Kimberly H. Palkowetz

Interleukin-10 in human milk.

ABSTRACT: The concentrations of immunoreactive IL-10 in the aqueous fraction of 20 specimens of human milk obtained during the first 80 h of lactation and stored at –60°C ranged from 66 to 9301 pg/mL (mean ± SD, 3304 ± 3127 pg/mL). IL-10 was present also in the lipid layer of milk. Gel filtration revealed that IL-10 was located in a high molecular weight fraction, where certain other cytokines in human milk have been found. In addition, immunoreactive IL-10 in milk increased after treatment with sodium taurocholate. Bioactive IL-10 was demonstrated by the finding that human milk inhibited [3H]thymidine uptake by human blood lymphocytes and that inhibition was partly overcome by concomitant incubation with antibodies to human IL-10. IL-10 mRNA but no protein product was found in cultured human mammary epithelial cells. Some IL-10 was associated with preparations of human milk leukocytes, but the data did not suggest that the cells were producing the cytokine. Bioactive IL-10 in a possible protected compartment suggests that IL-10 in human milk may have immunomodulating, antiinflammatory effects on the alimentary tract of the recipient infant.

Decreased Interleukin-10 Production by Neonatal Monocytes and T Cells: Relationship to Decreased Production and Expression of Tumor Necrosis Factor-α and Its Receptors

The production of IL-10 by human neonatal blood mononuclear leukocytes(BML) stimulated with lipopolysaccharide (LPS), tumor necrosis factor-α(TNF-α), antibodies to CD3, or phorbol 12-myristate 13-acetate (PMA) was measured. The production of IL-10 by neonatal BML cultured with LPS or TNF-α was ≈20 and ≈15%, respectively, of adult BML. The combination of human recombinant TNF-α and LPS failed to augment IL-10 production in neonatal BML. The decreased production of IL-10 by neonatal leukocytes was not due to an autocrine feedback mechanism because only low concentrations of IL-10 were found in newborn sera. A connection with TNF-α could not be ruled out, because TNF-α production by LPS-stimulated newborn BML and the expression of TNF-α receptors on newborn monocytes were reduced. Mean ± SD of concentrations of IL-10 in supernatants from adult and neonatal BML after stimulation with antibodies to human CD3 for 48 or 72 h were 914 ± 386 and 178 ± 176 pg/mL, respectively (p < 0.0001). In experiments with enriched populations of neonatal T cells, the addition of PMA failed to augment IL-10 production. This suggested that newborn T cells may be in a different state of activation than adult T cells Thus, IL-10 production in neonatal monocytes and T cells is reduced and this study suggests that the reduction may be secondary in part to regulatory processes involving TNF-α and its receptors.

Tumor necrosis factor-α in human milk

ABSTRACT: We previously demonstrated that certain biologic activities in human milk were partially blocked by antibodies directed against human tumor necrosis factor-α (TNF-α). In this study, immunochemical methods were used to verify the presence of TNF-α in human milk obtained during the first few days of lactation. Gel filtration revealed the presence of TNF-α by RIA in molecular weight fractions between 80 and 195 kD. TNF-α could not be detected consistently by conventional Western blotting or cytotoxic assays. Although immunoreactive bands were detected by a Western blot-125I protein A technique in TNF-α-positive fractions from gel filtration, those bands proved to be nonspecific. TNF-α in milk was reliably quantified by the competitive RIA. Those studies revealed that the concentrations of TNF-α in milk were 620 ± 183 pg/mL. Although RNA to TNF-α was detected in milk leukocytes by Northern blotting, little TNF-α was found in those cells before or after stimulation with N-formyl-l-methionyl-l-leucyl-l-phenylalanine or 4β-phorbol-12β-myristate-13α-acetate. The origin of this cytokine in human milk remains unclear. Nevertheless, this study suggests that TNF-α is present in early human milk in sufficient quantities to exert possible biologic effects upon the mammary gland of the mother or the immune system of the infant.

Production of interleukin-6 and interleukin-8 by human mammary gland epithelial cells

The production of transforming growth factor-beta 2 (TGF-beta 2), interleukin-1 beta (IL-1 beta), IL-6, IL-8, and tumor necrosis factor-alpha (TNF-alpha) by spontaneously immortalized human mammary gland epithelial cells of non-malignant origin and the effect of prolactin upon the production of those cytokines were investigated. Cells were cultured on plastic with epithelial growth factor, insulin, and hydrocortisone. Cytokines were quantified by enzyme-immunoassays. The cells produced IL-6 and IL-8, but no detectable TGF-beta 2, IL-1 beta, or TNF-alpha. Although prolactin enhanced the uptake of [3H]thymidine, it did not alter the production of cytokines/interleukins. Because of the marked production of IL-8 by mammary epithelium and a past report of TGF activity in human milk, those agents were quantified in human milk. The mean +/- S.D. concentrations of IL-8 and TGF-beta 2 in human milk obtained in the first 3 days of lactation were 3684 +/- 2910 and 130 +/- 108 pg/ml, respectively. Thus, IL-8 and TGF-beta 2 are normal constituents in human milk, and human mammary gland epithelium may be responsible for producing some of the IL-6 and IL-8 in human milk.

Interleukin-6 in human milk

Interleukin-6 (IL-6) in human milk collected during the first 2 days of lactation was investigated by a competitive radioimmunoassay (RIA) and column chromatography. The concentrations of IL-6 in the aqueous phase of fresh human milk were 151 +/- 89 pg/ml. The concentrations of IL-6 in milk increased after storage at 4 degrees C and decreased after storage at -20 degrees C (P < 0.01). Column chromatography revealed two molecular weight peaks of IL-6 in human milk, the first > or = 100 kDa and the second between 25 and 30 kDa. The 25-30-kDa peak corresponded to known isoforms of human IL-6 and to the elution pattern for 125I-labeled recombinant human IL-6, whereas the higher molecular weight peak may be in keeping with a bound or compartmentalized form of that cytokine. The precise molecular forms of this protein, the compartmentalization of or binding proteins for this cytokine and the in vivo effects of IL-6 in human milk upon the mammary gland or the recipient infant remain to be explored.

Heat Susceptibility of Interleukin-10 and Other Cytokines in Donor Human Milk

OBJECTIVE Holder pasteurization renders donor human milk safe for consumption. Because human milk reduces the risk of necrotizing enterocolitis in preterm infants, we tested whether Holder pasteurization affects certain factors in human milk that protect the intestines: epidermal growth factor (EGF), transforming growth factor (TGF)-beta1, erythropoietin (EPO), and interleukin (IL)-10. Donor human milk from a milk bank was examined. METHODS The aqueous phase of 17 samples of donor term human milk (mean duration of lactation, 8 +/- 3.5 months) was examined before and after Holder pasteurization. In the case of IL-10, lesser degrees of pasteurization were also evaluated. The agents were quantified using enzyme immunoassays. The function of IL-10 was also tested. RESULTS Concentrations of EGF and IL-10 were markedly lower than previously reported values in human milk from earlier phases of lactation. Holder pasteurization significantly reduced the concentrations of EPO and IL-10, whereas lesser degrees of heating increased the detection of IL-10. The immunosuppression of T-cell proliferation by human milk, thought to be attributed to IL-10 alone, persisted after Holder pasteurization. CONCLUSIONS Holder pasteurization greatly decreased concentrations of EPO and IL-10 in human milk. These decreases may impact the ability of human milk to protect against necrotizing enterocolitis. Evidence of possible binding of IL-10 to other proteins in human milk was also found. Experiments to test whether Holder pasteurization affects the function of IL-10 in human milk produced evidence for an agent in human milk other than IL-10 that inhibits T-cell proliferation and resists Holder pasteurization.

Arteriovenous CO2 Removal Improves Survival Compared to High Frequency Percussive and Low Tidal Volume Ventilation in a Smoke/Burn Sheep Acute Respiratory Distress Syndrome Model

Objectives and Summary Background:Low tidal volume ventilation (LTV) has improved survival with acute respiratory distress syndrome (ARDS) by reducing lung stretch associated with volutrauma and barotrauma. Additional strategies to reduce lung stretch include arteriovenous carbon dioxide removal (AVCO2R), and high frequency percussive ventilation (HFPV). We performed a prospective, randomized study comparing these techniques in our clinically relevant LD100 sheep model of ARDS to compare survival, pathology, and inflammation between the 3 ventilator methods. Methods:Adult sheep (n = 61) received smoke inhalation (48 breaths) and a 40% third-degree burn. After ARDS developed (Pao2/FiO2 <200), animals were randomized. In experiment 1, animals were killed at 48 hours after randomization. Hemodynamics, pulmonary function, injury scores, myeloperoxidase (MPO) in lung tissues and neutrophils, IL-8 in lung tissues, and apoptosis were evaluated. In experiment 2, the end point was survival to 72 hours after onset of ARDS or end-of-life criteria with extension of the same studies performed in experiment 1. Results:There were no differences in hemodynamics, but minute ventilation was lower in the AVCO2R group and Paco2 for the HFPV and AVCO2R animals remained lower than LTV. Airway obstruction and injury scores were not different among the 3 ventilation strategies. In experiment 1, lung tissue MPO and IL-8 were not different among the ventilation strategies. However, in experiment 2, lung tissue MPO was significantly lower for AVCO2R-treated animals (AVCO2R < HFPV < LTV). TUNEL staining showed little DNA breakage in neutrophils from experiment 1, but significantly increased breakage in all 3 ventilator strategies in experiment 2. In contrast, AVCO2R tissue neutrophils showed significant apoptosis at 72 hours post-ARDS criteria as measured by nuclear condensation (P < 0.001). Survival 72 hours post-ARDS criteria was highest for AVCO2R (71%) compared with HFPV (55%) and LTV (33%) (AVCO2R vs. LTV, P = 0.05). Conclusions:Significantly more animals survived AVCO2R than LTV. In experiment 2, Lung MPO was significantly lower for AVCO2R, compared with LTV (P < 0.05). This finding taken together with the TUNEL and neutrophil apoptosis results, suggested that disposition of neutrophils 72 hours post-ARDS criteria was different among the ventilatory strategies with neutrophils from AVCO2R-treated animals removed chiefly through apoptosis, but in the cases of HFPV and LTV, dying by necrosis in lung tissue.

Interleukin-8, aquaporin-1, and inducible nitric oxide synthase in smoke and burn injured sheep treated with percutaneous carbon dioxide removal.

We previously showed that a percutaneous arteriovenous gas exchanger was effective in removing CO2 and reversing respiratory failure in an ovine model of adult respiratory distress syndrome (ARDS) produced by smoke inhalation and burn injury (Alpard et al., Ann Surg 230:215–224, 1999). In this study, we tested the hypothesis that arteriovenous CO2 removal (AVCO2R) lessened endogenous inflammation in the lung. Myeloperoxidase activity, aquaporin-1 (AQP-1), interleukin-8 (IL-8), and inducible nitric oxide synthase mRNAs as well as aquaporin-1, and IL-8 protein were measured in ovine lung tissue. Lung tissue was taken at 96 h (time of sacrifice) from animals with combined smoke inhalation and 40% third degree dermal burn and subsequently treated with AVCO2R or sham (ventilator alone) after onset of ARDS (PaO2:FiO2 ratio of < 200). Myeloperoxidase activity was 1.862 ± 0.302 U/mg protein in the ventilator group and 0.830 ± 0.141 in the AVCO2R plus ventilator group. AQP-1 mRNA was 140,482 ± 31,702 copies/&mgr;g total RNA in the ventilator group and 61,854 ± 22,433 copies/&mgr;g total RNA in the AVCO2R plus ventilator group (p = 0.076). mRNA for IL-8 mRNA in the ventilator alone treated animals was 74,000 ± 3,300 copies/&mgr;g total RNA compared to < 1,000 copies/&mgr;g total RNA in the ventilator plus AVCO2R group. This result was highly significant (p < 0.001) Inducible nitric oxide synthase mRNA was 7,853 ± 2,229 copies/&mgr;g total RNA for the AVCO2R group and 5,854 ± 2,070 copies/&mgr;g total RNA for the ventilator managed animals. These differences were not statistically significant (p = 0.54). Percutaneous AVCO2R produced a specific decrease in IL-8 in the smoke and burn injured animals. Furthermore, this effect was consistent with cell signaling mechanisms that increase the expression of IL-8 by cyclic stretching and the observed reduction in the number of neutrophils in the lung parenchyma. Therefore, we speculate that the mechanism by which CO2 removal exerts a beneficial effect may be due to both decreases in ventilatory requirements, with an accompanying reduction in alveolar stretching, and reduction of neutrophil numbers in lung tissue.

Deficient quantitative expression of CD45 isoforms on CD4+ and CD8+ T cell subpopulations and subsets of CD45RAlowCD45ROlow T cells in newborn blood

Deficiencies in the quantitative expression of CD45RA and CD45RO on CD4+ and CD8+ T cells and in a population of CD45RA(low)CD45RO(low) T cells in blood from term newborn infants were found by flow cytometry. The relative frequencies of CD45RO on CD4+ T cells from adults and newborn infants were 72 and 58%, respectively. However, in newborn infants greater than 70% of T cells expressing CD45RO also expressed CD45RA. In addition, the quantitative expression of CD45RA and CD45RO on newborn T cells was significantly less than that found on adult blood T cells.

Genesis of Progressive T‐Cell Deficiency Owing to a Single Missense Mutation in the Common Gamma Chain Gene

Patients with a moderate X‐linked combined immunodeficiency (XCID) owing to a single missense mutation in the common gamma chain (γc) gene (L→Q271) were found to have a progressive T‐cell deficiency. Blood T cells from four older subjects with XCIDL→Q271 were studied to ascertain the basis of that progression. Few CD4+ T cells displayed the phenotype (CD45RA+ CD62L+) or deletion circles from T‐cell receptor (TCR) Vβ‐gene rearrangements found in recent thymic emigrants. These deficiencies were more severe in older males with XCIDL→Q271. Relative frequencies of fresh CD4+ and CD8+ T cells that bound annexin V, an early indicator of programmed cell death, or propidium iodide, an indicator of cell necrosis, were greater in XCIDL→Q271 T cells than in normal fresh T cells. The binding of annexin V and propidium iodide to XCIDL→Q271 T cells increased marginally after stimulation with anti‐CD3, but binding by fresh or stimulated XCIDL→Q271 T cells exceeded that found in normal stimulated T cells. Also, telomeres from XCIDL→Q271 CD4+ T cells were shortened in these patients compared to normal young adults. It therefore appears that the thymus is dysfunctional and that mature T cells are not effectively rescued from apoptosis or replication senescence via γc‐mediated pathways in XCIDL→Q271.