As targets for the immunosuppressive drugs cyclosporin A and FK506, transcription factors of the NFAT (nuclear factor of activated T cells) family have been the focus of much attention. NFAT proteins, which are expressed in most immune-system cells, play a pivotal role in the transcription of cytokine genes and other genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the calcium/calmodulin-dependent phosphatase calcineurin, a primary target for inhibition by cyclosporin A and FK506. Calcineurin controls the translocation of NFAT proteins from the cytoplasm to the nucleus of activated cells by interacting with an N-terminal regulatory domain conserved in the NFAT family. The DNA-binding domains of NFAT proteins resemble those of Rel-family proteins, and Rel and NFAT proteins show some overlap in their ability to bind to certain regulatory elements in cytokine genes. NFAT is also notable for its ability to bind cooperatively with transcription factors of the AP-1 (Fos/Jun) family to composite NFAT:AP-1 sites, found in the regulatory regions of many genes that are inducibly transcribed by immune-system cells. This review discusses recent data on the diversity of the NFAT family of transcription factors, the regulation of NFAT proteins within cells, and the cooperation of NFAT proteins with other transcription factors to regulate the expression of inducible genes.

TRANSCRIPTION FACTORS OF THE NFAT FAMILY:Regulation and Function

Group A streptococci are model extracellular gram-positive pathogens responsible for pharyngitis, impetigo, rheumatic fever, and acute glomerulonephritis. A resurgence of invasive streptococcal diseases and rheumatic fever has appeared in outbreaks over the past 10 years, with a predominant M1 serotype as well as others identified with the outbreaks. emm (M protein) gene sequencing has changed serotyping, and new virulence genes and new virulence regulatory networks have been defined. The emm gene superfamily has expanded to include antiphagocytic molecules and immunoglobulin-binding proteins with common structural features. At least nine superantigens have been characterized, all of which may contribute to toxic streptococcal syndrome. An emerging theme is the dichotomy between skin and throat strains in their epidemiology and genetic makeup. Eleven adhesins have been reported, and surface plasmin-binding proteins have been defined. The strong resistance of the group A streptococcus to phagocytosis is related to factor H and fibrinogen binding by M protein and to disarming complement component C5a by the C5a peptidase. Molecular mimicry appears to play a role in autoimmune mechanisms involved in rheumatic fever, while nephritis strain-associated proteins may lead to immune-mediated acute glomerulonephritis. Vaccine strategies have focused on recombinant M protein and C5a peptidase vaccines, and mucosal vaccine delivery systems are under investigation.

Pathogenesis of Group A Streptococcal Infections

Lipopolysaccharide (LPS) is lethal to animals because it activates cytokine release, causing septic shock and tissue injury. Early proinflammatory cytokines (e.g., tumor necrosis factor [TNF] and interleukin [IL]-1) released within the first few hours of endotoxemia stimulate mediator cascades that persist for days and can lead to death. High mobility group 1 protein (HMG-1), a ubiquitous DNA-binding protein, was recently identified as a “late” mediator of endotoxin lethality. Anti–HMG-1 antibodies neutralized the delayed increase in serum HMG-1, and protected against endotoxin lethality, even when passive immunization was delayed until after the early cytokine response. Here we examined whether HMG-1 might stimulate cytokine synthesis in human peripheral blood mononuclear cell cultures. Addition of purified recombinant HMG-1 to human monocyte cultures significantly stimulated the release of TNF, IL-1α, IL-1β, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1α, and MIP-1β; but not IL-10 or IL-12. HMG-1 concentrations that activated monocytes were within the pathological range previously observed in endotoxemic animals, and in serum obtained from septic patients. HMG-1 failed to stimulate cytokine release in lymphocytes, indicating that cellular stimulation was specific. Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation. Computer-assisted image analysis demonstrated that peak intensity of HMG-1–induced cellular TNF staining was comparable to that observed after maximal stimulation with LPS. Administration of HMG-1 to Balb/c mice significantly increased serum TNF levels in vivo. Together, these results indicate that, like other cytokine mediators of endotoxin lethality (e.g., TNF and IL-1), extracellular HMG-1 is a regulator of monocyte proinflammatory cytokine synthesis.

https://rupress.org/jem/article-pdf/192/4/565/1129482/000676.pdf

High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes.

Respiratory exposure to allergen induces T cell tolerance and protection against the development of airway hyperreactivity and asthma. However, the specific mechanisms by which tolerance is induced by respiratory allergen are not clear. We report here that pulmonary dendritic cells (DCs) from mice exposed to respiratory antigen transiently produced interleukin 10 (IL-10). These phenotypically mature pulmonary DCs, which were B-7hi as well as producing IL-10, stimulated the development of CD4+ T regulatory 1–like cells that also produced high amounts of IL-10. In addition, adoptive transfer of pulmonary DCs from IL-10+/+, but not IL-10−/−, mice exposed to respiratory antigen induced antigen-specific unresponsiveness in recipient mice. These studies show that IL-10 production by DCs is critical for the induction of tolerance, and that phenotypically mature pulmonary DCs mediate tolerance induced by respiratory exposure to antigen.

Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen.

Toxic epidermal necrolysis (TEN, Lyell's syndrome) is a severe adverse drug reaction in which keratinocytes die and large sections of epidermis separate from the dermis. Keratinocytes normally express the death receptor Fas (CD95); those from TEN patients were found to express lytically active Fas ligand (FasL). Antibodies present in pooled human intravenous immunoglobulins (IVIG) blocked Fas-mediated keratinocyte death in vitro. In a pilot study, 10 consecutive individuals with clinically and histologically confirmed TEN were treated with IVIG; disease progression was rapidly reversed and the outcome was favorable in all cases. Thus, Fas-FasL interactions are directly involved in the epidermal necrolysis of TEN, and IVIG may be an effective treatment.

https://science.sciencemag.org/content/sci/282/5388/490.full.pdf

Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin.

Cytokine production needs to be evaluated by multiple approaches since all existing methods for quantification are hampered by various drawbacks. Bioassays measure functional properties, but they are rarely monospecific and the results are influenced by inhibitory factors. Techniques based on ELISA and RIA systems are highly specific but do not distinguish between biologically active and inactive substances. Furthennore, these assays determine the extracellular presence of a secreted interieukin, reflecting the net outcome of produced, absorbed and degraded cytokine. The demonstration of a receptor-directed focusing of lymphokine release by helper T cells (Poo et al. 1988) argues for direct cellular approach to cytokine assessment. The use of in situ hybridization offers a highly sensitive method to study cytokine production in individual cells, but the technqiue is fairly time-consuming and the presence of cytokine mRNA does not guarantee that it will be translated.

Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure

Previous studies have suggested a central role for superantigen-induced immune responses in the pathogenesis of streptococcal toxic shock syndrome. The production of streptococcal superantigens by clinical group A streptococcal (GAS) isolates was studied, and the ability of plasma collected from patients with severe invasive GAS infections to neutralize the proliferative- and cytokine-inducing activities of these superantigens was investigated. Overnight culture supernatants from all GAS isolates obtained from patients with invasive disease were found to contain superantigenic activity, as evident from their ability to drive potent T cell proliferation, induce high production of cytokines, and stimulate T cells in a V beta-specific manner. Twelve patients with severe invasive GAS infections, including 11 streptococcal toxic shock syndrome cases and one necrotizing fasciitis without shock, were treated with i.v. infusions of normal polyspecific Ig (IVIG). Plasma samples collected from each patient before and after IVIG administration were analyzed for their ability to neutralize the activity of streptococcal superantigens produced by the GAS isolate that caused their disease. In all IVIG-treated patients, the capacity to neutralize the superantigenic activity, produced by their respective GAS isolate or by purified streptococcal pyrogenic exotoxins, increased in plasma following IVIG administration. Of particular clinical relevance, post-IVIG plasma from each patient completely blocked cytokine production elicited by their respective GAS culture supernatants or by purified streptococcal pyrogenic exotoxins. This study shows that IVIG treatment confers in vivo inhibitory activity against a large array of streptococcal superantigens and suggests that IVIG may be useful in the treatment of severe invasive streptococcal infections.

Plasma from patients with severe invasive group A streptococcal infections treated with normal polyspecific IgG inhibits streptococcal superantigen-induced T cell proliferation and cytokine production.

Bacterial toxins constitute one group of many virulence factors by which bacteria cause disease. Advances in our knowledge of bacterial toxins concern their interaction with the host's immune system, in particular by inducing release of cytokines. The latter effect has been studied both in vitro and in vivo particularly in regard to the actions of endotoxin, the lipopolysaccharide (LPS) component of Gramnegative bacteria. It is capable of inducing fever, shock and the acute-phase response in human patients {Peavy et al. 1970, Berry 1977). Many of these effects are caused by the capacity of endotoxin to induce cytokines with pyrogenic activity {Dinarello 1984, Lasfargues et al. 1987, Beutler & Cerami 1989, Cannon et al. 1990). Structurally, LPS consists of three regions: Hpid A, core oligosaccharide and 0-polysaccharide (Luderitz et al. 1984, Warner et al. 1985). LPS is a potent activator of macrophages in their production of monokines (Hamilton & Adams 1987, Prpic et al. 1987). Though there are many candidates for LPS ligands on the cellular surface, the mechanism of endotoxicity remains unclear One receptor for LPS complexted with its binding protein is the CD14 structure (Wright et al. 1990). Exotoxins produced by Gram-positive bacteria have long been recognized as human pathogens, but plausible mechanisms of toxicity have only recently been revealed. The toxins comprise a large group of proteins produced by several different types of bacteria including staphylococci, streptococci and mycoplasma and are involved in different clinical manifestations. A group of toxins produced by various strains of Staphylococcus aureus, staphyloccocal enterotoxin A, B, C]_3, D, E (SEA-E), exfoliating toxins A + B and TSST-1, caused food poisoning,

Bacterial toxin-induced cytokine production studied at the single-cell level.

The production of interleukin 2 (IL 2), IL 4 and interferon-gamma (IFN-gamma) by in vitro activated unselected human blood mononuclear cells was studied at a single-cell level. Individual lymphokine-synthesizing cells were identified by intracellular immunofluorescent staining using cytokine-specific monoclonal or polyclonal antibodies. Cultures from adult blood donors revealed a biphasic kinetic production pattern for IL 2 and IFN-gamma with peaks occurring 4-6 and 24-30 h after initiation of the cultures. Approximately 20%-40% of the lymphocytes produced IL 2 and IFN-gamma. In contrast, only 1%-3% of the lymphocytes synthesized IL 4 with maximal frequency after 6 h of culture. CD4+ as well as CD8+ T cells contributed to the synthesis of all three lymphokines studied. CD4+CD45R- T cells were the major producers of IL 2 and IL 4, while CD8+CD45R- T cells were the most common phenotype of IFN-gamma-synthesizing cells. By performing two-color immunofluorescence studies we observed that among IL 4-producing cells every second one made simultaneously IL 2 and every fourth one made IFN-gamma. Mononuclear cells from umbilical cord blood could be stimulated to make IL 2 to the same extent as cells from adult blood donors. No IL 4 production and a strikingly reduced frequency of IFN-gamma producers were noted in cell cultures from neonates. IL 2, IL 4 and IFN-gamma accumulated in the Golgi system, which resulted in a characteristic morphology of the staining, eliminating problems with evaluation of background signals.

Simultaneous production of interleukin 2, interleukin 4 and interferon-γ by activated human blood lymphocytes

Mononuclear cells obtained from human blood were mitogen or antigen activated in vitro in the presence or absence of FK506 or cyclosporin A (CsA). Cytokine production was studied at a single-cell level by ultraviolet (UV) microscopy of fixed permeabilized cells using cytokine-specific monoclonal antibodies (mAb). Phenotypic characterization of the monokine-producing cells was achieved by two-colour immunofluorescent staining. Cytokine production after antigen activation with Staphylococcus aureus enterotoxin A (SEA) was significantly reduced. FK506 or CsA inhibited SEA-induced tumour necrosis factor-alpha (TNF-alpha) production both in monocytes (P less than 0.01) and in lymphocytes (P less than 0.001), at a drug concentration of 1-25 ng/ml for FK506 and 100-500 ng/ml for CsA. Lymphocyte synthesis of interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and TNF-beta after SEA activation was also significantly reduced by either of the drugs. In contrast, endotoxin-induced monokine production (TNF-alpha and IL-6) after lipopolysaccharide (LPS) stimulation was unaffected by FK506 or CsA even when added in concentrations as high as 1000 ng/ml. When the cells were stimulated by phorbol ester (phorbol 12-myristate 13-acetate, PMA) plus calcium ionophore (ionomycin), FK506 and CsA inhibited, in a dose-dependent manner, the production of IL-2, IL-4, IL-5, IFN-gamma and TNF-alpha. The 50% inhibitory concentration (IC50) for FK506 or CsA on the cellular synthesis of the various cytokines varied between 0.6 and 1.0 ng/ml and 20 and 60 ng/ml, respectively. Further stimulation by addition of anti-CD28 mAb to the cultures resulted in an augmented IL-2 and IFN-gamma production which was resistant to both FK506 and CsA. This report delineates extensive similarities between the two drugs in mechanisms of immunosuppression by blockade of identical interleukin production. Depending on the mode of cell activation the two drugs inhibited not only cytokine production in lymphocytes but also antigen-induced monokine (TNF-alpha) production in macrophages, although the optimal immunomodulatory effect of FK506 was achieved at a concentration approximately 50-fold lower than that of CsA.

Jan Andersson

Papers

Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure

Plasma from patients with severe invasive group A streptococcal infections treated with normal polyspecific IgG inhibits streptococcal superantigen-induced T cell proliferation and cytokine production.

Bacterial toxin-induced cytokine production studied at the single-cell level.

Simultaneous production of interleukin 2, interleukin 4 and interferon-γ by activated human blood lymphocytes

Effects of FK506 and cyclosporin A on cytokine production studied in vitro at a single-cell level.