Characterization of Clozapine-Responsive Human T Cells.

Use of the atypical antipsychotic clozapine is associated with life-threatening agranulocytosis. The delayed onset and the association with HLA variants are characteristic of an immunological mechanism. The objective of this study was to generate clozapine-specific T cell clones (TCC) and characterize pathways of T cell activation and cross-reactivity with clozapine metabolites and olanzapine. TCC were established and characterized by culturing PBMCs from healthy donors and patients with a history of clozapine-induced agranulocytosis. Modeling was used to explore the drug-HLA binding interaction. Global TCC protein changes were profiled by mass spectrometry. Six well-growing clozapine-responsive CD4+ and CD8+ TCC were used for experiments; activation of TCC required APC, with clozapine interacting directly at therapeutic concentrations with several HLA-DR molecules. TCC were also activated with N-desmethylclozapine and olanzapine at supratherapeutic concentrations. Marked changes in TCC protein expression profiles were observed when clozapine treatment was compared with olanzapine and the medium control. Docking of the compounds into the HLA-DRB1*15:01 and HLA-DRB1*04:01 binding clefts revealed that clozapine and olanzapine bind in a similar conformation to the P4-P6 peptide binding pockets, whereas clozapine N-oxide, which did not activate the TCC, bound in a different conformation. TCC secreted Th1, Th2, and Th22 cytokines and effector molecules and expressed TCR Vß 5.1, 16, 20, and 22 as well as chemokine receptors CXCR3, CCR6, CCR4, and CCR9. Collectively, these data show that clozapine interacts at therapeutic concentrations with HLA-DR molecules and activates human CD4+ T cells. Olanzapine only activates TCC at supratherapeutic concentrations.

Brief Report: Drugs Implicated in Systemic Autoimmunity Modulate Neutrophil Extracellular Trap Formation.

OBJECTIVE: Aberrant neutrophil extracellular trap (NET) formation has been implicated as a mechanism to induce autoreactivity in individuals at risk of autoimmune diseases. The objective of this study was to assess whether medications implicated in cases of drug-induced autoimmunity (hydralazine and procainamide) and medications less commonly associated with drug-induced autoimmunity (minocycline and clozapine) induce NET formation and/or prevent NET degradation. METHODS: Human neutrophils were incubated with the drugs of interest and resultant NET formation was quantified by fluorescent microscopy. The ability of these drugs to interfere with NET degradation by serum nuclei was assessed. Pathways of drug-induced NET formation were studied with pharmacologic inhibitors of reactive oxygen species (ROS), peptidylarginine deiminases (PADs), and muscarinic receptors, and by assessment of intracellular calcium levels by flow cytometry. To determine if NET protein cargo varies by drug stimulus and/or neutrophil source, proteomic analysis of NET lysates induced by specific medications was compared using neutrophils from healthy donors and from patients with autoimmune diseases. RESULTS: Hydralazine and procainamide significantly induced NET formation while minocycline and clozapine did not. None of the medications significantly impaired NET degradation. NETosis induced by these drugs required NADPH oxidase and PAD4 activation. Procainamide triggered NETs via muscarinic receptor engagement on neutrophils, while hydralazine modulated calcium release from intracellular stores. Differences in protein cargo, particularly histone content, were observed in NETs induced by hydralazine and procainamide. CONCLUSION: Medications commonly implicated in drug-induced autoimmunity trigger NET formation displaying distinct protein cargo, via common and specific pathways. NETosis may play a role in the pathogenesis of drug-induced autoimmunity.

Assessment of possible immunotoxicity of the antipsychotic drug clozapine.

OBJECTIVES: The immunomodulatory effects of clozapine (CLZ), antipsychotic drug, were investigated in vivo using female Balb/c mice. The main aim of this study was to evaluate the immunomodulatory effects of CLZ, antipsychotic drug, following daily intraperitoneal injection to female Balb/c mice over a period of 21 days. METHODS: Mice were divided into five groups, eight animals per group. Group I, served as a control group, received only the vehicle. Groups II-V received a daily intraperitoneal dose of CLZ (1, 5, 10 and 20 mg/kg, respectively) over a period of 21 days. KEY FINDINGS: CLZ has shown a significant decrease in the animal body weight, and it showed a significant decrease in the percentage of circulating neutrophils and lymphocytes while circulating monocytes were increased. The immunotoxicity has been also assessed by evaluating spleen cellularity, humoral immune response to a foreign antigen using sheep red blood cells and delayed-type hypersensitivity reaction. The results showed a marked suppression in these responses in CLZ-treated mice compared with the control group. Detectable changes have also been noticed in the histology of the footpad tissue and spleen. CONCLUSIONS: Results showed significant immunomodulatory effects of CLZ when used in Balb/c mice.

Increased serum interleukin-8 and interleukin-10 in schizophrenic patients resistant to treatment with neuroleptics and the stimulatory effects of clozapine on serum leukemia inhibitory factor receptor.

There is now evidence that schizophrenia may be accompanied by an activation of the monocytic and T-helper-2 (Th-2) arms of cell-mediated immunity (CMI) and by various alterations in the Th-1 arm of CMI. There is also evidence that repeated administration of typical and atypical antipsychotics may result in negative immunomodulatory effects. This study was carried out to examine (1) the serum concentrations of interleukin-8 (IL-8), IL-10, the soluble CD8 (sCD8) and the leukemia inhibitory factor receptor (LIF-R) in nonresponders to treatment with typical neuroleptics as compared with normal volunteers and responders to treatment; and (2) the effects of atypical antipsychotics on the above immune variables. The latter were determined in 17 nonresponders to treatment with neuroleptics and in seven normal volunteers and 14 schizophrenic patients who had a good response to treatment with antipsychotic agents. The nonresponders had repeated measurements of the immune variables before, and 2 and 4 months after treatment with clozapine or risperidone. Serum IL-8 and IL-10 were significantly higher in schizophrenic patients than in normal controls. The serum concentrations of the sCD8 were significantly increased 2 months, but not 4 months, after starting treatment with atypical antipsychotics. Serum LIF-R concentrations were significantly increased 2 and 4 months after starting treatment with atypical antipsychotics. It is concluded that: (1) schizophrenia is characterized by an activation of both pro-inflammatory and anti-inflammatory aspects of cell-mediated immunity; (2) prolonged treatment with atypical antipsychotics may increase the anti-inflammatory capacity of the serum in schizophrenic patients by increasing serum LIF-R concentrations; and (3) short-term treatment with clozapine may induce signs of immune activation which disappear upon prolonged treatment.

Bioactivation and covalent binding of hydroxyfluperlapine in human neutrophils: implications for fluperlapine-induced agranulocytosis.

The use of fluperlapine and the structurally related clozapine has been associated with the induction of agranulocytosis in humans. Unlike clozapine, fluperlapine is relatively resistant to chemical and biochemical oxidations. In this study we demonstrated that 7-hydroxyfluperlapine, the major metabolite of fluperlapine in humans, is oxidized to a reactive intermediate by HOCl and by myeloperoxidase in the presence of H(2)O(2) and Cl(-). This reactive intermediate was identified as an iminoquinone species with a M + 1 ion at m/z 324 by mass spectrometry. The iminoquinone intermediate was trapped by N-acetyl-L-cysteine (NAC) as well as GSH. NMR spectra of the NAC adducts indicated that the NAC was bound to the 6 and 9 positions of the aromatic ring. This is the same orientation as the binding of nucleophiles to the reactive metabolite of clozapine. We were able to use an antibody against clozapine to demonstrate that 7-hydroxyfluperlapine, but not fluperlapine itself, covalently modifies human myeloperoxidase. Furthermore, we demonstrated that 7-hydroxyfluperlapine is metabolized by activated neutrophils to a reactive intermediate that covalently binds to neutrophils. In the presence of NAC or GSH, such covalent binding was inhibited and the NAC or GSH adducts were formed. Thus, the reactivity and even the orientation of the binding of the reactive metabolite of 7-hydroxyfluperlapine is very similar to that of clozapine. These results provide a mechanism for the formation of a reactive metabolite of fluperlapine similar to clozapine that may explain its ability to induce agranulocytosis.

51Cr release assay of clozapine-induced cytotoxicity: evidence for immunogenic mechanism.

Serum drawn from patients during clozapine-induced agranulocytosis was toxic to human polymorphonuclear leukocytes (PMNs). Toxicity was produced by an immunoglobulin fraction, predominantly of the IgM class. The offending drug was not necessary at this stage to produce cytotoxicity. These effects were observed by inhibiting post-phagocytosis glycolysis, by ejection of trypan blue, or by enhanced release of 51Cr from lysed-labeled PMNs. Direct chemical toxicity, produced by clozapine or its metabolites, was tested by similar procedures. At a concentration of 10(-5) M in a colloidal milieu produced by dilution with AB serum, no cytotoxicity was observed; however, in aqueous medium. N-desmethylclozapine was toxic to PMNs and proliferating lymphocytes. Post-recovery serum appeared to be inert, but cytotoxicity was restored by adding clozapine or N-desmethylclozapine to the sensitive patient's serum. At this stage, cytotoxicity as measured by 51Cr release was abrogated by anti-IgG or anti-IgM. These relationships favor an immunologic mechanism that damages peripheral PMNs. Development of colony-forming units-granulocyte (CFU-G) was similarly inhibited in normal marrow cultures by cytotoxic serum alone, whereas no metabolite had such an effect at the same concentration (10(-5) M).

Clozapine-induced agranulocytosis in a Native American: HLA typing and further support for an immune-mediated mechanism.

BACKGROUND: Approximately 30% of schizophrenic patients defined as treatment refractory significantly improve with clozapine. However, clozapine produces agranulocytosis in approximately 1% to 2% of patients in the United States. The mechanism of clozapine-induced agranulocytosis has not been established, but evidence suggests an immune-mediated mechanism. METHOD: Human leukocyte antigen (HLA) typing was performed in a native American with clozapine-induced agranulocytosis. RESULTS: Our findings support previous observations of a role of the HLA-B16, DR4, DQw3 haplotype in predicting susceptibility to agranulocytosis in clozapine-treated patients. CONCLUSION: We suggest that HLA typing of clozapine candidates may be useful for predicting the risk for clozapine-induced agranulocytosis.

Clozapine-induced agranulocytosis: non-cross-reactivity with other psychotropic drugs.

Clozapine, an atypical neuroleptic with unique clinical and preclinical properties, represents a potentially valuable addition to the psychopharmacopeia. Its development and use have been limited by its higher frequency, compared with other pharmacologic treatments, of the potentially fatal side effect of agranulocytosis. This article describes the natural history of five cases of agranulocytosis that occurred in the course of clozapine treatment. The cases were generally uniform as to onset, recovery, and hematologic features. No patient had hematologic reactions to treatment with psychotropic agents before or after clozapine treatment. These findings, along with other work in progress, suggest that clozapine's granulocytoxic effects are produced by a highly specific immune-mediated mechanism.