| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 27, 2001 as doi:10.1096/fj.00-0583fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Department of Pharmacology and Toxicology,
Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada;
Department of Medical Biochemistry, Arabian Gulf University, Manama, Bahrain; and
|| Department of Paediatrics, Children’s Hospital of Western Ontario, University of Western Ontario, Gene Therapy and Molecular Virology Group, John P. Robarts Research Institute, London, Ontario, Canada
2Correspondence: Departments of Paediatrics and Pharmacology and Toxicology, Children’s Hospital of Western Ontario, University of Western Ontario, 800 Commissioners Rd. E., London, Ontario, Canada, N6J 1Y5. E-mail: mrieder{at}julian.uwo.ca
SPECIFIC AIMS
The hydroxylamine derivative of sulfamethoxazole (SMX-HA) is a critical determinant in the pathogenesis of serious hypersensitivity reactions to the sulfonamides; SMX-HA been shown to produce suppression of T cell activation in concentrations achieved during therapy and alterations in immunity associated with sulfonamide reactive metabolites have been postulated as key elements in the pathogenesis of sulfonamide hypersensitivity. We studied the hypothesis that sulfonamide reactive metabolites in sublethal concentrations alter cytokine production and interfere with cytokine receptor-mediated signal transduction in vitro and that this response favored a Th1 type cell-mediated response.
PRINCIPAL FINDINGS
1. SMX-HA does not reduce cytokine message and significantly
reduced the production of tumor necrosis factor 
(TNF-),
interleukin ß (IL-1ß), and IL4 protein without inhibiting the
production of interferon 
(IFN-)
Incubation of mitogen-stimulated human peripheral blood
mononuclear cells (PBMCs) with sublethal concentrations of SMX-HA (<25
mm) did not inhibit mRNA expression for IL-2, IL-4, IL-5, IL-10, IL-13,
or IFN- (Fig. 1
). This was quantified by densitometry and confirmed with RT-PCR
detection of cytokine mRNA expression (data not shown). Incubation at
these concentrations had no effect on cell viability or on the
steady-state production of message for ß-actin. However, sublethal
concentrations of SMX-HA produced significant reductions in supernatant
cytokine concentrations of TNF- and IL-1ß measured by
cytokine-specific ELISA (22 and 8% of control, respectively).
Production of the Th2 type cytokine IL-4 was also significantly reduced
(25% of control) at an SMX-HA concentration of 25 mm, whereas
production of IFN- was not affected except at lethal concentrations
(Fig. 1a
, b
).
|
These results indicated that reactive sulfonamide metabolites do not
inhibit message cytokines but significantly inhibit cytokine
production, notably the inflammatory cytokines TNF-, IL-1ß, and
the Th2 type cytokine IL-4. This inhibition occurs at concentrations
that exist in patients during therapy. In contrast, there was no
inhibition of the Th1 type cytokine IFN- at these concentrations,
suggesting a shift in cytokine balance to favor a cell-mediated
response.
2. SMX-HA inhibits phosphorylation of Jak 1 and Jak 3 and
downstream phosphorylation of Stat3 and Stat5a
Incubation of mitogen-stimulated human PBMCs with SMX-HA from 0 to
25 mm produced concentration-dependent inhibition of tyrosine
phosphorylation of IL-2 receptor chain (Fig. 2a
) without interfering with IL-2 R–Jak 3 association
(Fig. 2b
). When SMX-HA from 0 to 25 mm was incubated with
mitogen-stimulated PBMCs, there was a concurrent
concentration-dependent inhibition of tyrosine phosphorylation of Jak 3
(Fig. 2c
), which occurred without a decrease in the amount
of Jak 3 immunoprecipitated (Fig. 2d
). Similarly, incubation
of 0 to 25 mm of SMX-HA with mitogen-stimulated human PBMCs produced
inhibition of Stat 3 phosphorylation (Fig. 2e
) with no
effect on the amount of Stat 3 immunoprecipitated (Fig. 2f
). Densitometry of Jak 3 and Stat 3 phosphorylation
(Fig. 2g
, h
) to correct for the total amount of Jak 3 and
Stat 3 protein present confirmed the concentration-dependent inhibition
of phosphorylation by SMX-HA. Similar reductions in the tryosine
phosphorylation of Jak 1 and Stat 5a were also observed (data not
shown).
|
CONCLUSIONS
Adverse events related to drug therapy are among the top six causes of death in North America. The pathogenesis of many serious adverse drug reactions (ADRs) has been poorly understood, notably for events such as severe idiosyncratic hypersensitivity reactions. Sulfonamide antibiotics are among the most common inducers of idiosyncratic hypersensitivity, especially in the setting of AIDS. Studies investigating sulfonamide ADRs have identified the hydroxylamine (SMX-HA) metabolite, produced in vivo by oxidative metabolism, as a major component of sulfonamide hypersensitivity.
Progression of sulfonamide ADRs after drug bioactivation appears to be modulated by the immune system. Clinically relevant concentrations of SMX-HA have been associated with significant immunomodulatory effects in vitro, including concentration-dependent inhibition of mitogen-stimulated PBMC proliferation. The precise mechanisms by which SMX-HA induces immunomodulatory effects on cells of the immune system have been unclear, especially with respect to effector cytokine production and/or interference with cytokine signal transduction pathways. Due to the effects of SMX-HA in combination with other immunosuppressive agents and the lack of inhibition of IL-2 production previously reported, we postulated that SMX-HA may be interfering with the lymphocyte response to IL-2.
The Th1 and Th2 dichotomy provides an important functional division in
the immune system. SMX-HA significantly reduced mitogen-stimulated PBMC
production and/or secretion of the Th2 type cytokine IL-4 and was
equally effective at inhibiting the production of inflammatory
cytokines IL-1ß and TNF-. However, sublethal concentrations of
SMX-HA did not reduce PBMC production of the Th1 type cytokine IFN-,
which suggests that exposure of cells to reactive sulfonamide
metabolites may lead to a heightened Th1 type cell-mediated response on
exposure to antigen.
These results and previous studies documenting the antiproliferative
effects of SMX-HA support the role of SMX-HA as an immune-modulating
agent capable of interfering with cytokine production at sublethal
concentrations in vitro and provide background for defining the role of
reactive metabolites in the propagation of sulfonamide hypersensitivity
reactions in a clinical setting (Fig. 3
). Under conditions of high-dose therapy, clinically relevant
concentrations of reactive metabolites may be reached in vivo. Cells of
the immune system exposed to SMX-HA would be anticipated to remain
viable and at the same time functionally compromised in terms of
intercellular cytokine communication. Interference in normal immune
cell function may have deleterious effects on special populations of
cells of immunocompromised individuals (such as patients with AIDS) and
may be important in the increased rates of adverse reactions to
sulfonamide therapy in these patients. SMX-HA binding to cellular
constituents may provide a mechanism by which an immune response is
initiated against hydroxylamine labeled proteins, leading to adverse
reactions to sulfonamides in sensitized individuals.
|
Traditional immunosuppressive agents act by blocking production of
message for stimulatory cytokines. Unlike these agents, SMX-HA did not
significantly alter the accumulation of major stimulatory cytokine
mRNAs (Fig. 1a
), suggesting that SMX-HA induces
antiproliferative effects through an action after T cell
receptor-mediated up-regulation of cytokine transcription. Until this
report, the specific T cell targets for hydroxylamine binding have
remained an unknown component of the mechanism of SMX-HA immune
suppression. Here we illustrate for the first time that SMX-HA prevents
IL-2R-mediated Jak-Stat phosphorylation in human PBMCs in vitro.
The Jak-Stat pathway is an essential component for type I cytokine receptor signaling and represents an extremely rapid membrane to nucleus system for the transcriptional activation of target genes involved in cell survival, growth, differentiation, and proliferation. Thus, SMX-HA-induced interference in activation of the IL-2R-mediated Jak-Stat pathway would provide a molecular mechanism to explain the profound effect SMX-HA treatment has on cytokine production and lymphocyte proliferation.
Jak1 and Jak3 associate with the IL-2Rß and IL-2R chain,
respectively, via the kinase amino-terminal region and the receptor
membrane proximal box1/box2 domain. SMX-HA did not interfere with Jak1
or Jak3 association with the IL-2Rß or IL-2R chains, respectively,
in PHA-stimulated PBMCs. Despite the availability of both Jak1 and Jak3
to the receptor, rIL-2 stimulation of SMX-HA-treated cells displayed
greatly decreased levels of Jak3 phosphorylation and an absence of Jak1
phosphorylation at 25 mM SMX-HA. Reduced Jak phosphorylation occurred
whereas high levels of Jak1 and Jak3 were available for phosphorylation
in rIL-2-stimulated, SMX-HA-treated samples.
Phosphorylated tyrosine on the IL-2Rß and IL-2R chains provide
docking sites for the recruitment of Stat molecules to the IL-2R
complex. Stat3 and Stat5a/b molecules are most consistently and
prominently phosphorylated by the Jak molecules involved in IL-2R
signaling. SMX-HA at 25 mM significantly inhibited phosphorylation of
both Stat3 and Stat5a molecules, respectively. This observation
directly links Jak activation to Stat phosphorylation and implicates
SMX-HA as a potential inhibitor of gene expression involved in cell
proliferation. Transcriptional regulation of genes is typically
achieved by the coordinated effects of multiple factors, making it
necessary to understand how inhibition of the Jak-Stat pathway or other
pathways affected by SMX-HA integrate into the complex program of cell
proliferation.
This work demonstrates that reactive sulfonamide metabolites inhibit
production of signals through the IL-2R-mediated Jak-Stat pathway,
which could lead to the declines observed in the production of specific
secondary cytokines. These changes may then favor an immune adaptation,
including selective apoptosis, leading to tolerance of therapy (Fig. 3)
; in the case of imbalances in production and detoxication of
reactive metabolites, this provides a mechanistic framework for
understanding how these can lead to altered immunity expressed
clinically as sulfonamide hypersensitivity.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0583fje ; to cite this article, use FASEB J. (June 27, 2001) 10.1096/fj.00-0583fje 
This article has been cited by other articles:
|
|
 |
|
  D. Lin, M J. Tucker, and M. J Rieder Increased Adverse Drug Reactions to Antimicrobials and Anticonvulsants in Patients with HIV Infection Ann. Pharmacother., September 1, 2006; 40(9): 1594 - 1601. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
