The effects of iron deficiency on lymphocyte cytokine production and activation: preservation of hepatic iron but not at all cost.

Worldwide, over 40% of children have iron deficiency anaemia, frequently associated with infections. Certain cytokines are involved in both immune activation/response to infection and iron transport/metabolism. We therefore assessed the relations among iron deficiency, cytokine production and lymphocyte activation markers in 142 hospitalized Malawian children. We examined peripheral blood lymphocyte antigens/cytokine production using four- colour flow cytometry and serum transferrin receptor (TfR) levels, an inverse measure of iron status unaffected by acute illness or infection, with an enzyme-linked immunosorbent assay. Wilcoxon rank sum tests and logistic regression analyses (LRA) were performed. Iron deficiency (TfR > or = 10 microg/ml) versus TfR < 10 microg/ml, was associated with higher percentages of lymphocytes producing: (a) induced or spontaneous IL-6 (medians: induced, 15.9% for iron-deficient children versus 8.8% for iron-replete children, P = 0.002; spontaneous, 24.4% versus 13.0%, P < 0.001) and (b) induced IFN-gamma (medians:18.4% versus 12.4%, P = 0.006). The percentages of CD8(+) T cells spontaneously producing IL-6 and of all lymphocytes producing induced TNF-alpha and IFN-gamma in the same cell had the strongest relationships to iron deficiency (b = + 0.0211, P = 0.005 and b = + 0.1158, P = 0.012, respectively, LRA) and were also positively related to the co-expression of the T cell activation markers HLA DR and CD38. Severe iron deficiency (TfR > or = 30 microg/ml) was associated with the percentage of lymphocytes producing induced IL-4 (medians: 0.5% versus 1.6%, P < 0.010). The cytokine patterns associated with iron deficiency in our study would preserve iron stores but also preferentially retain the activation capabilities of T cells, albeit not necessarily other immune cells, until a critical level of iron depletion is reached.

Cytokines and malaria parasitemia.

The balance between pro- and antiinflammatory cytokines may be important in malaria presentation and outcome. Malaria tends to be more severe in children than in adults, presumably because partial immunity develops with age. However, the full nature of, and age-related differences in, anti-malarial immunity are unknown. We compared: (1) serum and cell-specific cytokines of patients with acute malaria to those of patients with other acute illnesses and to those of healthy adults and (2) the cytokine responses of parasitemic children and parasitemic adults. Flow cytometry was done on the peripheral blood mononuclear cells of 148 hospitalized children, 161 febrile hospitalized adults, and 20 healthy adults in Malawi, Africa, a malaria-endemic country. Serum cytokines were also assessed for 80 of these patients. Thirty-eight participants were parasitemic with Plasmodium falciparum. Serum interleukin (IL)-10 (an antiinflammatory, immunoregulatory, and type 2 cytokine) levels were higher in malaria patients than in other patients (medians 502 pg/mL vs 16 pg/mL, P = 0.002), and the percentages of various lymphocyte populations making IL-6 (a proinflammatory, type 2 cytokine regulating iron distribution) were lower in malaria patients than in other patients (e.g., for spontaneous production by children's CD8(+) T cells: medians 1.4% vs 33.1%, P = 0.004). For adult patients, the percentages of lymphocytes spontaneously making IL-4 (a type 2 cytokine) were significantly lower in those with malaria than in those without malaria (medians 0.9% vs 2.1%, P = 0.005). The percentages of monocytes spontaneously making IL-8 (a chemotactic, proinflammatory chemokine) were higher in parasitemic children than in parasitemic adults (medians 5.8% vs 1.7%, P = 0.003). A number of cellular proinflammatory, type 1 parameters were significantly higher in all children (with or without malaria) than in all adults; these included the percentages of various lymphocyte populations making IL-6, both IL-6 and interferon-gamma, or IL-8. These data support the importance of IL-10 in malaria parasitemia. Given the lack of an IL-4 (type 2) response, IL-10's primary role may be immunoregulatory rather than type 2 in nature. In this study, the immune response to malaria was more proinflammatory in children than in adults. This difference, if corroborated by other studies, could be related to malaria's greater severity in children.

Effects of HIV-1 peptides on T-cell receptor variable beta chain families.

Superantigens (SAGs) selectively stimulate expansion and then deletion of specific T cell antigen receptor (TCR) variable beta chain (Vbeta) families. We investigated six synthetically produced HIV-1-related peptides for evidence of SAG activity: three derived all or in part from the transmembrane gp41 protein and three from the genetic sequence of the tRNA binding region. The first three were chosen because they are highly immunogenic; the second three, because their genetic sequence is completely homologous to a region of the mouse mammary tumor virus, a known superantigen. We cultured peripheral blood mononuclear cells (PBMC) of HIV-negative, healthy human donors with each of these six HIV-1 peptides. Resting and blastic CD4(+) and CD8(+) lymphocytes were assessed pre- and post-culture using 3-color cytofluorometry and monoclonal antibodies to CD4, CD8, and 14 human TCR Vbeta families. Significance testing was done using a Student t-test. Two of the HIV-1 peptides showed possible SAG activity, one from gp41 transmembrane protein, and one from tRNA binding region. Peptide JJ1, from gp41, was associated with an increased percentage of resting and blastic Vbeta 5, 8, and 21 in CD4(+), but not CD8(+) lymphocytes (3/3 donors, p = 0.014, p = 0.011, and p = 0.019, respectively, for blastic CD4(+) lymphocytes). Peptide JJ5, from the tRNA binding region, was associated with an increased percentage of resting and blastic Vbeta 5, 12, 16, and 17 in CD8(+) but not CD4(+) lymphocytes (4/4 donors for blastic CD8(+) lymphocytes, 3/4 for resting CD8(+) lymphocytes, p < 0.05 for each Vbeta family, for blastic CD8(+) lymphocytes). These results suggest that peptide JJ1 may have SAG activity restricted to CD4(+) lymphocytes and that peptide JJ5 may have restricted cytotoxic activity, associated with CD8(+) cell responsiveness. For both, the activities would lead to increased localized cytokine production and work to the advantage of the virus. These antigens might thus represent potential targets for future antiretroviral therapy.

Immune determinants of organism and outcome in febrile hospitalized Thai patients with bloodstream infections.

Opportunistic infections (OI) and the human immunodeficiency virus (HIV) cause significant morbidity and mortality in developing countries. Immune cell and cytokine profiles may be related to the type and course of OI and to the OI-HIV interaction. Examining cell-specific cytokine production ex vivo has only recently become feasible. In Thailand, 53 febrile, hospitalized adults were enrolled in a study of the immune correlates of bloodstream infections (BSI). On site, blood cells were stimulated ex vivo. Cell-surface antigens and eight intracellular cytokines were subsequently analyzed using flow cytometry to determine associations with mortality and the organism causing the BSI. By logistic regression analysis, the percentage of CD3(+) CD16/56(+) cells making tumor necrosis factor alpha (TNF-alpha) (P = 0.033) and the percentage of CD3(-) CD16/56(+) cells (NK) (P = 0.032) were related to HIV positivity. Lymph node enlargement with HIV infection and the percentage of CD3(+) CD16/56(+) making TNF-alpha were predictive of death. A lower percentage of CD3(+) CD8(+) lymphocytes making interleukin-8 (IL-8) (P = 0.005), fewer monocytes expressing CD14 (P = 0.009), and the percentage of CD3(+) CD8(+) cells producing gamma interferon (P = 0. 011) were associated with blood culture positivity and the causative organism. For every one point decrease in the percentage of CD3(+) CD8(+) cells making IL-8, the likelihood of a positive culture increased 23%; for every one point decrease in the percentage of monocytes expressing CD14, the likelihood of a positive culture increased by 5%. Only a few immune cell types and three of their related cytokines were significantly associated with HIV disease outcome or the BSI organism. These cell types did not include CD3(+) CD8(-) cells (a surrogate for CD4(+) cells), nor did they involve cytokines associated with a type I to type II cytokine shift, which might occur with advancing HIV infection. These associations support the premise that CD8(+) and CD16/56(+) lymphocytes play significant roles in HIV and type I infections.

Single-cell cytokine profiles in normal humans: comparison of flow cytometric reagents and stimulation protocols.

Cytokines are produced and function at a micro environmental level: intracellular assessment has only recently become practically feasible. We used 3-color flow cytometry to examine surface and cytoplasmic antigens on peripheral blood lymphocytes of 18 normal donors, assessing the applicability/comparability of various directly conjugated anti-human cytokine reagents and stimulation protocols using separated cells or whole blood preparations. Interdonor variability far exceeded variability due to reagent or stimulation and separation techniques. Based on all results with various reagents, post 4-5.5 h stimulation with PHA/PMA/ionomycin, the range of the percents of T lymphocytes producing various cytokines included: gamma-IFN-13.2-65.0%, IL-2-10.0-56.7%, and TNF-alpha-17.1-79.2%. Compared to CD8+ cells, CD4+ cells more often expressed IL-2 (mean 45.7% of CD4 + vs. 21.4% of CD8+ p < 0.0001), less often expressed gamma-IFN (18.5% vs. 55.3%, p < 0.0001), and did not differ in TNF-alpha expression (52.9% vs. 59.4%). Of T cells producing gamma-IFN, 64.8-100.0% also produced TNF-alpha 3.5-100.0%, IL-2. Of T cells producing IL-2, 6.0-63.9% also produced gamma-IFN and 37.6-100.0%, TNF-alpha. These results demonstrate the broad spectrum of cytokine patterns in normal human adults, as well as the usefulness and limitations of various currently available cytokine products.

Plasma ascorbate concentrations and blood cell dehydroascorbate transport in patients with diabetes mellitus.

Rates of dehydroascorbate uptake by blood granulocytes and mononuclear cells are slower, and plasma ascorbate concentrations are lower, among persons with diabetes mellitus than in nondiabetic subjects. These measurements do not correlate with one another or with simultaneously measured plasma glucose or glycosylated hemoglobin; they do not differ with type of diabetes or mode of treatment. In those diabetic granulocytes that exhibit slow dehydroascorbate uptake, maximal velocity (Vmax) transport rates for dehydroascorbate, 2-deoxyglucose, and 3-O-methylglucose are decreased, each to the same degree, while Km values for transport of these ligands are not different from those observed in nondiabetic cells. Since diffusion of these ligands is facilitated by a common transporter, these observations may reflect decreased numbers of glucose transporters in the plasma membranes of some diabetic leukocytes.