| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Modulation by Penicillins
*
Max Planck Institute for Immunobiology, Freiburg, Germany; and
Institute of Immunology and Allergology, Inselspital, Bern, Switzerland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß+ cells was detected. A panel of T cell clones was
then prepared from Pen G-specific lines after two cycles of
restimulation with the hapten. For the majority of these clones, we
found that high doses of Pen G induced optimal IL-4 secretion, whereas
the amount of IFN-
secreted was inversely correlated with the dose
of Pen G, thus leading to a hapten-inducible shift of the fuctional
phenotypes for some of the clones. Finally, Pen V and Amp were used to
modulate different Ag-induced immune responses. We found that Amp had
no influence on the cytokine pattern induced by specific Ag or
mitogens. In contrast, Pen V inhibited the secretion of IFN-, but
not IL-4, most likely by Ag-independent mechanisms. This last finding
may open new applications for immune intervention in those diseases in
which polarized Th1 responses are involved in the development of the
pathology. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recent advances in our understanding of the molecular mechanims inducing these adverse immune responses have pointed to hapten-modified peptides as Ags (5). Using the ß-lactam antibiotic penicillin G (Pen G)2 as a model, we demonstrated that penicillin-specific CD4+ T cells react to synthetic MHC class II-binding peptides that contain appropriate HLA-DR anchor residues and penicilloylated lysine in selected positions along a polyalanine backbone. These responses are highly dependent on the precise position of the hapten, but independent from the carrier sequence (6). Both the side chain and the backbone structure of the drug molecule are crucial in activating penicillin-specific TCR (7).
Upon initial exposure to a foreign Ag, T cells can differentiate
into type 1 or type 2 phenotypes with different functional capacities.
Type 1 cells secrete mainly IFN- and are involved in cell-mediated
adverse reactions; type 2 cells produce IL-4 and drive immediate type
hypersensitivity reactions (8). In cases in which polarized responses
are dominant, Ag dose appears as one of the critical parameters in
determining naive T cell differentiation (9, 10, 11).
We investigated the extent to which hapten doses can influence human immune responses to drugs in vitro, focusing on allergic hypersensitivity reactions to ß-lactam antibiotics and using Pen G, Pen V, and Amp as model haptens.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Pen G, Pen V, and Amp were obtained from Sigma (St. Louis, MO). PHA was obtained from Murex Diagnostics (Dartford, U.K.). The peptide tetanus toxoid (TT) 616–630 (VRDIIDDFTNESSQK) was synthesized by continuous-flow solid-phase peptide synthesis.
For all Ags, a 10-fold concentrated-stock solution was freshly prepared in PBS and added to the wells at the time of the experiment.
Culture media
The complete culture medium used in this study was RPMI 1640 supplemented with 2 mM L-glutamine, 5 x 10-5 M 2-ME, 1 mM sodium pyruvate, and 1x mixture of nonessential amino acids (Life Technologies, Paisley, Scotland). The medium was prepared without penicillins or any other antibiotics. For growing T cell clones, the complete RPMI medium was used with the addition of 5% pooled human serum (HS; Swiss Red Cross, Bern, Switzerland) and 250 U/ml of human rIL-2 (proleukin; Eurocetus, Ratingen, Germany). EBV-transformed B lymphoblastoid cell lines (EBV-B) were grown in complete RPMI supplemented with 10% heat-inactivated FCS.
Primary culture
Donors BAM, CAS, and ES had a positive history of penicillin
allergy as summarized in Table I
. Primary
cultures were prepared from freshly isolated PBMC in two parallel
96-well plates. Cells at a concentration of 3 x
105/well were stimulated with 3-fold dilutions of Pen G
from 3 mg/ml to 0. 1 µg/ml. After a 5-day incubation at 37°C in 5%
CO2, the proliferative response was determined in one of
the plates as the [3H]thymidine incorporation after a
16-h incubation with 1 mCi = 37 kBq [3H]thymidine
(Dupont, Boston, MA). Cells were harvested on GF/A-filters (Dunn
Labortechnik, Asbach, Germany), and the incorporation was measured in
an automatic beta counter (Inotech, Asbach, Germany). The second plate
was further expanded in IL-2-containing medium for 4 days. Cells were
then pooled for any given Pen G concentration, washed, and rested in
the absence of IL-2. After another 4 days, cells were harvested and
analyzed for TCR expression by cell surface staining and for
IL-4/IFN- production by intracellular staining.
|
T cell clones were generated from Pen G-specific T cell lines. Freshly isolated PBMC were stimulated with 1, 0.3, and 0.1 mg/ml of Pen G at a density of 2 x 105 cells/well in a final volume of 200 µl of complete RPMI medium containing 5% HS in 96-well flat-bottom culture plates. After 5 days at 37°C and 5% CO2, 25 U/ml of IL-2 was added to the wells. After another 4 days, the cultures were pooled, rested for 3 days in the absence of IL-2, and subjected to a second round of restimulation using irradiated autologous PBMC as APC and the same dose of Pen G given during the first restimulation. After 3 days, the different cultures were again expanded in IL-2-containing medium and then cloned by limiting dilution. T cell blasts were seeded at 0.3 cell/well in Terasaki plates (Nunc, Roskilde, Danmark) in the presence of 1 µg/ml PHA and 104 irradiated allogeneic PBMC in complete RPMI medium containing 5% HS and 250 U/ml IL-2. The T cell clones obtained were maintained in culture by periodic stimulation in the presence of irradiated-allogeneic PBMC, PHA, and IL-2.
EBV-B cells
Autologous EBV-B lymphoblastoid cell lines were prepared by culturing freshly isolated PBMC in complete RPMI medium containing 10% FCS, 30% supernatant of the EBV-producing marmoset cell line B95–8 (American Type Culture Collection, Manassas, VA), and 600 ng/ml cyclosporin A (Sandoz, Basel, Switzerland). After overnight incubation, cells were washed and cultured in complete culture medium.
Flow cytometric analysis of intracellular IFN- and IL-4
production
Intracellular staining for cytokines was performed following the
protocol described by Murphy et al. (12), with a few modifications.
Briefly, cells were resuspended at 106/ml in 96-well plates
in a final volume of 200 µl/well of culture medium and stimulated
with PMA at 50 ng/ml plus ionomycin at 500 ng/ml for 5 h at
37°C. Brefeldin A at 10 µg/ml was then added to the wells and
incubation was prolonged for another 4 h. Cells were then washed
and resuspended in 100 µl/well of PBS before adding an equal volume
of 4% formaldeyde fixative. After fixing for 20 min at room
temperature, cells were either stored at 4°C overnight or stained
immediately for intracellular cytokines. For intracellular staining,
all reagents and washes contained 1% BSA and 0.5% saponin, and all
incubations were at room temperature. Cells were first washed and
permeabilized for 30 min. After washing, blocking-mouse IgG (Jackson
ImmunoResearch, West Grove, PA) at a concentration of 300 µg/ml was
given to the well for 10 min. Then, phycoerythrin (PE)-conjugated
anti-human IL-4 Ab (clone 8D4-8; PharMingen, San Diego, CA) plus
FITC-conjugated anti-human IFN- Ab (clone 4S.B3; PharMingen) or
isotype-matched controls (PE- and FITC-conjugated mouse IgG1,
PharMingen) at a final concentration of 5 µg/ml were directly added
to the wells. After 20 min, cells were washed twice in PBS/BSA/saponin
and then with PBS/BSA to allow membrane closure. Samples were
resuspended in PBS/BSA and analyzed on a FACScan flow
cytometer (Becton Dickinson, San Jose, CA). Thresholds were set
on control stainings and for every sample 50,000 cells were acquired.
Results were analyzed using Cellquest software (Becton
Dickinson).
T cells were detected by standard surface staining using a
PE-conjugated anti-human TCRß Ab (clone BMA03; Immunotech,
Marseille, France).
Cytokine measurement
For cytokine measurement, 105 T cells and 5 x
104 autologous, irradiated-EBV-B cells were mixed in 200
µl of complete RPMI-FCS medium containing appropriate Ag
concentration. After overnight incubation, supernatants were collected
and tested for the concentration of IL-2, IFN-, and IL-4. IL-2 was
quantified on a IL-2-dependent CTLL line. IFN- was measured by a
sandwich ELISA as described (12). IL-4 was measured using the Duo-Set
ELISA kit provided by Genzyme Diagnostics (Cambridge, MA). Eventually,
cells were kept in culture for another 24 h, and cellular
proliferation was measured by thymidine uptake as described above.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Patients BAM, CAS, and ES participating in this study developed
allergic reactions after treatment with ß-lactam antibiotics with
clinical features as summarized in Table I
. At the time of diagnosis,
they all presented different symptoms after treatment with ß-lactams
as well as positive transformation test in vitro (Table I; Refs. 2 and
4, and data not shown). The Pen G specificity of their allergic
reactions was assessed by radioallergosorbent test and skin tests which
gave clear positive results for donors BAM and ES but results were
negative for donor CAS. For all three donors, an immediate type of
hypersensitivity was diagnosed, associated with delayed-type reactions
for donor BAM.
Primary in vitro cultures were prepared by stimulating purified PBMC
with different doses of Pen G. The hapten was given in 3-fold dilutions
from 3 mg/ml to 0. 1 µg/ml in two parallel cultures; controls were
without addition of Pen G. After 5 days of incubation, the
proliferative response was measured in one of the two cultures by
thymidine incorporation. As shown in Fig. 1, PBMC proliferated in response to Pen G
in the range from 1 to 0.01 mg/ml, depending on the donor. The
intensity of the response was dose-dependent, with maximal
proliferation at 0.3 mg/ml of Pen G for all donors.
|
and IL-4 expression
by flow cytometry. TCRß+ cells constituted 82–90% of
the cell population in all cell lines (Fig. 1). As shown in the right
panels of Fig. 1, PMA and ionomycin induced IFN- secretion even in
cells that had not been stimulated with Pen G. If anything, these
IFN- responses were somewhat reduced at high Pen G concentrations.
In contrast, the number of IL-4-producing cells increased with rising
Pen G concentrations. Comparing left and right panels of Fig. 1, the
expansion of IL-4-producing cells directly correlated with the
intensity of the proliferative response. This indicates that, in our
system, Pen G preferentially induced the recruitment of IL-4-producing
cells from peripheral blood lymphocytes and the extent of their
expansion depended on the dose of hapten used for their induction in
vitro. Interestingly, the preferential expansion of Pen G-specific
IL-4-producing cells was associated with immediate type
hypersensitivity reactions in all the patients analyzed (Table I). Effect of hapten doses on T cell clones
To evaluate the role of Pen G concentration in inducing functional
differentiation of established T lymphocytes, a panel of independent
Pen G-specific T cell clones was prepared. PBMC from donors BAM and ES
were subjected to two rounds of stimulation in vitro with Pen G at
concentrations of 1, 0.3, and 0.1 mg/ml in three independent T cell
lines for each donor. After expansion in IL-2-containing medium, each
line was independently cloned by limiting dilution. A total of 18 Pen
G-specific T cell clones were obtained from donor BAM and 24 from donor
ES. The hapten-specific T cell phenotype in terms of IL-4 and IFN-
production was determined for each T cell clone upon stimulation with
3-fold dilutions of Pen G from 3 mg/ml to 0.03 mg/ml in the presence of
irradiated autologous EBV-B cells. After 24-h incubation, supernatants
were harvested and processed for cytokine measurement, while cells were
kept in culture for another 24 h and T cell proliferation was
measured by thymidine uptake. We then examined IL-4 and IFN-
secretion induced in the presence of the highest and the lowest Pen G
concentration that still triggered cellular proliferation considering
SI values 
2.0 as positive responses. Results are summarized in Table II.
|
at high and low hapten concentration. Only one clone (G0.1F3)
secreted IL-4 in the absence of IFN-. Interestingly, lower Pen G
concentrations induced higher levels of IFN- in 11 of the 18 clones
analyzed. For four of these clones (G1B2, G1D6, G0.3A1, G0.1G4) a
dose-dependent alteration of the cytokine pattern resulted in a
remarkable shift from type 0 to type 1 phenotypes.
Clones from donor ES were less sensitive to the hapten, being
unable to proliferate to Pen G at concentrations <0.3 mg/ml. For these
clones the lowest Pen G doses able to trigger cellular proliferation
were 0.3 mg/ml with SI never exceeding 8.6. The majority of clones ES
mounted type 2 responses and secreted measurable amount of IL-4 only at
the highest dose of Pen G tested (3 mg/ml). None of these clones
produced a detectable amount of IFN-.
The dose of Pen G employed for the priming induction of peripheral
blood T cells did not influence the type of T cell clones obtained.
Rather, it appeared that the individual blood donors imposed a major
bias on the system. Thus, almost all T cell clones from donor BAM, who
suffered from delayed as well as immediate type hypersensitivity
reactions, produced IFN- either alone or together with IL-4. In
contrast, PBMC from donor ES, who was characterized by an immediated
type of hypersensitivity, mainly gave rise to IL-4-producing T cell
clones. However, the concentration of Pen G was indeed able to modulate
the T cell phenotypes. In fact, very high doses of Pen G were required
to induce IL-4 production by ES clones, whereas low doses of hapten
induced higher level of IFN- secretion resulting eventually in a
shift of T cell phenotypes.
Pen G can modulate the T cell phenotype of fully differentiated hapten-specific T cell clones
The data in Table II revealed that the dose of Pen G used for in
vitro restimulation of established clones modulated the T cell
phenotype in some cases. This resulted from the fact that IL-4
secretion increased with rising doses of Pen G, whereas IFN-
production was optimal at low hapten concentrations. This phenomenon
was further examined with two previously established CD4+ T
cell clones.
Clone BAM25 was isolated 2 yr ago in our laboratory and has been already described as Pen G specific (7). Clone 1ES4 was derived from the stimulation of peripheral blood cells of donor ES with the TT peptide sequence 616–630. The two clones were stimulated in vitro with different concentrations of either their specific Ag or the T cell mitogen PHA and cytokines released in the culture supernatant were measured after 24 h incubation.
As shown in Fig. 2, the cytokine pattern
of clone BAM25 strongly depended on the dose of Pen G used for
stimulation. This clone was raised with a dose of Pen G of 1 mg/ml (7)
and behaved in this condition as a Th2 clone, secreting IL-4 but not
IFN-. However, the same clone simultaneously secreted IL-4 and
IFN- in the presence of lower doses (<0.3 mg/ml) of Pen G (Fig. 2A). In contrast, PHA stimulation induced a dose-dependent
production of both IL-4 and IFN- (Fig. 2B), indicating
the importance of the hapten-specific stimulation in defining Th0 or
Th2 profiles.
|
in response
to both the specific Ag as well as the mitogen in a dose-dependent way
(Fig. 2, C and D). Thus, the modulation of the T cell phenotype observed seems to be a particular characteristic of our hapten system. It appears feasible to influence the cytokine profile of long-term established Pen G-reactive T cell clones by altering the stimulatory hapten concentration.
Ag-independent IFN- down-modulation by Pen V
The possibility to modulate the phenotype of even fully differentiated T cell clones could certainly introduce new strategies to manipulate immune responses. However, the data presented above apply exclusively to Pen G specificities. To find a system of more general application, we investigated the possibility of using "nonreactive" penicillin derivatives to modulate different Ag-specific immune reactions.
In a first set of experiments, we studied the effect of Pen V and Amp on the T cell phenotype induced by Pen G in two Pen G-specific CD4+ T cell clones.
Upon stimulation with Pen G, clone BAM25 revealed a dose-dependent
secretion of IL-2, IL-4, and IFN- (Fig. 3, A-C). However,
IL-2 and IL-4 reached plateau values at 0.5–1 mg/ml, and IFN-
secretion was completely shut off at Pen G concentrations 0.3 mg/ml,
as already shown in Fig. 2. Clone ES5.13 produced IL-2 and IL-4 (Fig. 4, A and B) upon
stimulation with Pen G. Neither clone responded to Pen V nor Amp (Fig. 3, A–C and Fig. 4, A and
B, respectively). We then used graded concentrations of Pen
V and Amp together with a fixed dose of 0.25 mg/ml of Pen G for T cell
stimulation and measured the cytokine pattern secreted in the culture
supernatants after 24 h incubation.
|
|
, D–F. Addition of Amp did not affect the Pen
G-induced cytokine pattern except for a slight reduction of IFN-
secretion at high concentration (Fig. 3, D–F).
In contrast, Pen V reduced the amount of IL-2 of about 50% (Fig. 3D) and completely abrogated IFN- production at
concentrations 0.1 mg/ml (Fig. 3F) without affecting IL-4
secretion (Fig. 3E). The same type of assay, performed on
clone ES5.13, revealed no interference of either Pen V or Amp on Pen
G-induced IL-2 secretion (Fig. 4C) and only a marginal
reduction of IL-4 production at high Pen V concentration (Fig. 4D).
Considering the structural similarities of the different penicillin
derivatives, one might expect that the strong effect of Pen V on
IFN- production was due to antagonistic interactions of Pen V with
the Ag-binding sites of the Pen G-specific TCR. To test this
possibility, we performed the same assay described above with the
TT-specific T cell clone 1ES4. Upon stimulation with either the peptide
sequence TT 616–630 at 1 µg/ml and PHA at 0.3 µg/ml, this clone
has a classical Th1 phenotype (Fig. 2). Surprisingly, even for these
reactivities, the addition of Pen V at 0.1 mg/ml completely abrogated
IFN- secretion in response to either the specific Ag (TT peptide
616–630; Fig. 5B) and the
mitogen (PHA; Fig. 5D), whereas IL-2 production was
unaffected (Fig. 5, A and C). Again, addition of
Amp had no effect (Fig. 5).
|
secretion could be induced by
the addition of Pen V on two T cell clones of totally different
specificities, i.e., Pen G (clone BAM25) or TT (clone 1ES4). Therefore,
the effect observed is apparently independent of interactions of Pen V
with the TCR, and due to a direct interference of Pen V with IFN-
secretion pathways. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we focused on the human allergic response to ß-lactam antibiotics, investigating the influence of hapten doses first on the recruitment in vitro of drug-specific cells from PBMC of allergic donors and also on the induction of T cell phenotype of established-T cell clones.
All the donors participating in this study suffered from immediate type hypersensitivity to ß-lactams and in particular to Pen G. Upon in vitro stimulation with different doses of Pen G, we found a preferential recruitment of IL-4-producing peripheral blood-derived T cells. This expansion was maximal for high to middle doses of hapten (around 1–0.1 mg/ml concentration) and directly correlated with the proliferative responses of the cultures. This suggests that the drug-specific T cells contributing to the adverse reactions were mainly of type 2 phenotype, as expected from the clinical characteristics of all the donors. Remarkably, this effect was particularly evident for donor CAS who developed anaphylactic shock after treatment with ß-lactams.
The role of Pen G doses in inducing functional differentiation of
established T lymphocytes was analyzed on a panel of 42 independent T
cell clones isolated from PBMC of penicillin allergic donors after two
cycles of restimulation with Pen G. We found that the concentration of
Pen G during in vitro induction did not affect the type of clones
isolated; the concentration depended only on the individual
characteristics of the donor studied. However, once T cell clones had
been isolated and expanded, the dose of Pen G used for stimulation was
indeed a critical factor, determining their functional, hapten-specific
phenotype. In fact, high doses of Pen G (3 mg/ml) induced optimal IL-4
secretion and the isolated type 2 clones behaved as "inert" cells
at low hapten concentrations. On the other hand, low Pen G
concentrations induced higher levels of IFN- secretion; as a
consequence, 20% of the clones isolated from donor BAM shifted their
phenotype from type 0 to type 1 at low Pen G concentrations (Table II).
Remarkably, such a phenotype shift was not observed with a TT-specific
CD4+ clone (i.e., clone 1ES4) and not even by PHA
stimulation (Fig. 2). Moreover, the phenomena observed depended mainly
on the modulation of IFN- secretion, as shown in detail for clone
BAM25 that could secrete IFN- only at a Pen G concentration
0.3 mg/ml (Fig. 2).
For a correct interpretation of these data, one should consider how hapten-derived epitopes are formed and how the strength of their interaction with specific TCR is determined, in comparison to nominal peptidic Ag (13, 14). On a given APC, the density of the epitopes formed by hapten modification will depend on at least two factors: the number of modifiable sites and the concentration of reactive-hapten molecules. At saturation, the density of the epitopes will not increase with the concentration of the hapten. On the other hand, multiple modifications might interfere with the strength of TCR ligation. We already found that a peptide sequence carrying multiple penicilloyl groups was a poor stimulator compared with designer penicilloyl-peptides that carry a single modified lysine residue for the same clone (15). Taken together, these considerations suggest that for drug-specific immune responses, there is no direct correlation between the concentration of hapten molecules, epitope density, and strength of TCR engagement, because reactive haptens may not only create, but also destroy antigenic epitopes.
Several recent studies focus on the identification of factors and signals able to reverse Th responses (16, 17). In our system, the phenotype modulation observed for Pen G-specific immune responses is most probably TCR mediated.
In the last set of experiments presented, we could also identify at
least one penicillin derivative able to induce a modulation of the T
cell phenotype, most likely with a TCR-independent mechanism. In fact,
a dose-dependent inhibition of IFN- production, was observed when
Pen V was added to manipulate a Pen G-specific immune response (clone
BAM25 in Fig. 3), a TT-specific response, or even a mitogen-induced
phenotype (clone 1ES4 in Fig. 5). In all these systems, we observed a
down-regulation of IFN- secretion with no effect on IL-4 production.
The different specificities that could be manipulated speak in favor of
TCR-independent mechanisms for Pen V-mediated IFN- down-modulation.
A similar effect has very recently been described for other compounds
such as pyridinyl imidazole drugs. One of these compounds, SB203580,
induces selective IFN- down-modulation in mouse Th1 cells through
specific inhibition of p38 mitogen-activated protein kinases (18). It
is tempting to speculate that similar targets could also be modified by
Pen V.
Taken together, the data presented indicate that penicillins can
modulate the T cell phenotype of fully differentiated T cell clones in
vitro with Ag-dependent and -independent mechanisms (Fig. 6), thus potentially presenting new tools
for immune intervention. In particular, the possibility to selectively
down-modulate IFN- production via TCR-independent pathways offers a
broad application for therapeutic intervention in autoimmune diseases
and other pathological situations in which a predominant Th1 profile is
found.
|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: Pen G, penicillin G; Pen V, penicillin V; Amp, ampicillin; TT, tetanus toxoid; HS, human serum; EBV-B, EBV-transformed B lymphocytes.
Received for publication July 13, 1998. Accepted for publication September 24, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß-transgenic model. J. Exp. Med. 182:1579. expression by Th1 effector T cells mediated by the p38 MAP kinase signalling pathway. EMBO J. 17:2817.[Medline]
This article has been cited by other articles:
|
|
 |
|
  T. Shimizu, Y. Osaka, C. Banri-Koike, M. Yoshida, K. Endo, K. Furukawa, M. Oda, A. Murakami, S. Ogawa, R. Abe, et al. T cells specific to hapten carrier but not to carrier alone assist in the production of anti-hapten and anti-carrier antibodies Int. Immunol., October 1, 2007; 19(10): 1157 - 1164. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
