Introduction

T cell independent type-1 (TI-1) antigens, such as lipopolysaccharide (LPS), are intrinsically stimulatory for B cells^{1, 2, 3, 4, 5, 6}. However, antibodies made in response to these antigens are antigen specific.^{1, 7} This specificity is thought to be achieved through specific B cell Ig receptors binding to epitopes within the TI-1 antigen, leading to the focusing of the non-specific stimulatory signals to the surface of antigen-specific cells.^{5, 6, 8, 9} When high concentrations of LPS are focused to the B cell surface by surface (s)Ig, however, the specific response is inhibited. Coutinho and Möller's one-signal model assumed that this inhibition was due to high-dose paralysis,⁹ a phenomenon that our accompanying paper disputes.¹⁰ As an alternative, we have proposed the hypothesis that the inhibition of ASC formation at high concentrations is due to B cells receiving a sIg-mediated signal at the same time as the TI-1 activation signal.¹¹ This alternative hypothesis, and not that of Coutinho and Möller, is consistent with the ability of anti-Ig reagents to inhibit LPS-induced antibody secreting cell (ASC) formation.^{12, 13, 14, 15} The important point of difference between the two models is that in one an antigen signal transmitted by sIg is not involved, whereas in the other this signal is essential.

One potential way to distinguish experimentally between our model and that described by Coutinho and Möller⁹ was to measure the hapten-specific dose–response curves in the presence of an inhibitor of the sIg-mediated antigen signal that does not also affect LPS stimulation. If our model is correct, the curve would convert from being bell shaped to being conventionally sigmoidal. In contrast, the Coutinho and Möller model would expect no change to the dose curve. A possible candidate for an inhibitory drug with these characteristics was cyclosporine A (CsA), because this drug has been shown in vitro to inhibit sIg-mediated signals, but not LPS-induced B cell responses, over a broad concentration range.^{16, 17, 18} To examine whether CsA could be used in this way, it was important to first show that this drug would prevent the ability of anti-Ig reagents to inhibit LPS-induced ASC formation, as originally shown by Andersson et al.¹² Experiments that set out to test this possibility are described below.

Materials and Methods

Experimental animals

B cells were prepared from CBA/H mice aged between 6 and 14 weeks. Mice were obtained and housed at the animal facility at the John Curtin School of Medical Research (Canberra, ACT, Australia).

Reagents and antibodies

Lipopolysaccharide isolated by phenol extraction from Salmonella typhosa was purchased from Sigma (St Louis, MO, USA). The LPS solutions were prepared in B cell media (BCM; RPMI-1640 containing 2 mmol/L L-glutamine and supplemented with 10% heat-inactivated (HI) FCS that had been screened for low intrinsic B cell mitogenicity, 5 10^-5 mol/L mercaptoethanol, 10 mmol/L HEPES pH 7.4, 0.1 mmol/L non-essential amino acids, 1 mmol/L sodium pyruvate, 60 g/mL benzyl penicillin and 100 g/mL streptomycin), the solution sterilized by filtration through 0.2 m membranes (Millipore, Bedford, MA, USA) and stored at 4°C.

Goat antimouse IgM (gIgM) was purchased from Cappel (West Chester, PA, USA) and diluted in BCM.

B cell preparation

B cells were prepared from mouse spleen as described previously.^{10, 19} Briefly, a single cell suspension of murine spleen cells was prepared and consecutively depleted of red blood cells, with a lysis buffer (10 mmol/L potassium bicarbonate, 0.15 mol/L ammonium chloride, 0.1 mmol/L EDTA and 5% HI FCS, pH 7.3); adherent cells, by allowing cells to adhere to plastic Petri dishes; and T cells, using a cocktail of anti-T cell antibodies (antimouse CD4, RL172 anti-CD8, 31M and anti-Thy1, 30H-12²⁰) followed by rabbit complement (Cederlane Laboratories, Hornby, Ontario, Canada). Following T cell depletion, cells were washed and resuspended in BCM in preparation for experiments.

Measuring B cell proliferation

B cell proliferation was determined by [³H]-methyl 1,2, thymidine (TdR) incorporation during a 4 h pulse. In a typical experiment, B cells were cultured in 96-well flat-bottom plates (Nunc, Roskilde, Denmark) in a final volume of 200 L BCM/well and incubated in a humidified atmosphere at 37°C and 5% CO₂. Four hours prior to harvesting the cells, 37 kBq [³H]-TdR (Amersham, Uppsala, Sweden) was added to each well. Following [³H]-TdR incorporation, the cells were harvested from the wells and transferred to glass fibre filter mats using a 96-well cell harvester (Pharmacia Wallac 1295–004 Betaplate^TM). The filter mats were dried, placed in bags and scintillant added according to the manufacturer's instructions. The level of [³H]-TdR incorporation per well was determined using a Betaplate^TM liquid scintillation counter (Pharmacia Wallac 1205).

Enzyme-linked immunosorbent spot assays

Enzyme-linked immunosorbent spot (ELIspot) assays were used to detect Ig-secreting B cells²¹ using a modification of the original method.^{10, 19} Briefly, B cells were cultured as for [³H]-TdR incorporation and following the desired incubation period, cells were harvested, washed and transferred into microtitre 96-well filtration plates (ELISpot plates; Millipore, Bedford, MA, USA), which had previously been coated with sheep antimouse Ig antibody (sIg, obtained from Silenus, Melbourne, Vic., Australia). B cells were incubated undisturbed in ELIspot plates for 4 h in a humidified atmosphere at 37°C and 5% CO₂, before the supernatant was removed and the ELIspot plates washed. Antibody secreting cells (ASC) were visualized by adding sIg coupled to horseradish peroxidase (Silenus) and incubating overnight at 4°C before washing and adding substrate. The plates were monitored for the formation of spots and the reaction was stopped by washing the plates with water. After drying, the number of spots per well were counted using a low-power microscope.

Results

Anti-Ig-induced B-cell proliferation: Dose-dependent effects

To explore the role of the antigen signal in TI-1 activation, B cells were stimulated with a combination of LPS and gIgM. The anti-Ig antibody was used to trigger a signal from sIg and LPS was used as a TI-1 B cell activator. The gIgM could induce a measurable B cell proliferative response when added to B cell cultures at concentrations > 10–20 g/mL (Figure 1). An additional feature of the dose–response curve was the inhibition of B cell proliferation at low to moderate concentrations (1 ng–1 g/mL) of gIgM (Figure 1). The inclusion of 40 ng/mL CsA in these cultures completely inhibited gIgM-induced [³H]-TdR incorporation, while having only a small inhibitory effect on LPS-induced proliferation (Figure 2). At higher concentrations, CsA began to inhibit LPS-induced [³H]-TdR incorporation, as previously reported,¹⁸ whereas lower doses did not completely inhibit the B cell response to gIgM (data not shown). Therefore, a concentration of 40 ng/mL CsA was used in all subsequent experiments. Having established a CsA dose that would inhibit gIgM-induced proliferation, it was important for our experimental aims to determine whether this dose could also prevent the ability of gIgM to inhibit LPS induction of ASC.

Figure 1.

Goat anti-IgM (gIgM)-induced B cell proliferation is inhibited by cyclosporine A (CsA). B cells were cultured in B cell media (5 10⁴ cells/200 L well) containing varying concentrations of gIgM (). Replicate cultures were also prepared which contained gIgM and 40 ng/mL CsA (). After 3 days, B cell proliferation was measured by [³H]-thymidine incorporation. The data illustrated represents the mean SEM of triplicate cultures. Background proliferation: cells only () and CsA only ().

Full figure and legend (15K)

Figure 2.

Cyclosporine A (CsA) inhibits goat anti-IgM (gIgM)- but not LPS-induced B cell proliferation. B cells were cultured as described in Figure 1, in the presence of 10 g/mL gIgM (a) or 50 g/mL LPS (b). Replicate cultures were prepared, which contained CsA (40 ng/mL) in combination with LPS, or gIgM. After 3 days, B cell proliferation was measured by [³H]-thymidine incorporation. The data illustrated represents the mean SEM of triplicate cultures. Background proliferation: cells only and CsA only.

Full figure and legend (35K)

Anti-Ig-mediated inhibition of LPS-induced ASC formation is CsA insensitive

The gIgM antibody was tested for its ability to inhibit LPS-induced B cell differentiation to ASC. Figure 3a shows a dose-dependent inhibition by gIgM antibody, as previously described by Andersson et al.^{12, 14} and Kearney et al.^{13, 15} Given the inhibitory effect of CsA on gIgM-induced proliferation, it was anticipated that CsA may also block the anti-Ig mediated decline in ASC number. However, as illustrated in Figure 3a, CsA had almost no effect on gIgM-mediated inhibition of LPS-induced ASC formation. Rather, the addition of CsA to cultures containing gIgM and LPS caused a slight further reduction in the overall number of ASC (Figure 3a). These results revealed that the sIg-mediated signal(s) responsible for ASC inhibition were CsA insensitive and that the plan to use this drug to address the role of the antigen signal in regulating TI-1 antibody responses could therefore not be pursued.

Figure 3.

Coculture with goat anti-IgM (gIgM) and LPS reveals different effects of surface Ig-mediated signals, which vary in their sensitivity to cyclosporine A (CsA). B cells were cultured as described in Figure 1 in the presence of varying concentrations of gIgM and 50 g/mL LPS. Replicate cultures were also prepared, which contained gIgM, LPS and 40 ng/mL CsA. After 3 days, total number of antibody secreting cells (ASC) determined by ELIspot assay (a): LPS only, (); LPS and CsA only, (); LPS and gIgM, (); LPS, gIgM and CsA, (); cells only, (). Proliferation was measured by [³H]-thymidine (TdR) incorporation (b): LPS only, (); LPS and CsA only, (); LPS and gIgM, (); LPS, gIgM and CsA, (); cells only, (). (c) Overlay of the data illustrated in (a) and (b) to demonstrate the similarity in dose response between ASC number and proliferation. In the absence of CsA (upper panel), the dose–response curves for ASC formation () and [³H]-TdR incorporation () diverge at gIgM concentrations > 3g/mL. However, in the presence of CsA a similar dose–response curve is generated for both ASC number and proliferation. Background antibody production: LPS CsA () and cells CsA () and background proliferation LPS CsA () and cells CsA (). In each case, the data illustrated represents the mean SEM of triplicate cultures.

Full figure and legend (54K)

Goat anti-IgM has opposing dose-dependent effects on LPS-induced proliferation that differ in their sensitivity to CsA

In addition to monitoring the generation of ASC, the proliferation of B cells stimulated with LPS and gIgM was also measured in the presence and absence of CsA (Figure 3b). In these cultures, gIgM altered LPS-induced proliferation in two apparently contradictory ways. As illustrated in Figure 3b, low doses (< 1 g/mL) of gIgM inhibited LPS-induced [³H]-TdR incorporation, whereas higher antibody concentrations induced a dose-dependent increase in proliferation. The addition of CsA to these cultures did not affect low-dose inhibition of proliferation by gIgM, but prevented the increase in [³H]-TdR incorporation at high antibody doses (Figure 3b). Furthermore, in the presence of CsA, the inhibition of LPS-induced proliferation by gIgM followed approximately the same dose–response curve as obtained for the reduction in ASC number (Figure 3c). It was also of interest to note that the low concentrations of gIgM that inhibited LPS-induced B cell proliferation (Figure 3b) were similar to those which significantly reduced the level of spontaneous [³H]-TdR incorporation (Figure 1). Thus, gIgM appeared to induce two distinct dose-dependent effects on B cells. Low concentrations of gIgM inhibited spontaneous or LPS-induced proliferation and ASC formation, whereas high antibody concentrations promoted proliferation without affecting ASC development.

Different preparations of gIgM differ in their signalling capacity

Through the time period of these experiments, two different batches of gIgM were tested. As indicated by the dose– response curves for B cell proliferation (Figure 4), the two antibody preparations differed significantly. Whereas at low concentrations of gIgM the dose–response curves inhibiting proliferation were similar (Figure 4), the concentration at which the different preparations of gIgM were able to enhance proliferation above the background differed approximately 10-fold (Figure 4). These results indicate that the relative efficacy of gIgM in triggering the growth stimulatory signals differed between the two preparations, even though the efficiency at which the inhibitory signals were transmitted were similar. These results imply that the two sIg- mediated signals may be independently regulated.

Figure 4.

The response to different batches of goat anti-IgM (gIgM) suggests that the inhibitory and stimulatory signals are independent of each other. B cells were cultured as described in Figure 1 in the presence of varying concentrations of different batches of gIgM. (), Batch 1; (), batch 2. After 3 days, B cell proliferation was measured by [³H]-thymidine incorporation. The data illustrated represents the mean SEM of triplicate cultures. (), Background proliferation.

Full figure and legend (16K)

Discussion

A full understanding of humoral immunity will require a theory to explain the significance of the B cell antigen signal in regulating the outcome of B cell responses. Much experimental work to date on antigen stimulation using a variety of reagents has revealed that the signal is not always obligatory or even positive in promoting B cell activation. This point is particularly evident from studies of the role of antigen signalling in TI-1 responses using LPS as the model. Coutinho and Möller originally determined that the antigen signal played no positive role in this system and much evidence has accumulated since that antigen stimulation plays an inhibitory role for antibody production, but not for B cell proliferation. We have attempted to incorporate this data into a logical framework by suggesting that antigen stimulation may play a role in dampening the affinity of the antibody response to this group of antigens, although the logic of such an arrangement is not yet apparent.¹⁰ To explore the role of the antigen signal in LPS responses further, we attempted to use the drug CsA to inhibit the signal specifically. The results revealed an additional complexity within a system that already appeared complex.

Our results clearly indicate that the previous conclusion that anti-Ig reagents do not affect LPS-induced proliferation^{12, 13} is only approximately correct. Engaging the receptor in the presence of CsA, a drug which blocks Ca⁺ mediated responses and inhibits many attributes of anti-Ig-mediated activation,^{16, 22, 23} revealed that a signal is transmitted following stimulation that prevents LPS-induced proliferation. However, this inhibition is usually hidden, because a CsA-sensitive signal will act in concert with LPS to promote and 'rescue' proliferation. Thus, the complex dose curve seen in Figure 3b, in which anti-Ig first inhibits and then enhances LPS proliferation, is a net effect of the two signals. An important and noticeable difference is that the inhibitory signal was triggered at lower concentrations of anti-Ig to those that promoted proliferation. Furthermore, the identical dose curve of inhibition of ASC formation and proliferation and the observation that both responses are CsA insensitive suggests that the two responses are under control of the same sIg-mediated signal (Figure 3c). It is also important in interpreting the significance of this result that similar inhibitory and stimulatory features were apparent even in the absence of LPS. Thus, low concentrations of anti-Ig inhibit background proliferation of B cells, whereas higher concentrations are required to stimulate proliferation in the manner of Ca⁺-dependent TI-2 activation.^{24, 25} This result implies that concomittant LPS stimulation is not essential for triggering either the positive or the inhibitory signal. Rather, the additional signals being provided to the B cell at the time of receptor engagement determine the functional consequence of the two signals once generated.

When taken at face value, the results reported here suggest a modification to our previous model incorporating a role of antigen in TI-1 responses. In the new version, the antigen signal can inhibit LPS-induced ASC formation, as before. However, in B cells that strongly bind antigen (and that presumably have high affinity for the antigen), this signal induces the cells to divide more rapidly while not secreting antibody. This model would still result in a bell-shaped curve for LPS-induced antibody production, as observed by Coutinho and Möller⁹ and which was the original motivation for our model making. If this interpretation is correct, it is not obvious why this mechanism has evolved. It is possible that the antigen-specific B cells expand to generate a memory cell population available for subsequent antigen exposure. Alternatively, the progeny of these higher-affinity cells may be activated to secrete antibody once epitope binding and signal strength is reduced, either due to the presence of soluble antibody sequestering antigen or by antigen clearance. The appearance of highly antigen-specific antibodies late in the response could expedite the complete eradication of the antigen. This possibility could be tested by examining whether LPS-stimulated B cells cultured in the presence of anti-Ig to inhibit development of ASC go on to become ASC after the anti-Ig is removed.

The above model assumes a fixed relationship between signal strength and B cell behaviour. By this view, there will always be a sequence in which antigen stimulation will first inhibit and then enhance B cell proliferation in the presence of LPS. However, another interesting possibility to explain the appearance of two signals with different activation thresholds can be suggested. It is well described that the physical characteristics of the antigen are an important variable in triggering the TI-2 form of activation. Thus, antigens or sIg-binding reagents presented in a form that can cross-link the receptor are much more potent than equivalent mono- or pauci-valent interactions.^{24, 26, 27, 28, 29} Furthermore, this cross-linking-dependent signal is inhibited by CsA and is not required for all forms of B cell activation, including that provided by T cells.^{30, 31, 32} Thus, the transmission of this signal appears to have specifically evolved to provide information about the physical form of the antigen, which in turn is a key determinant of which antibodies of what affinity will most rapidly clear the antigen when secreted into serum.³³ By this argument, therefore, the existence of this positive TI-2 signal can easily be accommodated into a logical B cell activation scheme that demonstrates a clear survival advantage for the host.

The inhibitory signal transmitted by receptor cross-linking presumably also has requirements for physical interaction with the ligand. Rather than being the same as the TI-2 activation requirements, it is possible they are quite different and may even be independent of each other. If this were the case, then the ability to trigger one signal would not be a predictor of agonism for the other. Although our data preented here are too preliminary to reach a firm conclusion, the strikingly different dose–response curves for inhibition and enhancement of LPS-induced proliferation may be taken as some support for this possibility. Furthermore, the contrasting behaviour of two batches of anti-Ig, which induced the inhibitory signal at similar concentrations but differed 10-fold in their ability to stimulate proliferation directly, is also consistent with this alternative view. Independent triggering of two distinct signals based on antigen structure, with the consequence for B cell behaviour determined by the sum or ratio of the signals, could explain our observations reported here and many of the disparate results earlier workers have found in using anti-Ig reagents.^{12, 13, 24, 26, 29, 34, 35, 36} We are currently testing this possibility further.

The use of CsA in these experiments has provided some indication as to the identity of the intracellular components involved in the two signals. Cyclosporine A inhibits a number of signalling events that occur as a result of calcium mobilization: these include the activation and migration from the cytoplasm to the nucleus of transcription factors, such as NF-AT^{23, 37, 38, 39} and NF-kB.³⁷ The inhibitory action of CsA appears to be the result of the inhibition of the Ca²⁺/calmodulin-dependent phosphatase, calcineurin.^{22, 40, 41, 42} By inhibiting the phosphatase activity of calcineurin, CsA can prevent the dephosphorylation and subsequent migration into the nucleus of NF-AT.^{23, 37, 38, 39, 43} By preventing the migration of NF-AT and other transcription factors, which promote the expression of a number of genes,³⁹ CsA may be able to inhibit gIgM-induced B cell proliferation. In contrast, the failure of CsA to prevent gIgM-mediated inhibitory effects and the inhibition of LPS-induced proliferation^{44, 45} and ASC formation by phorbol esters^{45, 46} suggests that the sIg-mediated inhibitory signal could involve the activation of protein kinase C (PKC). However, the regulation of B cell responses by sIg-mediated signals cannot be quite this simple, because PKC is also a component of the intracellular signalling pathways that result in the activation of B cells by both LPS^{17, 44, 47} or anti-Ig reagents.^{17, 48, 49, 50, 51} In addition, the combination of phorbol esters and calcium ionophores has been shown to induce B cell proliferation.⁵² Therefore, the B cell response may not be simply due to the activation of PKC or the mobilization of calcium, but could depend on the strength of, and interaction between, these and other intracellular messengers. While discussion of the role of calcium or PKC is speculative, the results do suggest that sIg-mediated signals provide more information to the B cell about the nature of the bound antigen than has previously been anticipated.

Finally, the inhibitory effects of gIgM are unlikely to be due to coligation of sIg and the FcRIIB, which results in Fc-mediated inhibition,^{53, 54} because the Fc region of goat antibodies are reported not to bind to FcRIIB.^{55, 56, 57} Furthermore, we have shown that trinitrophenol (TNP)-Ficoll can inhibit the production of LPS-induced anti-TNP ASC, presumably through a sIg-mediated signal.¹⁰ These results are similar to those obtained in mice that express the transgene for anti-TNP Ig, because LPS-induced anti-TNP ASC formation is inhibited by both TNP-BSA and anti-Ig antibodies.⁵⁸ Therefore, gIgM-mediated inhibition of proliferation and ASC formation would appear to be caused by sIg-mediated, rather than Fc-mediated, signals.

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