Danazol suppresses the production of interleukin-1 beta and tumor necrosis factor by human monocytes

The effects of estradiol (E2), progesterone (P), and danazol on the production of interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) by OK-432 (a streptococcal preparation)-stimulated monocytes were examined. E2 and P at physiologic concentrations enhanced IL-1 beta and TNF production by monocytes from donors with lower control levels (without steroids added) of IL-1 beta and TNF. However, E2 and P at physiologic concentrations did not affect IL-1 beta and TNF production by monocytes from donors with higher control levels of IL-1 beta and TNF. Danazol inhibited IL-1 beta and TNF production by monocytes in a dose-dependent manner from not only donors with lower control levels of IL-1 beta and TNF but also donors with higher control levels of IL-1 beta and TNF. Danazol at a concentration of 10(-6) M significantly suppressed IL-1 beta and TNF production in the presence of E2 and/or P at concentrations giving peak responses of IL-1 beta production. These findings suggest possible new mechanisms of action for danazol in the treatment of endometriosis and infertility associated with immune abnormalities.     

Mycoplasma hyorhinis molecules that induce tumor necrosis factor alpha secretion by human monocytes.

Mycoplasma hyorhinis has been shown to induce the secretion of tumor necrosis factor alpha (TNF-alpha) from monocytes. To identify the molecules responsible for this activity, we separated sonicated M. hyorhinis lysate material by centrifugation at 100,000 x g into soluble (S) and particulate (P) fractions. The fractions were assayed for TNF-alpha-inducing activity by the L929 bioassay. Both the soluble and particulate fractions were able to induce TNF-alpha in roughly equal amounts. The optimum dose for both fractions was 1 micrograms/ml. Proteinase K treatment of either fraction eliminated the activity, suggesting that a protein component is involved in induction. Phase partitioning into Triton X-114 aqueous (A) and detergent (D) phases showed that the soluble fraction was composed of 80% aqueous-phase proteins, while the particulate fraction was > 75% detergent-phase proteins. All four fractions (SA, SD, PA, and PD) were able to induce TNF-alpha release. Treatment with NaIO4 to remove carbohydrate reduced the inducing activity of the SA phase by 80%, whereas that of the other fractions was unaffected by this treatment. The M(r)S of the inducing activity were determined by the monocyte Western (immunoblot) technique. The SA phase activity was associated with a single periodate-sensitive peak of 69 to 75 kDa. The two detergent phases had similar profiles of inducing activity, containing four peaks of activity. These peaks corresponded to 48 to 52, 43 to 45, 39 to 40, and 31 to 32 kDa. The PA fraction also contained four peaks of activity, 69 to 75, 55 to 57, 48 to 52, and 39 to 40 kDa. Thus, both a protein and glycan moiety from M. hyorhinis are capable of inducing TNF-alpha release from human monocytes.     

Pneumolysin stimulates production of tumor necrosis factor alpha and interleukin-1 beta by human mononuclear phagocytes.

Human peripheral blood monocytes and a human monocyte cell line were exposed to the toxin pneumolysin. Pneumolysin-exposed cells produced significantly larger amounts of tumor necrosis factor alpha and interleukin-1 beta than cells not exposed to the toxin. The viability of cells was not affected by the concentrations of pneumolysin used in the experiments.     

Role of the adhesion molecule lymphocyte function associated antigen 1 in toxic shock syndrome toxin 1-induced tumor necrosis factor alpha and interleukin-1 beta secretion by human monocytes.

We previously demonstrated that the induction by staphylococcal toxic shock syndrome toxin 1 (TSST-1) of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion by human monocytes requires direct T cell-monocyte contact. In the present study, a role for the adhesion molecule lymphocyte function associated antigen 1 (LFA-1) in TSST-1-induced cytokine secretion by human monocytes among 12 normal healthy donors was investigated. Monoclonal antibodies to the alpha chain (anti-CD11a) and to the beta chain (anti-CD18) of LFA-1 significantly inhibited TSST-1-induced TNF-alpha and IL-1 beta secretion (P < 0.025; Wilcoxon signed-rank test, two tailed), while a control monoclonal antibody directed against the monocyte CD14 antigen had no effect. These results suggest that LFA-1 may play an important role in the secretion of TNF-alpha and IL-1 beta by TSST-1-stimulated human monocytes, likely by promoting cell-cell adhesion between monocytes and lymphocytes.     

Fibronectin fragments stimulate tumor necrosis factor secretion by human monocytes.

Fibronectin (Fn) is a high molecular-weight glycoprotein that can influence many aspects of monocyte function. The purpose of this study was to determine whether Fn could stimulate monocyte tumor necrosis factor (TNF) secretion. Monocytes were isolated from the peripheral blood of healthy volunteers by density gradient centrifugation and adherence to plastic (2 h). Plasma Fn was purified from the blood by gelatin-Sepharose affinity chromatography. Monocytes were stimulated with Fn for 18 h and the supernatants were assayed for TNF activity using the L929 bioassay. Intact Fn stimulated the secretion of TNF in a dose-dependent manner. Intact Fn-induced TNF secretion by monocytes was inhibited (50%) but not eliminated by the addition of the R-G-D-containing peptide GRGDSP. Limited proteolysis of the Fn molecule using insoluble chymotrypsin resulted in a fragment preparation that was dramatically more stimulatory than the intact Fn preparation. High-performance liquid chromatographic (HPLC) purification of the fragments demonstrated that at least two fragments were capable of stimulating TNF secretion. Further purification by affinity chromatography and HPLC localized the stimulatory activity to the 120-kd cell-binding fragment. The possibility that the stimulatory activity was the result of endotoxin contamination was ruled out using macrophages from C3H/Hej mice. These results suggest that Fn fragments are potentially important molecules for activation of monocytes and may stimulate monocyte cytotoxic activity.     

Gram-positive cell walls stimulate synthesis of tumor necrosis factor alpha and interleukin-6 by human monocytes.

Purified cell walls representing a wide variety in teichoic acid and peptidoglycan structure prepared from eight different gram-positive bacterial species induced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 from human monocytes in the presence of 10% plasma or serum. Significant amounts of cytokines began to be produced at concentrations above 100 ng to 1 microgram of cell walls per ml, with maximal production requiring 10 to 100 micrograms of cell wall material per ml. In the absence of plasma, the cytokine-inducing capacity of cell wall preparations was lower by at least an order of magnitude. The serum-derived cofactor was inactivated by heating at 90 degrees C for 30 min, suggesting that the activity is associated with a protein. On the other hand, replacement of normal with hypogammaglobulinemic plasma, inactivation of complement (at 56 degrees C), and blockade by the monoclonal antibody MY4 of the CD14 receptors on monocytes did not inhibit the production of TNF-alpha induced by whole cell walls. Cell walls also stimulated production of TNF-alpha induced by whole cell walls. Cell walls also stimulated production of TNF-alpha in the presence of polymyxin B, and macrophages derived from the lipopolysaccharide-insensitive cell line of C3He/HeJ mice also produced this cytokine when stimulated by cell walls. Both peptidoglycan and the soluble glycan-teichoic acid component prepared by an enzymatic method from the same wall preparation exhibited a serum-dependent induction of TNF-alpha from monocytes, while stem peptides and disacharride peptides had only poor, if any, activity. Cell walls may contribute to the septic shock induced by gram-positive bacteria.     

Staphylococcal toxic shock syndrome toxin 1-induced tumor necrosis factor alpha and interleukin-1 beta secretion by human peripheral blood monocytes and T lymphocytes is differentially suppressed by protein kinase inhibitors.

The signal transduction pathways by which staphylococcal toxic shock syndrome toxin 1 (TSST-1) induces tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion were examined with various protein kinase inhibitors. TNF-alpha secretion by normal human monocytes and T cells in response to TSST-1 was suppressed by inhibitors of protein kinase C (H7) and tyrosine kinases (genistein). In contrast, the secretion of IL-1 beta was blocked by a cyclic AMP- and cyclic GMP-dependent kinase inhibitor (HA1004) as well as by H7 and genistein. These results suggest that the secretion of TNF-alpha and IL-1 beta may be differentially regulated by TSST-1 and that protein kinases play an important role in mediating cytokine responses to the toxin.     

Alpha 1-acid glycoprotein potentiates lipopolysaccharide-induced secretion of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha by human monocytes and alveolar and peritoneal macrophages.

Although the physiological role of alpha 1-acid glycoprotein (AGP), an acute-phase protein, is poorly understood, several lines of evidence support a modulatory action on the immune response. In this study, we investigated the effect of AGP on the production of interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF)-alpha by human monocytes, macrophages and the monocytic THP-1 cell line. AGP significantly enhanced (2- to 7-fold) the production of these cytokines in monocytes induced by suboptimal concentrations of lipopolysaccharide [E. coli lipopolysaccharide (LPS): 100 ng/ml] in serum-free conditions, whereas it had little or no effect in the absence of LPS. The potentiating effect of AGP was inhibited by specific antibodies. It was concentration dependent and the greatest enhancement was observed with 250-500 micrograms/ml. Moreover, AGP only potentiated the effect of suboptimal concentrations of LPS. AGP did not alter the time course of LPS-induced IL-1 beta, IL-6 or TNF-alpha secretion. AGP acts as a co-inducer and could also potentiate cytokine secretion triggered by Neisseria meningitidis LPS and muramyl dipeptide. The glycan moiety of AGP did not seem to be involved in its potentiating effect, since both its major glycoforms and asialo-AGP potentiated the effect of LPS to the same extent as native AGP. Possible differences in the effect of AGP according to cell maturation were investigated using isolated human macrophages: AGP potentiated LPS-induced cytokine production by both peritoneal and alveolar macrophages. These data suggest that AGP can modulate monocyte/macrophage functions, thereby contributing to the amplification and regulation of immune and inflammatory responses.     

Mycoplasma arginini TUH-14 membrane lipoproteins induce production of interleukin-1, interleukin-6, and tumor necrosis factor alpha by human monocytes.

Mycoplasma arginini TUH-14 partially purified membrane lipoproteins (TUH-14-pp) directly induce secretion of the cytokines involved in the inflammatory response, namely, interleukin 1 (IL-1), tumor necrosis factor alpha, and IL-6, by human monocytes cultured in the absence of serum. The biological activity of each cytokine correlates with its immunoreactivity. Upon stimulation with either TUH-14-pp or lipopolysaccharide, most tumor necrosis factor alpha and IL-6 is secreted in the extracellular compartment, whereas a significant amount of IL-1 remains cell associated. Finally, polymyxin B does not affect secretion of cytokines induced by TUH-14-pp, indicating that mycoplasma lipopolysaccharide does not account for their effects on monocytes. Altogether, our data show that direct interaction of mycoplasma membrane components with human blood monocytes induces secretion of high levels of cytokines known to trigger inflammatory responses. This new concept of membrane-bound active components of mycoplasma may explain its ability to efficiently initiate inflammatory reactions.     

The detection of intracytoplasmic interleukin-1 alpha, interleukin-1 beta and tumour necrosis factor alpha expression in human monocytes using two colour immunofluorescence flow cytometry.

Two colour flow cytometry was used to analyse in situ cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis. Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86-99% (n = 33)). After fixation with 4% PF and permeabilisation with 1% saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF-alpha), or control rabbit IgG. Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC). FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte IL-1 beta and TNF-alpha expression following LPS stimulation, with early peaks in TNF-alpha (2 h), compared with IL-1 beta (4 h), and IL-1 alpha (12 h). Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines. The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.     

Priming and stimulation of bovine neutrophils by recombinant human interleukin-1 alpha and tumor necrosis factor alpha.

We have examined the in vitro effects of two recombinant human monokines, interleukin-1 alpha (rHuIL-1 alpha) and tumor necrosis factor alpha (rHuTNF alpha), on bovine neutrophil functions. Both rHuIL-1 alpha (10 to 1,000 ng/ml) and rHuTNF alpha (5 to 50 ng/ml) directly stimulated the oxidative burst of bovine neutrophils as measured by Luminol-dependent chemiluminescence, superoxide anion generation, and hydrogen peroxide production. In addition, both rHuIL-1 alpha (1 to 1,000 ng/ml) and rHuTNF alpha (0.5 to 50 ng/ml) primed bovine neutrophils for an enhanced oxidative burst to subsequent stimulation with opsonized zymosan. Neutrophils pre-treated with either monokine exhibited an earlier, as well as stronger, zymosan-stimulated Luminol-dependent chemiluminescence response, as compared to untreated neutrophils. Exposure of bovine neutrophils to combinations of suboptimal doses of rHuIL-1 alpha (10 and 100 ng/ml) and rHuTNF alpha (0.5 and 5 ng/ml) resulted in a synergistic stimulation of Luminol-dependent chemiluminescence, whereas, no synergism was observed when using optimal doses of each monokine. Pre-incubation of bovine neutrophils with an optimal concentration of recombinant bovine interferon gamma (100 U/ml), and either rHuIL-1 alpha or rHuTNF alpha, further augmented the maximal oxidative response of neutrophils stimulated with opsonized zymosan. Bovine neutrophils released both primary and secondary granules in response to rHuIL-1 alpha and rHuTNF alpha, and also exhibited enhanced adherence in the presence of either monokine.     

Glomerular basement membrane-containing immune complexes stimulate tumor necrosis factor and interleukin-1 production by human monocytes.

The ability of human peripheral blood monocytes to produce tumor necrosis factor (TNF) and interleukin-1 (IL-1) in an in vitro model of immune complex-mediated glomerulonephritis was investigated. When isolated monocytes were incubated with human glomerular basement membrane (GBM) containing anti-GBM immune complexes, both TNF and IL-1 were produced and secreted into the medium. The time course of secretion differed, with IL-1 production being maximal after approximately 8 hours, whereas TNF levels continued to rise for 30 hours. The activities of the monocyte-derived TNF and IL-1 were inhibitable by specific antibodies. No effect was seen when monocytes were incubated separately with either GBM alone or anti-GBM IgG. The levels of TNF and IL-1 released were comparable with those induced by high concentrations of LPS, indicating that production was close to the maximal levels reported for these cells. High levels of TNF and IL-1 also were produced in response to soluble immune complexes. The results show that monocytes can produce significant levels of TNF and IL-1 in response to both surface-bound and soluble immune complexes and provide support for the participation of these monokines in glomerulonephritis.     

Enhanced interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha production by LPS stimulated human monocytes isolated from HIV+ patients

Periodontal disease and tooth loss is a common finding among advanced HIV+ patients. In addition to local oral lipopolysaccharide (LPS) stimulation, systemic up-regulation of monocyte pro-inflammatory cytokine secretion may also be involved in the pathogenesis of HIV disease. A study was undertaken to investigate IL-1beta, IL-6 and TNF-alpha production by resting and LPS stimulated monocytes isolated from HIV+ patients and also to investigate the relationship of the patient's HIV viral load status to the cytokine production. Whole blood samples in EDTA were collected from 39 HIV-1 infected patients and 20 age and sex matched uninfected controls. Plasma was separated by centrifugation. Viral load was determined using a quantitative RT-PCR. Monocytes were isolated by Ficoll-hypaque gradient separation followed by overnight plastic adherence. Cultured monocytes (1x10(6)/ml) were stimulated with LPS (1 microg/ml) of either P. gingivalis or F. nucleatum for 2, 8, 24 and 48 h and supernatant fluids were collected. IL-1beta, IL-6, and TNF-alpha levels in supernatant fluids were estimated by ELISA. Increased overall production of IL-1beta, IL-6 and TNF-alpha by LPS stimulated monocytes isolated from HIV-1 infected patients was observed when compared to HIV-1 uninfected controls. LPS stimulated monocytes from HIV-1 infected patients with high viral load (HVL) produced significant (p<0.05) elevations in these pro-inflammatory cytokines when compared to HIV-1 uninfected controls. Both LPS of P. gingivalis and F. nucleatum produced a comparable cytokine production by monocytes after 8 h of stimulation. These data suggest that enhanced IL-1beta, IL-6 and TNF-alpha is produced by monocytes/macrophages isolated from HVL HIV+ patients and may be involved in the overall pathogenesis of HIV-1 infection.     

Activation of human monocytes by Streptococcus mutans serotype f polysaccharide: immunoglobulin G Fc receptor expression and tumor necrosis factor and interleukin-1 production.

Streptococcus mutans serotype f polysaccharide (poly f) was prepared from S. mutans whole cells by autoclaving. The poly f was purified by chromatography on DEAE Trisacryl M and Bio-Gel P100, treated with insoluble pronase, and resubjected to chromatography on DEAE Trisacryl M. Normal human blood monocytes, stimulated in vitro with purified poly f, produced extracellular tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) in a dose-dependent fashion as determined by a heterologous two-site sandwich enzyme-linked immunosorbent assay. Poly f also increased the expression of monocyte cell surface receptors for the Fc part of human immunoglobulin G, activity which is correlated with an increase of the phagocytic activity of the stimulated monocytes. Polymyxin B had no effect on TNF-alpha and IL-1 beta release. Neutralization assays with anti-recombinant human TNF-alpha and anti-recombinant human IL-1 beta immunoglobulin G confirmed the fact that the cytotoxic and mitogenic mediators released by the poly f-stimulated monocytes were mainly TNF-alpha and IL-1 beta.     

Production of tumor necrosis factor alpha by monocytes from patients with pulmonary tuberculosis.

We studied the production of tumor necrosis factor alpha (TNF-alpha) by peripheral blood monocytes taken from patients with pulmonary tuberculosis and from healthy controls. It was found that the monocytes from patients with newly diagnosed tuberculosis released significantly greater amounts of TNF-alpha in vitro in response to lipopolysaccharide than did those from healthy controls (P less than 0.05). However, the monocytes from patients with chronic refractory tuberculosis released significantly lower amounts of TNF-alpha than did those from patients with newly diagnosed tuberculosis (P less than 0.005). Even when the cells were primed for 24 h with 500 U of recombinant interferon gamma per ml, the same pattern of results was observed. The depressed TNF-alpha production by the monocytes from patients with chronic refractory tuberculosis was also shown in response to Mycobacterium bovis BCG. This depressed TNF-alpha production did not recover, even when cultured for 1 to 7 days in the sera of healthy individuals. The sera from patients with chronic refractory tuberculosis did not have any suppressive effect on the lipopolysaccharide-induced TNF-alpha production. Thus, it was demonstrated that the levels of TNF-alpha produced by monocytes were related to the disease states of pulmonary tuberculosis and that the depressed TNF-alpha production by monocytes in patients with chronic refractory tuberculosis might not be acquired.     

Production of tumor necrosis factor and interleukin-1 by macrophages from human atheromatous plaques.

The production of cytokines by atheromatous plaque macrophages from human endarterectomy tissue was assessed in vitro by short-term cell culture and in situ by immunohistology. Macrophages were isolated from plaques of 14 patients undergoing carotid endarterectomy and 7 patients undergoing reconstructive procedures on atheromatous distal aortic and femoral arteries. Tumor necrosis factor (TNF) and interleukin 1 (IL-1) production by plaque macrophages and blood monocytes isolated concurrently from these patients was assessed. TNF release by macrophages from carotid plaques (0.39 +/- 0.12 ng/10(6) cells/24 hours) was significantly augmented compared to the release by corresponding blood monocytes (0.014 +/- 0.011 ng/10(6) cells/24 hours, P = 0.03), and by macrophages from noncarotid lesions (0.038 +/- 0.036 ng/10(6) cells/24 hours, P < 0.04). Cellular TNF expression by macrophages within carotid plaques was also more prominent than in noncarotid lesions. By contrast, IL-1 production by plaque macrophages from both carotid and noncarotid plaques was not augmented compared to blood monocytes, and only infrequent and low-intensity labeling for IL-1 was present on macrophages within plaques from either group. These results provide functional and immunohistological evidence for increased production of TNF but not IL-1 by activated macrophages, indicating local and selective augmentation of cytokine production within carotid plaques. This suggests that macrophages play an active role in the inflammatory response within atheromatous carotid plaques.     

Interleukin-8 is a major neutrophil chemotactic factor derived from cultured human gingival fibroblasts stimulated with interleukin-1 beta or tumor necrosis factor alpha.

Inflammatory mediators produced by cells in the gingiva have been implicated in the initiation and progression of periodontal disease, a common infectious disease. In this study, we examined the biological activity of neutrophil chemotactic factors and the kinetics of expression of interleukin-8 (IL-8) mRNA derived from normal gingival fibroblasts in response to inflammatory mediators in an in vitro model. Gingival fibroblasts stimulated by either recombinant human interleukin-1 beta or recombinant human tumor necrosis factor alpha produced neutrophil chemotactic factors after 4 h, whereas expression of cell-derived IL-8 mRNA was detected within 1 h after stimulation. Furthermore, in a neutralization assay, rabbit anti-recombinant human IL-8 antiserum inhibited neutrophil chemotactic activity to basal levels. These results provide evidence that gingival fibroblasts synthesize potent chemotactic factors such as IL-8 in the presence of the inflammatory mediators interleukin-1 beta and tumor necrosis factor alpha. The activity of these factors may contribute to neutrophil-mediated processes in the pathogenesis of periodontal disease.     

Production of interleukin-1 but not tumor necrosis factor by human monocytes stimulated with pneumococcal cell surface components.

While there is considerable evidence that both interleukin-1 (IL-1) and tumor necrosis factor (TNF) are central mediators of inflammation caused by gram-negative bacteria and endotoxin, the roles of these two mediators in gram-positive infection are unknown. Pneumococcal infections are characterized by an intense inflammatory reaction in infected tissues. Current evidence suggests that the component of the pneumococcus which causes this inflammation in many body sites is the cell wall. We determined the ability of native pneumococcal cell wall, lipoteichoic acid, and cell wall subcomponents to stimulate secretion of IL-1 and TNF from human monocytes. Each pneumococcal cell surface component was found to have a different specific activity for induction of IL-1. Teichoication was an important determinant of this activity: teichoicated species were at least 10,000-fold more potent than endotoxin and 100-fold more potent than teichoic acid-free peptidoglycan. IL-1-inducing activity was greatly reduced by chemical alteration of the teichoic acid. In contrast to endotoxin, cell wall did not induce production of TNF. This dissociation of the production of IL-1 and TNF during the response of the human monocyte to pneumococcal surface components suggests that, in at least some circumstances, the mechanisms for generation of an inflammatory response to infection may be fundamentally different between gram-positive and gram-negative disease.     

Differential elevation of circulating interleukin-1 beta, tumor necrosis factor alpha, and interleukin-6 in AIDS-associated cachectic states.

Elevation of serum interleukin-1 beta (IL-1 beta) levels, and to a lesser degree tumor necrosis factor alpha levels, was found in cachectic human immunodeficiency virus (HIV)-infected African patients without concurrent opportunistic infection or neoplasia (HIV wasting syndrome). A heterogeneous pattern of elevations of cytokine levels, including mild elevations of IL-1 beta and pronounced elevations of IL-6 levels, was found in other cachectic states.