Nikolai Petrovsky

The Chronobiology of Human Cytokine Production

Cytokine production in human whole blood exhibits diurnal rhythmicity. Peak production of the pro-inflammatory cytokines IFN-gamma, TNF-alpha, IL-1 and IL-12 occurs during the night and early morning at a time when plasma cortisol is lowest. The existence of a causal relationship between plasma cortisol and production is suggested by the finding that elevation of plasma cortisol within the physiological range by the administration of cortisone acetate results in a corresponding fall in pro-inflammatory cytokine production. Cortisol may not be the only neuroendocrine hormone that entrains cytokine rhythms; other candidates include 17-hydroxy progesterone, melatonin and dihydroepiandrostene dione (DHEAS). The finding of diurnal cytokine rhythms may be relevant to understanding why immuno-inflammatory disorders such as rheumatoid arthritis or asthma exhibit night-time or early morning exacerbations and to the optimisation of treatment for these disorders. Diurnal rhythmicity of cytokine production also has implications for the timing of blood samples drawn for diagnostic T-cell assays. Finally, diurnal rhythmicity of immune function suggests that the nature of an immune response, for example in response to vaccination, may be modified by the time of day of antigen administration and raises the possibility that immune responses could be therapeutically manipulated by co-administration of immuno-regulatory hormones such as glucocorticoids.

Cytokine-based human whole blood assay for the detection of antigen-reactive T cells

The measurement of cytokines produced by activated T cells refines assessment of cellular immune function and facilitates whole blood T cell assays. The latter approximate conditions in vivo and obviate the need to purify blood mononuclear cells. We have investigated the parameters of the whole blood assay in humans to standardize and optimize the detection of tetanus-specific T cell cytokine responses. Optimal conditions include the use of undiluted whole blood, an incubation time of 36-48 h and a minimum of delay between venesection and incubation of the blood with antigen. Blood should be drawn at a standard time of day to minimize inter-assay variation due to diurnal rhythmicity in cytokine production. Interferon-gamma or interleukin-2 are specific and reliable readouts; other cytokines can be measured to further characterize the TH1 and TH2 elements of the T cell responses, although tetanus-stimulated IL-4 production is detected in only a minority of healthy individuals. The whole blood assay is a potentially valuable tool for assessing cellular immune function and screening for antigen-reactive T cells in humans.

HLA Class II-associated polymorphism of interferon-γ production implications for HLA-disease association

One of several possible mechanisms for the HLA-disease association is HLA-related polymorphism of cytokine expression. However, with the exception of the tumor necrosis factors, no evidence has been found for a relationship between HLA alleles and cytokine expression. This may be because cytokine responses to commonly employed mitogens are neither antigen nor HLA dependent, and responses to recall antigens are dominated by the effect of prior antigen exposure. We reasoned that responses to alloantigens would be independent of prior antigen exposure and may therefore reveal subtle HLA-related variations in cytokine production. Here we demonstrate HLA Class II-related polymorphism of IFN-gamma production in the MLR performed between 32 subjects by a novel whole-blood method. HLA DR1, 2, and 6 were associated with high, whereas DR 3, 4, 5, and 7 were associated with low IFN-gamma production. Interestingly, DQ alleles with which these DR alleles are in linkage dysequilibrium, DQ1 and DQ2 and 3, were also associated with high and low IFN-gamma production, respectively. Ranking of HLA alleles according to whole-blood IFN-gamma production in response to mitogen or recall antigens was similar to that in the MLR, although individual allele-related differences did not reach statistical significance. TNF-alpha production was significantly higher in DR3-positive than in DR3-negative subjects, in accord with previous studies. These findings suggest that HLA Class II alleles, particularly at the DQ locus, or alternatively, genes in linkage with them, regulate IFN-gamma expression by T cells. The finding of HLA allele-related polymorphism of IFN-gamma production corroborates other lines of evidence that regulation of IFN-gamma expression contributes to HLA-associated susceptibility to immunoinflammatory diseases, in particular insulin-dependent diabetes and multiple sclerosis.

Evidence from twins for acquired cellular immune hyperactivity in type 1 diabetes

Type 1 diabetes has been associated with an increased frequency of activated T cells and T‐cell hyperactivity to non‐specific and disease‐specific stimuli including the islet autoantigen glutamic acid decarboxylase 65 (GAD). To address whether T‐cell hyperactivity is genetic or acquired we measured whole blood cytokines in vitro in response to GAD or tetanus in 18 identical twin pairs, nine discordant for type 1 diabetes. In addition, the activity of 2′, 5′ oligoadenylate synthetase (OAS) in blood mononuclear cells was measured as a marker of viral infection. Interleukin‐2 (IL‐2) basally and IL‐2 and interferon‐γ (IFN‐γ) in response to GAD, were detected more frequently and at higher levels in diabetic compared to non‐diabetic twins. IL‐10 was not different between groups. OAS activity was increased in diabetic compared to non‐diabetic twins and showed a correlation with basal IL‐2 and GAD‐stimulated IFN‐γ and IL‐10. These findings suggest that T‐cell hyperactivity in type 1 diabetes is an acquired trait and could reflect persisting virus expression.

HLA-matched control subjects are essential in studies of susceptibility to IDDM

Dear Sir, With considerable enthusiasm I have followed the recent interest in the possibility that insulin-secreting tissue contains the liver type of pyruvate kinase. The latest interest appears to have begun with the paper by Marie and co-workers [1] whose experiments, primarily in the INS-1 cell, provided evidence for glucose induction of L-type pyruvate kinase mRNA. They also showed that refeeding of starved rats induces L-pyruvate kinase mRNA in pancreatic islets. This is of interest because Lpyruvate kinase possesses sequences similar to those in the 5 flanking region of the insulin gene and, of course, transcription of the insulin gene is stimulated by glucose. However, before conclusions about islet metabolism can be drawn, the presence of L-pyruvate kinase protein and enzyme activity must be demonstrated in the islet not that these authors made any inferences about metabolism. However, their interesting finding has caused others to do this. Several years ago, we put considerable effort into demonstrating that L-pyruvate kinase was present in normal islets and failed. We were eager to show that L-pyruvate kinase was present in islets because we found a phosphoprotein in islets with a size and a pI similar to pyruvate kinase [2]. Three lines of evidence indicated that normal rat islets and rabbit islets did not contain L-pyruvate kinase, but did contain the M 2 or K type of pyruvate kinase [3], the isozyme that is present in many internal organs. First, the enzyme activity of pyrnvate kinase from liver was inhibited (due to phosphorylation) by incubating liver cytosol with ATR However, the enzyme in islet cytosol was not inhibited. Second, antisera to L-pyruvate kinase inhibited pyruvate kinase enzyme activity in liver cytosol, but did not inhibit the activity of the enzyme in islet cytosol. Antisera to the skeletal muscle M 1 pyruvate kinase (which crossreacts with the M; enzyme) inhibited pyruvate kinase in islet cytosol, but not in liver cytosol. Third, L-pyruvate kinase, M1 and M 2 pyruvate kinase isozymes each show distinct patterns of activation in the presence of fructose bisphosphate and inhibition in the presence of phenylalanine and alanine. These patterns for the enzyme in islet cytosol were almost identical to those in kidney cytosol, which possesses primarily the M 2 isozyme, but were dissimilar to those of liver and skeletal muscle. The INS-1 cell, because of its glucose-responsiveness [4J is an important and convenient tool for diabetes research. However, it is a relatively new addition to the diabetes research armamentarium. Studies of pyruvate kinase in betacell lines, as well as islets from other genera besides the rat, such as humans and mice, should be conducted. I predict that all normal islets would not possess L-pyruvate kinase protein or enzyme activity because inhibition by phosphorylation is a characteristic required for the enzyme in liver, which is, of course, a gluconeogenic tissue. The insulin cell appears to have no need for gluconeogenesis, and furthermore, islets lack enzyme activity of, and mRNA for, phosphoenolpyruvate carboxykinase [5, 6] the enzyme in the gluconeogensis pathway that catalyses part of the reverse reaction of pyruvate kinase in glycolysis.