD3 enhanced the proliferation of LN cells from WT and
IL-6-/- PBS (data not shown) and egg-injected mice (Fig. 7), although it did not restore proliferation of the
IL-6-/- cultures to WT levels. Decreased proliferation
was also seen with Ag-specific stimulation (data not shown). The
difference in proliferative responses of WT versus
IL-6-/- LN cells was not due to a difference in cell
types present in the LN at day 1 or 7 (Table I) because the small differences in
cellular composition could not account for the dramatic difference in
proliferation. Interestingly, although the LN cells from the WT
PBS-injected mice showed 5-fold greater proliferation on day 1, by day
7 the proliferation had dropped to the level of the
IL-6-/- LN cells (Fig. 6C). This result
suggests that on day 1 the PBS-injected mice are nonspecifically
activated by the injection, but by day 7 the activating stimulus has
dissipated. Overall, these findings indicate that after activation of
LN cells either through introduction of schistosome eggs or immediately
after a trauma induced by injection, the proliferation of the
IL-6-/- cells in vitro is seriously impaired and this
proliferative defect can be partially corrected by the addition of
rIL-6.
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the percentage of proliferating CD4
and CD8 T and B lymphocytes from IL-6-/- mice injected
with eggs 7 days previously was significantly reduced compared with
cells from WT mice. Similar results were seen with the T and B cells
from PBS-injected mice (data not shown). The addition of rIL-6 to the
LN cultures from egg-injected IL-6-/- mice primarily
enhanced proliferation of CD8 T cells and, to a lesser extent, CD4 T
cells (Table II), indicating that exogenous IL-6 can partially overcome
the proliferative defect of IL-6-/- T cells. Although
addition of rIL-6 had only a subtle effect on CD4 proliferation, it had
a more pronounced effect on the percentage of CD4 cells in the LN
culture that may reflect a decrease in apoptosis rather than an
increase in proliferation. This finding is consistent with recent
findings that IL-6 prevents apoptosis of CD4 T cells in vitro by
maintaining levels of bcl-2 (29). In contrast to its effect
on T cells, addition of rIL-6 did not enhance IL-6-/- B
cell proliferation in vitro nor did it increase the percentage of B
cells in the LN cultures, suggesting that the problem in
IL-6-/- B cell proliferation may stem from a defect in
differentiation in vivo. This conclusion is further supported by
evidence that IL-6 enhances proliferation of only highly differentiated
B cells (9). Together these results reveal a role for IL-6 in
lymphocyte proliferation which is all the more striking given the
similar cytokine profiles.
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Since IL-2 is a well-characterized growth factor, a possible
explanation for the reduced proliferation of the IL-6-/-
cells in culture could be reduced production of IL-2. Following
anti-CD3 stimulation for 24 h, there are slightly higher
levels of IL-2 in culture supernatants of LN cells from WT than
IL-6-/- mice that had been injected with eggs 7 days
previously (Fig. 8A). However,
after 72 h of in vitro stimulation, high levels of IL-2 were
detected in the IL-6-/- cultures, whereas no IL-2 was
found in the WT LN cultures (Fig. 8B; please note the
difference in scales). Comparable results were found using LN cells
isolated day 1 after egg injection (data not shown). Addition of rIL-6
to the LN cultures stimulated with anti-CD3 resulted in a dramatic
decrease in the levels of IL-2 detected in the WT and
IL-6-/- culture supernatants after 24 or 72 h (Fig. 8, A and B). Similar results were seen with LN
cells from either PBS or egg-injected mice (data not shown). The
increased levels of IL-2 in the culture supernatants of the
IL-6-/- mice point toward a reduced utilization of IL-2
as the cause of the proliferative defect. Furthermore, the reduced
levels of IL-2 detected in culture supernatants after the addition of
rIL-6 suggests that IL-6 promotes an increased utilization of IL-2,
which in turn enhances the proliferative responses in both
IL-6-/- and WT mice.
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). Culture with medium alone
resulted in a low level of IL-2R expression in both the WT and
IL-6-/- cultures whereas addition of anti-CD3
markedly up-regulated the levels on both WT and IL-6-/-
cells (Fig. 9). As hypothesized, a lower percentage of
IL-6-/- cells expressed the IL-2R and at a lower level
compared with WT cells. This reduced expression on
IL-6-/- cells could be corrected by the addition of
exogenous IL-6 to the culture well (Fig. 9). The addition of rIL-6 to
WT cells further increased the expression of IL-2R and correlated with
increased usage of IL-2 (Fig. 8A). The reduced level of
IL-2R expression was seen on both CD4 and CD8 T cells as well as B
cells suggesting that the defect in IL-2R up-regulation is not specific
to one cellular subset (data not shown). Similar results were observed
with LN cells from mice injected 1 day previously (data not shown).
Furthermore, in contrast to WT, IL-2R expression was not up-regulated
on LN cells from egg-injected vs PBS-injected IL-6-/-
mice isolated on day 7 after injection and stained immediately after
isolation (WT, 21.3 vs 13.3%; and IL-6-/-, 13.3 vs
15.5%; egg vs PBS, respectively). Similar results were seen with LN
cells isolated at 1 day after egg injection (data not shown). These
results indicate that the impairment found in the proliferative ability
of the IL-6-/- cells corresponds to reduced IL-2R
expression in vitro and in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For IL-6 to play a role in early IL-4 production it must itself be produced rapidly following egg injection. Our studies confirm that this is the case, with significant levels of IL-6 being produced by LN cells recovered at 24 h postegg, though not PBS, injection. At present we do not know which cells are secreting this IL-6 nor what the stimulus for production is. As in previous studies, we were able to detect early IL-4 production following egg injection, though for the first time we show that at least part of the IL-4 is being produced by CD4 cells. It is our assumption that the cells producing this IL-4 are schistosome egg Ag-specific ThP cells responding for the first time and making IL-4, although this remains to be shown. Regardless, egg injection into WT and IL-6-/- mice results in the activation and/or recruitment of CD4 cells, not present in PBS-injected mice, that are able to make IL-4 when stimulated in vitro. This population of CD4 cells from IL-6-/- mice was found to produce less IL-4 than that from WT mice. We are not clear whether this is due to failure of the IL-4-producing cells to expand in vitro (see below) or to the absence of a more upstream effect of IL-6 on promoting IL-4 production, which would be consistent with the published report that IL-6 promotes IL-4 production by ThP cells (8). Nevertheless, the slight early differences in IL-4 production did not have a long-term effect on the outcome of the Th2 response as, in the absence of IL-6, the development following exposure to schistosome eggs of Th cells capable of making signature type 2 cytokines such as IL-4 and IL-5, was not impaired.
Although the ability to produce type 2 cytokines was unaffected in IL-6-/- mice, we found that in the absence of IL-6 proliferation of LN cells was dramatically impaired and that this defect in proliferation encompassed CD4 and CD8 T cells as well as B cells. The connection between IL-6 and proliferation of T cells, B cells, and other cells types has been described before (9, 28, 29, 32), but this is the first report of the in vitro impairment of proliferation in IL-6-/- mice as well as an associated in vitro and in vivo defect in IL-2R expression. We believe this defect in IL-2R expression accounts for the reduced proliferative response because addition of exogenous IL-6 enhanced proliferation of IL-6-/- LN and T cells and caused an up-regulation in IL-2R expression. Interestingly, addition of exogenous IL-6 did not augment proliferation of B cells from IL-6-/- mice. Because IL-6 is a late acting differentiation factor for B cells, the lack of effect of rIL-6 on IL-6-/- B cells may reflect an in vivo defect in B cell development (see below). These results complement recent observations that anti-IL-6 Ab blocks proliferation of WT cells (28), that Ab to IL-2 or the IL-2R blocked IL-6-induced T cell proliferation (32), and that IL-2R expression could be induced on T cell lines by IL-6 (33). Taken together, our data reinforce the view that IL-6, IL-2R expression and T cell proliferation are interlinked.
The relationship between IL-6 and proliferation is a possible explanation for the effects of IL-6 on Th2 differentiation in vitro. Addition of IL-6 may promote the production of IL-2 and, consequently, directly or indirectly influence IL-4 production by naive ThP cells. In studies by Seder (34), it was shown that IL-2 was required for IL-4 production and in vitro proliferation of naive CD4 T cells. The necessity for IL-2 production and proliferation before IL-4 production can occur may thus define the relationship between IL-4 and IL-6. However, these results do not exclude the role of other cytokines in promoting proliferation (e.g., IL-1ß) nor can they yet be extended to the in vivo situation. Therefore, while this proliferative defect in the IL-6-/- mice manifests itself in vitro, it is not clear that it occurs in vivo nor does it seem to effect the course of Th2 development in the mouse.
Although the lack of IL-6 failed to affect the development of Th cells capable of making the type 2 cytokines, it had a marked effect on B cell responses following immunization with decreased levels of total Ig and IgG2a as well as the complete absence of IgG1 compared with egg-injected WT mice. IL-6-/- mice have been shown to have defective germinal center formation characterized by diffusely structured follicles which lack the well-defined surrounding T cell areas (27). In previous studies this was linked to a deficit in T cell-dependent Ab production (27, 35) since Kopf et al. (27) reported defects in total IgG and IgG2a levels but found no significant differences on IgM and IgG1 production. The differences in our results may stem from the use of different Ag or differences in the timing of the response, for our studies are focused on early time points. It is possible that if the B cell response had been allowed to proceed longer, differences in IgG1 levels would have been less marked. Another indication of an in vivo B cell defect was the impaired in vitro B cell proliferation that could not be enhanced by the addition of rIL-6. Because IL-6 is a late stage B cell differentiation factor, a defect in in vivo B cell differentiation could result in the isolation and culture of undifferentiated B cells that are unable to proliferate in response to exogenous IL-6. Furthermore, since Th cells from immunized IL-6-/- mice are as capable as WT Th2 cells of producing cytokines, the defect in the B cell response is unlikely to stem from a lack of cytokine-mediated help. Other possible explanations for the impaired B cell response include a direct effect of IL-6 on B cells or the failure of T cells to express appropriate cell surface costimulatory molecules involved in B cell help (27, 35, 36). Regardless, our results point to a defect early in the development of the B cell response in IL-6-/- mice responding to schistosome eggs.
By using IL-6-/- mice, we have been able to better define the role of IL-6 in the promotion of T and B cell responses after the injection of a Th2 response-inducing Ag. Although egg injection resulted in the induction of IL-6 and Th2 development in WT mice, the absence of IL-6 did not prevent Th2 development in IL-6-/- animals. Despite the lack of effect of the absence of IL-6 on Th responses per se, B cell response development and LN cell proliferation were seriously impaired in the IL-6-/- mice. The defect in proliferation correlated with decreased IL-2R expression in vivo and in vitro and underscores the interconnections of IL-6, IL-2R, and proliferation. Further studies are needed to define the mechanism by which IL-6 controls IL-2R expression, whether proliferation is defective in IL-6-/- mice in vivo, and finally, if the reduced proliferation and/or IL-2R expression is responsible for the impaired B cell responses in vivo.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Edward J. Pearce, Department of Microbiology and Immunology, C5-165 Veterinary Medical Center, Cornell University, Ithaca, NY 14853. E-mail address:
3 Abbreviations used in this paper: ThP, precursor Th cell; WT, wild type; LN, lymph node; SEA, soluble egg Ag; CFSE, 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester; MFI, mean fluorescent intensity; HPRT, hypoxanthine phosphoribosyltransferase.
4 L. Rosa-Brunet, E. A. Sabin, A. W. Cheever, M. A. Kopf, and E. J. Pearce. IL-5 plays a role in the development of IL-4 producing non-T, non-B cells during murine schistosomiasis but is not required for the expression of a Th2 response or host resistance mechanisms. Submitted for publication.
Received for publication September 4, 1998. Accepted for publication February 26, 1999.
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References |
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 K. G.-J. Ooi, G. Galatowicz, V. L. Calder, and S. L. Lightman Cytokines and Chemokines in Uveitis - Is there a Correlation with Clinical Phenotype? Clin. Med. Res., December 1, 2006; 4(4): 294 - 309. [Abstract] [Full Text] [PDF]
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 S. Rose-John, J. Scheller, G. Elson, and S. A. Jones Interleukin-6 biology is coordinated by membrane-bound and soluble receptors: role in inflammation and cancer J. Leukoc. Biol., August 1, 2006; 80(2): 227 - 236. [Abstract] [Full Text] [PDF]
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S. A. Jones Directing Transition from Innate to Acquired Immunity: Defining a Role for IL-6 J. Immunol., September 15, 2005; 175(6): 3463 - 3468. [Abstract] [Full Text] [PDF]
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 R. M. McLoughlin, B. J. Jenkins, D. Grail, A. S. Williams, C. A. Fielding, C. R. Parker, M. Ernst, N. Topley, and S. A. Jones IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation PNAS, July 5, 2005; 102(27): 9589 - 9594. [Abstract] [Full Text] [PDF]
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 D. R. Boverhof, E. Tam, A. S. Harney, R. B. Crawford, N. E. Kaminski, and T. R. Zacharewski 2,3,7,8-Tetrachlorodibenzo-p-dioxin Induces Suppressor of Cytokine Signaling 2 in Murine B Cells Mol. Pharmacol., December 1, 2004; 66(6): 1662 - 1670. [Abstract] [Full Text] [PDF]
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Y.-W. Hsiao, K.-W. Liao, S.-W. Hung, and R.-M. Chu Tumor-Infiltrating Lymphocyte Secretion of IL-6 Antagonizes Tumor-Derived TGF-{beta}1 and Restores the Lymphokine-Activated Killing Activity J. Immunol., February 1, 2004; 172(3): 1508 - 1514. [Abstract] [Full Text] [PDF]
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I. L. Dodge, M. W. Carr, M. Cernadas, and M. B. Brenner IL-6 Production by Pulmonary Dendritic Cells Impedes Th1 Immune Responses J. Immunol., May 1, 2003; 170(9): 4457 - 4464. [Abstract] [Full Text] [PDF]
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 C.-M. Hogerkorp, S. Bilke, T. Breslin, S. Ingvarsson, and C. A. K. Borrebaeck CD44-stimulated human B cells express transcripts specifically involved in immunomodulation and inflammation as analyzed by DNA microarrays Blood, March 15, 2003; 101(6): 2307 - 2313. [Abstract] [Full Text] [PDF]
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A. C. La Flamme, A. S. MacDonald, C. R. Huxtable, M. Carroll, and E. J. Pearce Lack of C3 Affects Th2 Response Development and the Sequelae of Chemotherapy in Schistosomiasis J. Immunol., January 1, 2003; 170(1): 470 - 476. [Abstract] [Full Text] [PDF]
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 J. Mann, F. Oakley, P. W. M. Johnson, and D. A. Mann CD40 Induces Interleukin-6 Gene Transcription in Dendritic Cells. REGULATION BY TRAF2, AP-1, NF-kappa B, AND CBF1 J. Biol. Chem., May 3, 2002; 277(19): 17125 - 17138. [Abstract] [Full Text] [PDF]
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 A. C. La Flamme, E. A. Patton, and E. J. Pearce Role of Gamma Interferon in the Pathogenesis of Severe Schistosomiasis in Interleukin-4-Deficient Mice Infect. Immun., December 1, 2001; 69(12): 7445 - 7452. [Abstract] [Full Text] [PDF]
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 D. Markine-Goriaynoff, T. D. Nguyen, G. Bigaignon, J. Van Snick, and J.-P. Coutelier Distinct requirements for IL-6 in polyclonal and specific Ig production induced by microorganisms Int. Immunol., September 1, 2001; 13(9): 1185 - 1192. [Abstract] [Full Text] [PDF]
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R. G. Titus, G. K. DeKrey, R. V. Morris, and M. B. P. Soares Interleukin-6 Deficiency Influences Cytokine Expression in Susceptible BALB Mice Infected with Leishmania major but Does Not Alter the Outcome of Disease Infect. Immun., August 1, 2001; 69(8): 5189 - 5192. [Abstract] [Full Text] [PDF]
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