Characterization of a novel hemoglobin-glutathione adduct that is elevated in diabetic patients.

BACKGROUND: Typically, a diagnosis of diabetes mellitus is based on elevated circulating blood glucose levels. In an attempt to discover additional markers for the disease and predictors of prognosis, we undertook the characterization of HbA1d3 in diabetic and normal patients. MATERIAL AND METHODS: PolyCAT A cation exchange chromatography and liquid chromatography-mass spectroscopy was utilized to separate the alpha- and beta-globin chains of HbA1d3 and characterize their presence in normal and diabetic patients. RESULTS: We report the characterization of HbA1d3 as a glutathionylated, minor hemoglobin subfraction that occurs in higher levels in diabetic patients (2.26 +/- 0.29%) than in normal individuals (1.21 +/- 0.14%, p < 0.001). The alpha-chain spectrum displayed a molecular ion of m/z 15126 Da, which is consistent with the predicted native mass of the HbA0 alpha-globin chain. By contrast, the mass spectrum of the beta-chain showed a mass excess of 307 Da (m/z = 16173 Da) versus that of the native HbA0 beta-globin chain (m/z = 15866 Da). The native molecular weight of the modified beta-globin chain HbA0 was regenerated by treatment of HbA1d3 with dithiothreitol, consistent with a glutathionylated adduct. CONCLUSIONS: We propose that HbA1d3 (HbSSG) forms normally in vivo, and may provide a useful marker of oxidative stress in diabetes mellitus and potentially other pathologic situations.

HMG-1 as a late mediator of endotoxin lethality in mice.

Endotoxin, a constituent of Gram-negative bacteria, stimulates macrophages to release large quantities of tumor necrosis factor (TNF) and interleukin-1 (IL-1), which can precipitate tissue injury and lethal shock (endotoxemia). Antagonists of TNF and IL-1 have shown limited efficacy in clinical trials, possibly because these cytokines are early mediators in pathogenesis. Here a potential late mediator of lethality is identified and characterized in a mouse model. High mobility group-1 (HMG-1) protein was found to be released by cultured macrophages more than 8 hours after stimulation with endotoxin, TNF, or IL-1. Mice showed increased serum levels of HMG-1 from 8 to 32 hours after endotoxin exposure. Delayed administration of antibodies to HMG-1 attenuated endotoxin lethality in mice, and administration of HMG-1 itself was lethal. Septic patients who succumbed to infection had increased serum HMG-1 levels, suggesting that this protein warrants investigation as a therapeutic target.

Induction of the chemokine beta peptides, MIP-1 alpha and MIP-1 beta, by lipopolysaccharide is differentially regulated by immunomodulatory cytokines gamma-IFN, IL-10, IL-4, and TGF-beta.

The macrophage occupies a central role in the host response to invasion, exerting its control over the developing inflammatory response largely through the elaboration of an assortment of endogenous mediators including many cytokines. The beta chemokine peptides, macrophage inflammatory protein [MIP]-1 alpha and MIP-1 beta, are two such effectors markedly up-regulated in macrophages following exposure to bacterial lipopolysaccharide (LPS). These highly homologous peptides, like the other members of the beta chemokine family, exhibit diverse but partially overlapping biological activity profiles, suggesting that the cellular participants and intensity of an inflammatory response may in part be regulated by selective expression of these chemokines. Studies reported here demonstrate that, in contrast to the "balanced" MIP-1 alpha/MIP-1 beta chemokine responses of LPS-stimulated macrophage cultures in vitro, circulating levels of MIP-1 beta are significantly higher than those of MIP-1 alpha following LPS administration in vivo. Further studies have revealed that several immunomodulatory cytokines known to be up-regulated in vivo as a consequence of exposure to an invasive stimulus (gamma-IFN, IL-10, IL-4, and transforming growth factor [TGF]-beta) down-regulated the LPS-induced release of MIP-1 alpha by macrophages in vitro, but spared the MIP-1 beta response. This altered pattern of secretion may explain, at least in part, the high circulating levels of MIP-1 beta relative to MIP-1 alpha observed in vivo in response to LPS challenge.

Nuclear localization signal of HIV-1 as a novel target for therapeutic intervention.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) is a lentivirus and shares with other members of this retroviral subfamily the ability to replicate in nondividing cells, in particular, cells of the monocyte/macrophage lineage. This feature relies on the presence of a specific nuclear localization signal (NLS) within the viral matrix protein (MA p17), which to some degree can be complemented by the activity of the viral vpr gene product. The MA p17 NLS ensures efficient transportation of the viral preintegration complex into the nucleus of an infected macrophage and confers persistence of HIV-1 in quiescent T cells, and therefore presents an attractive target for therapeutic intervention. MATERIALS AND METHODS: Nuclear localization signals (NLS) in general and the HIV-1 MA p17 NLS in particular are characterized by a stretch of positively charged amino acids including one or more lysine residues. A series of compounds potentially capable of binding and reacting with lysine by forming Schiff base adducts was synthesized. Our special consideration was to make compounds that would preferentially bind to two closely contiguous amino functions, as opposed to isolated single lysine residues. We assumed that this approach might specifically target the compound to NLS while affecting other regions less, thus reducing nonspecific cytotoxicity. Antiviral activity was assessed in primary monocytes and in peripheral blood lymphocytes (PBL) infected with HIV-1ADA strain. Viral replication was monitored by reverse transcriptase (RT) activity in the supernatant. Efficiency of nuclear importation of the viral preintegration complex was estimated by the formation of 2-LTR circle forms of HIV-1 DNA and also by in situ PCR techniques. RESULTS: Arylene bis(methyl ketone) compounds with a nitrogenous third subsituent, especially a pyrimidinic side-chain, inhibited HIV-1 replication in human monocytes at an IC50 as low as 1 nM. These compounds did not block HIV-1 replication in peripheral blood lymphocyte cultures. The inhibitory effect observed in monocyte cultures appeared in the context of markedly reduced nuclear importation of viral DNA in the presence of the drug. No cytotoxic effects of the compounds was observed in vitro at concentrations as high as 10 microM. An amidinohydrazone derivative of the most active compound was about 100 times less active than the parent, indicating that carbonyl groups were instrumental in the antiviral effect. CONCLUSIONS: These early results suggest that retroviral replication in nondividing cells is susceptible to pharmaceutical intervention targeted against the NLS activity of HIV-1 proteins in the viral preintegration complex. The compounds described efficiently block translocation of viral DNA to the nuclei of infected primary monocytes, and inhibit viral replication. This inhibition is effective only in nondividing cells and is not seen in proliferating cultures, such as activated PBLs. Thus, drugs that target HIV-1 NLS may be useful to specifically block the macrophage arm of HIV infection and could thereby be of value in treating macrophage-specific manifestations of HIV disease, such as HIV-1 dementia. In combination with other drugs, potential therapeutics exploiting this target may also help to control the progression of HIV-1 infection and disease.

Reactive glycosylation endproducts in diabetic uraemia and treatment of renal failure.

In diabetes and ageing, glucose-derived advanced glycosylation endproducts (AGEs) cross-link proteins and cause vascular tissue damage. Elimination of circulating low-molecular weight AGE-modified molecules (LMW-AGEs) by the kidney is impaired in diabetic patients with end-stage renal disease, a group subject to accelerated atherosclerosis. We determined the effectiveness of current renal replacement treatments on elimination of serum LMW-AGEs in diabetic and non-diabetic patients with end-stage renal disease. Although diabetic patients receiving high-flux haemodialysis achieved 33% lower steady-state serum LMW-AGE than did those in conventional haemodialysis (p < 0.005), LMW-AGE concentrations remained 3.5-6 fold above normal, whether high-flux dialysis, conventional haemodialysis, or chronic ambulatory peritoneal dialysis were used. High-flux haemodialysis markedly reduced AGE during each treatment session (47.9% in the diabetic, p < 0.001 and 60.6% in the non-diabetic group, p < 0.001) but concentrations returned to pre-treatment range within 3 hours. In contrast, normal LMW-AGE concentrations were maintained in patients with functioning renal transplants. We found that LMW-AGEs with an apparent molecular weight of 2000-6000 circulate and retain strong inherent chemical reactivity--when exposed to collagen in vitro, up to 77% attached covalently to form AGE-collagen, and the AGE-crosslink inhibitor aminoguanidine completely inhibited this reaction. The results suggest that LMW-AGEs comprise a set of chemically-reactive molecules that are refractory to removal by current dialysis treatments. Through covalent reattachment onto vascular matrix or serum components, LMW-AGEs may exacerbate vascular pathology associated with end-stage renal disease.

MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia.

Cytokines are critical in the often fatal cascade of events that cause septic shock. One regulatory system that is likely to be important in controlling inflammatory responses is the neuroendocrine axis. The pituitary, for example, is ideally situated to integrate central and peripheral stimuli, and initiates the increase in systemic glucocorticoids that accompanies host stress responses. To assess further the contribution of the pituitary to systemic inflammatory processes, we examined the secretory profile of cultured pituitary cells and whole pituitaries in vivo after stimulation with bacterial lipopolysaccharide (LPS). Here we identify macrophage migration inhibitory factor (MIF) as a major secreted protein release by anterior pituitary cells in response to LPS stimulation. Serum analysis of control, hypophysectomized and T-cell-deficient (nude) mice suggests that pituitary-derived MIF contributes to circulating MIF present in the post-acute phase of endotoxaemia. Recombinant murine MIF greatly enhances lethality when co-injected with LPS and anti-MIF antibody confers full protection against lethal endotoxaemia. We conclude that MIF plays a central role in the toxic response to endotoxaemia and possibly septic shock.

Genomic cloning and promoter analysis of macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta, members of the chemokine superfamily of proinflammatory cytokines.

Macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta all belong to the newly recognized "chemokine" superfamily of structurally related, activation-inducible cytokines with inflammatory and growth regulatory activities. We report the isolation and sequencing of genomic clones for murine MIP-2 and murine MIP-1 beta, and analyze their regulatory sequences in comparison with each other and with several other members of the chemokine family. The murine (mu)MIP-2 genomic clone displays the canonical four exon/three intron structure typical of other genes in the chemokine alpha subfamily (e.g., IL-8). Potential cis regulatory elements in the proximal promoter region were highly conserved between muMIP-2 and its three most closely related human homologs: human (hu)GRO-alpha, huGRO-beta, and huGRO-gamma. A mouse macrophage cell line, RAW 264.7, was transfected with a growth hormone reporter construct driven by a proximal fragment of the muMIP-2 5' promoter, and nested deletion mutant analysis localized the LPS responsive element to a region that contains a conserved NF kappa B consensus motif and lies 51 to 70 bp 5' from the transcription start site. In contrast to that of MIP-2, the muMIP-1 beta genomic clone exhibited the three exon/two intron structure characteristic of the chemokine beta family members (e.g., MCP-1). A comparison of the promoters for muMIP-1 beta and muMIP-1 alpha reveals a conserved CK-1 element, but transient expression studies in RAW 264.7 macrophages with proximal fragments of either the muMIP-1 beta or the muMIP-1 alpha 5' promoter fused to a human growth hormone reporter gene link LPS-inducibility in both to promoter segments near to, but not identical with, the consensus CK-1 sequence. Proximal 5' promoter fragments cloned from both the MIP-1 alpha and MIP-1 beta genes unexpectedly conferred constitutive expression on the fused reporter gene sequences in macrophage-like cells, but initial 5' deletion analysis did not link this responsiveness to known sequence motifs. The muMIP-1 beta promoter, but not the muMIP-1 alpha promoter, was constitutively active in B16 mouse melanoma cells, and both promoters were active in the myelomonocytic cell line WEHI 3B(A)d-, the muMIP-1 alpha promoter being three times stronger.

Genomic structure of murine macrophage inflammatory protein-1 alpha and conservation of potential regulatory sequences with a human homolog, LD78.

The gene for a murine macrophage inflammatory cytokine, MIP-1 alpha, belongs to a newly recognized superfamily encoding small, inducible peptides shown to be up-regulated in association with cellular activation or transformation (tentatively designated the scy, or small cytokine, gene family). Secreted scy family peptides as a group, and MIP-1 alpha in particular, have inflammatory and mitogenic activities, and the family has been divided into CXC and CC subfamilies according to the spacing of conserved cysteine residues in the primary amino acid sequences. We have isolated and characterized a genomic clone encoding the CC subfamily member MIP-1 alpha. The organization of the murine MIP-1 alpha gene into three exons interrupted by two introns is identical to that found for other members of the CC subfamily (e.g., huLD78, muJE, huJE/MCP-1, muTCA3, and hul-309), which has been taken as evidence of evolution from a common ancestral gene. With the exception of the ratPF4 gene, which shares the two-intron/three-exon pattern typical of the CC subfamily, sequenced genes encoding CXC subfamily peptides (e.g., hulL-8 and hulP-10) include an additional intervening sequence that creates a fourth exon. Genomic nucleotide sequences 5' of the MIP-1 alpha cap site are highly homologous to corresponding regions of the human gene encoding a CC peptide variously designated as LD78/GOS19/pAT464, including consensus regulatory motifs in common, reinforcing the contention that MIP-1 alpha and LD78 may be interspecies homologs.

Metabolic effects of cachectin/tumor necrosis factor are modified by site of production. Cachectin/tumor necrosis factor-secreting tumor in skeletal muscle induces chronic cachexia, while implantation in brain induces predominantly acute anorexia.

We have developed a murine model of wasting by injecting intracerebrally cells which continuously secrete h-cachectin/TNF (CHO-TNF) to: (a) determine the effects of cachectin/TNF produced continuously in the central nervous system (CNS), and (b) compare the metabolic effects of cachectin/TNF-secreting tumor in the brain to the cachexia caused by CHO-TNF tumor in peripheral tissue (IM). Intracerebral CHO-TNF tumors produced increased serum h-cachectin/TNF levels with lethal hypophagia and weight loss (mean survival time of 11 d); these changes were not observed in association with nonsecretory control brain tumors. The metabolic consequences of intracerebral cachectin/TNF production were indistinguishable from acute, lethal starvation: whole-body lipid content was decreased significantly but protein was conserved. Although intramuscular cachectin/TNF-secreting tumors caused similar increases of serum h-cachectin/TNF levels, profound anorexia did not develop; wasting developed after a longer period of tumor burden (50 d) with classical signs of cachexia (i.e., anemia and depletion of both protein and lipid). These studies provide a reproducible animal model of site-specific cytokine production and suggest that, regardless of serum levels, cachectin/TNF produced locally in brain influences both the rate of development of wasting and its net metabolic effects.

TNF concentration in fatal cerebral, non-fatal cerebral, and uncomplicated Plasmodium falciparum malaria.

Plasma levels of tumour necrosis factor (TNF) were significantly higher in 178 Gambian children with uncomplicated malaria due to Plasmodium falciparum than in 178 children with other illnesses. 110 children with cerebral malaria were studied shortly after admission to hospital; 28 subsequently died. Compared with the children with uncomplicated malaria, mean plasma TNF levels were twice as high in cerebral malaria survivors and ten times as high in the fatal cases. Although high TNF levels were associated with high parasitaemia and with hypoglycaemia, they predicted fatal outcome in cerebral malaria independently of parasitaemia and glucose concentrations. Concentrations of interleukin-1 alpha, but not interferon gamma, were also related to the severity of malaria. We conclude that increased TNF production is a normal host response to P falciparum infection, but that excessive levels of production may predispose to cerebral malaria and a fatal outcome.

Processing of newly synthesized cachectin/tumor necrosis factor in endotoxin-stimulated macrophages.

The biosynthesis and processing of cachetin/tumor necrosis-factor (TNF) were examined in the murine macrophage-like cell line RAW 264.7. Lipopolysaccharide-stimulated cells secreted both glycosylated and nonglycosylated 17-kilodalton (kDa) mature cachectin/TNF into the culture medium. Secreted cachectin/TNF was derived from membrane-associated precursors that were precipitated by polyclonal antisera raised against either the mature protein or synthetic peptide fragments of the 79 amino acid cachectin/TNF prohormone sequence. About half of the precursors were N-glycosylated, apparently cotranslationally. The cachectin/TNF precursors were then proteolytically cleaved to release soluble mature cytokine into the medium, while the membrane-bound 14-kDa presequence remained cell associated. During the period of LPS stimulation, the amount of macrophage cell surface cachectin/TNF remained at a low level, suggesting that both nonglycosylated and glycosylated precursors of cachectin/TNF are efficiently cleaved by these cells. These findings suggest the presence of a unique mechanism for the secretion of cachectin/TNF.

Cerebrospinal fluid cachectin/tumor necrosis factor-alpha and platelet-activating factor concentrations and severity of bacterial meningitis in children.

In prospective studies, tumor necrosis factor (TNF alpha) was detected in cerebrospinal fluid (CSF) of 33 of 38 children with bacterial meningitis (BM) but in none of 15 with viral meningitis/encephalitis (P less than .001). BM CSF TNF alpha (less than 35 to greater than 25,500 pg/ml) correlated with CSF bacterial density (P less than .01), CSF protein (P less than .001), endotoxin (LPS) in gram-negative disease (P less than .01), and consecutive febrile hospital days (P less than .001); initial CSF TNF alpha greater than 1000 pg/ml was associated with seizures (P less than .05). Only 5 children with BM (13%) had detectable plasma TNF alpha activity on admission. A higher proportion who died had detectable plasma TNF alpha activity compared with survivors (3/4 vs. 2/34, P less than .005). Platelet-activating factor (PAF) in CSF was higher in 19 children with Haemophilus influenzae meningitis than in 17 controls (P less than .01) and correlated with bacterial density (P less than .01), CSF LPS (P less than .01), CSF TNF alpha levels (P less than .01), and the Herson-Todd severity score (P less than .01). Elevated CSF TNF alpha and PAF are often present in children with BM and are associated with seizures and severity of disease. Detectable CSF TNF alpha appears to distinguish BM from viral meningitis.

Tumour necrosis factor production in Falciparum malaria and its association with schizont rupture.

To investigate the involvement of tumour necrosis factor (TNF) in human malaria, we studied TNF production in patients infected with Plasmodium falciparum, and in co-cultures of human mononuclear cells and malaria parasites in vitro. In the examined sample, plasma TNF levels of over 39 pg/ml were detected in the plasma of 59% of Gambian children with acute malaria, 17% of convalescents, 9% of children with mild infections other than malaria, and 7% of healthy Gambian adults. Mononuclear cells of acute malaria patients, when stimulated with endotoxin in vitro, secreted twice as much TNF as did those of convalescent individuals, and three times that of healthy adult controls. Erythrocytic cultures of P. falciparum stimulated increased TNF secretion by mononuclear cells from uninfected individuals, and a sharp rise in the rate of secretion occurred shortly after schizont rupture. We suggest that malaria fever is mediated, at least in part, through paroxysmal TNF release associated with schizont rupture.

Infection and labor. IV. Cachectin-tumor necrosis factor in the amniotic fluid of women with intraamniotic infection and preterm labor.

A growing body of evidence supports a causal link between subclinical intrauterine infection and preterm labor. The mechanisms responsible for the onset of parturition in this setting have not been elucidated. The conventional view has been that bacterial products increase prostaglandin biosynthesis by intrauterine tissues and this, in turn, leads to the onset of labor. An alternative or complementary mechanism is that microbial products activate the host monocyte-macrophage system and that cytokines released during this process signal the initiation of parturition by stimulating prostaglandin biosynthesis by intrauterine tissues. This study was conducted to determine if cachectin-tumor necrosis factor is present in the amniotic fluid of women with intraamniotic infection and whether this cytokine can alter the rate of prostaglandin biosynthesis by intrauterine tissues. Amniotic fluid from 54 women was assayed for tumor necrosis factor. Tumor necrosis factor was not detectable in the amniotic fluid of women without intraamniotic infection regardless of the presence or absence of term or preterm labor. On the other hand, the amniotic fluid of 11 of 15 women with preterm labor and intraamniotic infection had measurable tumor necrosis factor. This cytokine stimulated prostaglandin E2 biosynthesis by amnion cells in monolayer culture in a dose-dependent fashion. These data support the concept that macrophage activation is involved in the onset of human parturition in the setting of infection. We propose that the host (fetus and/or mother) signals the onset of parturition through the secretion of inflammatory cytokines released in response to bacterial invasion.

Cachectin/tumor necrosis factor-alpha alters red blood cell kinetics and induces anemia in vivo.

Chronic inflammatory diseases are often associated with decreased red blood cell (RBC) mass. The cytokines cachectin/tumor necrosis factor-alpha (TNF) and interleukin 1 (IL 1) are produced by monocytes/macrophages in response to many inflammatory stimuli and have been implicated in the anemia of chronic disease. This study was undertaken to evaluate the mechanisms by which cachectin/TNF, IL 1, or endotoxin induce anemia. Hematologic parameters and RBC kinetics were quantitated in rats given chronic sublethal quantities of either recombinant human cachectin/TNF, recombinant human IL 1 alpha, or Salmonella endotoxin for 7 days. Cachectin/TNF or endotoxin treatment resulted in a 25 or 31% decrease, respectively, in total RBC mass, whereas RBC mass was unchanged by IL 1 administration. Anemia associated with either chronic cachectin or endotoxin administration was characterized by normal mean corpuscular volume, mean corpuscular hemoglobin content, and reticulocyte numbers. [59Fe]RBC survival was significantly shortened in animals given cachectin, IL 1 or endotoxin, but the magnitude of the response was greatest in cachectin/TNF-or endotoxin-treated rats. Although cachectin/TNF-IL 1-, or endotoxin treatment resulted in similar hypoferremia and shortened plasma iron half-life, endotoxin or cachectin/TNF treatment (but not IL 1) significantly reduced the incorporation of plasma 59Fe into newly synthesized RBCs. We conclude that chronic cachectin/TNF administration produces anemia by decreasing RBC synthesis and reducing the life span of circulating RBCs. An endogenous cachectin/TNF response during inflammatory disease may contribute to an associated anemic state, whereas the modestly reduced red cell life span induced by IL 1 does not lead to a net reduction in RBC mass, presumably owing to a preserved RBC synthetic rate.

Interleukin-2 initiates metabolic responses associated with critical illness in humans.

The cytokine interleukin-2 is a primary modulator of the immune response that occurs after infection, trauma, and transplant rejection, yet its role as a mediator of associated metabolic changes in surgical illness is unknown. We studied clinical and metabolic responses in eleven tumor-bearing humans with normal renal and hepatic function receiving bolus intravenous (I.V.) interleukin-2 (30,000 U/kg). Additional subjects (n = 6) were pretreated with the cyclooxygenase inhibitor, ibuprofen (1600 mg, orally), before interleukin-2 administration. Serial measurements were made of vital signs, symptoms, hematology, and plasma concentrations of pituitary and stress hormones and selected cytokines. Administration of interleukin-2 resulted in fever, tachyacardia, "flu-like" symptoms, and neurohormonal elaboration. The responses observed were quantitatively similar to those that occurred after endotoxin administration in healthy subjects (n = 13), but differed in the following manner: 1) the onset of fever and endocrine changes occurred after a longer latent interval (180-240 minutes vs. 60-90 minutes after endotoxin), 2) peak responses after the administration of interleukin-2 also occurred later, 3) no increased circulating tumor necrosis factor was detected after administration of interleukin-2 (peak plasma concentration was greater than 35 pg/ml vs. 270 +/- 70 pg/ml after endotoxin administration), and 4) administration of interleukin-2 but not of endotoxin was associated with increased circulating concentrations of gamma interferon (peak plasma concentration 1.7 +/- 0.2 NIH U/ml vs. less than 0.1 NIH U/ml after endotoxin administration). Fever and neurohormonal responses after interleukin-2 administration were greatly attenuated by ibuprofen administration. Interleukin-2 induces other cytokines that exert their effects largely through the cyclooxygenase pathway. Interleukin-2 may be an important signal, initiating the integrated host responses to infection and injury.

Tumor necrosis factor and endotoxin induce similar metabolic responses in human beings.

After injury, infection, or major operations a number of predictable metabolic responses occur. It has been proposed that the cytokine tumor necrosis factor (TNF)/cachectin is a primary mediator of these host responses. To test this hypothesis, we studied 16 tumor-bearing humans with normal renal and hepatic function, who received 24-hour continuous intravenous infusions of escalating doses of recombinant TNF (4 to 636/micrograms/m2/24 h). Serial measurements were made of vital signs and plasma concentrations of TNF, interleukin-1, adrenocorticotropic hormone, cortisol, iron, glucose, and C-reactive protein. Low doses of TNF had minimal metabolic effects, but infusions of greater than or equal to 545 micrograms/m2/24 hr (n = 8) resulted in fever, pituitary, and stress hormone release and acute phase changes. These alterations were compared with the changes that occurred in healthy humans (n = 13) receiving intravenous bolus injections of Escherichia coli endotoxin (4 ng/kg). TNF infusion in doses greater than or equal to 545 micrograms/m2/24 hr produced peak plasma TNF concentrations and metabolic responses that were similar to those after endotoxin injection. Interleukin-1 concentrations remained basal after TNF or endotoxin administration. TNF may represent the primary afferent signal that initiates many of the metabolic responses associated with sepsis and endotoxemia.

Detection of circulating tumor necrosis factor after endotoxin administration.

Cytokines, products of stimulated macrophages, are thought to mediate many host responses to bacterial infection, but increased circulating cytokine concentrations have not been detected consistently in infected patients. We measured plasma concentrations of circulating tumor necrosis factor alpha (cachectin), interleukin-1 beta, and gamma interferon, together with physiologic and hormonal responses, in 13 healthy men after intravenous administration of Escherichia coli endotoxin (4 ng per kilogram of body weight) and during a control period of saline administration. Eight additional subjects received ibuprofen before receiving endotoxin or saline. Plasma levels of tumor necrosis factor were generally less than 35 pg per milliliter throughout the control period, but increased 90 to 180 minutes after endotoxin administration to mean peak concentrations of 240 +/- 70 pg per milliliter, as compared with 35 +/- 5 pg per milliliter after saline administration. Host responses were temporally associated with the increase in circulating tumor necrosis factor at 90 minutes, and the extent of symptoms, changes in white-cell count, and production of ACTH were temporally related to the peak concentration of tumor necrosis factor. Ibuprofen pretreatment did not prevent the rise in circulating tumor necrosis factor (mean peak plasma level, 170 +/- 70 pg per milliliter) but greatly attenuated the symptoms and other responses after endotoxin administration. Concentrations of circulating interleukin-1 beta and gamma interferon did not change after endotoxin administration. We conclude that the response to endotoxin is associated with a brief pulse of circulating tumor necrosis factor and that the resultant responses are effected through the cyclooxygenase pathway.

Cachectin/TNF and IL-1 induced by glucose-modified proteins: role in normal tissue remodeling.

Proteins undergo a series of nonenzymatic reactions with glucose over time to form advanced glycosylation end products (AGEs). Macrophages have a receptor that recognizes the AGE moiety and mediates the uptake and degradation of AGE proteins. This removal process is associated with the production and secretion of cachectin (tumor necrosis factor) and interleukin-1, two cytokines with diverse and seemingly paradoxical biological activities. The localized release and action of these cytokines could account for the coordinated removal and replacement of senescent extracellular matrix components in normal tissue homeostasis.