Helen E. Rudloff

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.

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.

Missense mutation in exon 7 of the common gamma chain gene causes a moderate form of X-linked combined immunodeficiency.

Clinical and immunologic features of a recently recognized X-linked combined immunodeficiency disease (XCID) suggested that XCID and X-linked severe combined immunodeficiency (XSCID) might arise from different genetic defects. The recent discovery of mutations in the common gamma chain (gamma c) gene, a constituent of several cytokine receptors, in XSCID provided an opportunity to test directly whether a previously unrecognized mutation in this same gene was responsible for XCID. The status of X chromosome inactivation in blood leukocytes from obligate carriers of XCID was determined from the polymorphic, short tandem repeats (CAG), in the androgen receptor gene, which also contains a methylation-sensitive HpaII site. As in XSCID, X-chromosome inactivation in obligate carriers of XCID was nonrandom in T and B lymphocytes. In addition, X chromosome inactivation in PMNs was variable. Findings from this analysis prompted sequencing of the gamma c gene in this pedigree. A missense mutation in the region coding for the cytoplasmic portion of the gamma c gene was found in three affected males but not in a normal brother. Therefore, this point mutation in the gamma c gene leads to a less severe degree of deficiency in cellular and humoral immunity than that seen in XSCID.

Activated neutrophils and neutrophil activators in human milk: Increased expression of CD11b and decreased expression of L-selectin

Human milk neutrophils and macrophages were examined by flow cytometry to determine whether they displayed phenotypic markers of activation. The markers were CDllb and L‐selectin, which are increased or shed, respectively, from the surface of activated neutrophils. Phenotypic features of milk neutrophils and macrophages were similar to blood neutrophils stimulated with fMLP: plasma membrane expression of CDllb was increased and L‐selectin was decreased. After blood neutrophils were incubated in acellular milk, their expression of CDllb increased and L‐selectin decreased. The activation was not affected by trypsin but was significantly decreased by treating acellular milk with chloroform or ether. Sedimentation studies suggested that particulate fractions of milk were active. Further, the activation was partly blocked by treating target blood neutrophils with cytochalasin B. Thus, human milk neutrophils are activated, and the activation may be due partly to phagocytosis of membranous structures in milk.

A novel X-linked combined immunodeficiency disease.

A novel X-linked combined immunodeficiency disease was found in five living males in an extended family in the United States. The age of the affected males ranged from 2.5 to 34 yr. The most prominent clinical abnormalities were a paucity of lymphoid tissue; recurrent sinusitis, otitis media, bronchitis, and pneumonia; severe varicella; and chronic papillomavirus infections. The principal immunologic features of the disorder were normal concentrations of serum immunoglobulins but restricted formation of IgG antibodies to immunogens; normal numbers of B cells and NK cells but decreased numbers of CD4+ and CD8+ T lymphocytes, particularly the CD45RA+ subpopulations; diminished proliferative responses of blood T cells to allogeneic cells, mitogens and antigens; and decreased production of IL-2 by mitogen stimulated blood lymphocytes. Thus, affected males in this family carry an abnormal gene on their X chromosome that results in a combined immunodeficiency that is distinct from previously reported disorders.

Quantitation of intracellular Mac-1 (CD11b/CD18) pools in human neutrophils.

The adhesive glycoprotein Mac‐1 (CD11b/CD18) of the CD11/CD18 complex contributes to multiple neutrophil inflammatory functions. Activation of neutrophils by chemotactic stimuli results in a rapid, protein synthesis‐independent increase in surface Mac‐1 derived from incompletely defined intracellular compartments. Therefore, we developed a novel quantitative lectin immunoblot technique to define intracellular pools of Mac‐1 in subcellular neutrophil fractions resolved on discontinuous Percoll gradients. In cavitates of unstimulated neutrophils, 30% and 26% of total Mac‐1 was identified in β [1.10 gm/ml; vitamin B12 binding protein (vit B12 B.P.)‐rich] or pre‐γ (1.07 gm/ml; vit B12 B.P.‐poor) granular fractions, respectively, whereas 24% was associated with the plasma membrane‐rich γ (1.06 gm/ml) fractions. N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) stimulation (10−8 M, 15 min, 37°C) significantly diminished Mac‐1 in pre‐γ (‐18% of total, P < 0.05) but not β fractions (+6% of total). Under these conditions, the content of Mac‐1 in γ fractions increased 13% in association with four‐ to eightfold increase in surface Mac‐1 expression (OKM‐1 binding). These findings suggest that chemotactic stimuli increase plasma membrane and/or surface Mac‐1 on human neutrophils by mobilizing a novel intracellular granule pool.

Complex aphthosis: A forme fruste of Behçet's syndrome?

The evaluation of the rare patient who presents with oral and genital aphthae or almost constant, multiple (greater than 3) oral aphthae, but no systemic signs or symptoms (i.e., complex aphthosis), is difficult because no laboratory test is available to exclude Behçets syndrome. Six patients with complex aphthosis were evaluated. In addition, patients with simple aphthosis, those with seronegative arthritis, and normal controls were assessed for circulating immune complexes (CIC) by in vitro and in vivo assays and for neutrophil migration by subagarose methods, since these tests have given significant results in patients with Behçets syndrome. Patient 1, with complex aphthosis, had Raji cell evidence for CIC (51.2 mg aggregated human gamma globulin Eq/ml), C1q, and C3 in dermal blood vessels 4 hours post intradermal histamine injection and had a Sweets syndrome-like vasculitis 24 hours post histamine injection. In addition, her serum enhanced the migration of patient neutrophils (3.6 +/- 0.6 to 4.6 +/- 0.5; N = 6, p less than or equal to 0.01). All other test and control patients had negative or normal CIC and neutrophil migration determinations. Sixteen-month clinical follow-up has confirmed that Patient 1, but not Patients 2 to 6, has developed overt manifestations of Behçets syndrome.

Neuronal nitric oxide synthase inhibition attenuates cardiopulmonary dysfunctions after combined burn and smoke inhalation injury in sheep.

Objective:We hypothesized that nitric oxide derived from the neuronal nitric oxide synthase (NOS) is responsible for much of the injury resulting from skin burn and smoke inhalation. Therefore, we aimed to examine the effects of selective neuronal NOS inhibition on cardiopulmonary functions and cellular injury in sheep with acute respiratory distress syndrome secondary to combined burn and smoke inhalation injury. Design:Prospective, randomized, controlled laboratory experiment. Setting:Investigational intensive care unit. Subjects:A total of 22 chronically instrumented adult ewes. Interventions:Sheep were randomly assigned to either healthy controls (sham), injured controls (40% third-degree flame burn; 48 breaths of cotton smoke), or an injury group treated with the specific neuronal NOS inhibitor 7-nitroindazole (1 mg·kg−1·hr−1) from 1 hr postinjury to the end of the 48-hr study period. Hypoxic pulmonary vasoconstriction was assessed as decrease in left pulmonary blood flow in response to single-lung hypoxic challenges (100% nitrogen) at baseline, 24 hrs, and 48 hrs. Measurements and Main Results:The combination injury contributed to a ∼90% loss of hypoxic pulmonary vasoconstriction and was associated with significant pulmonary shunting and death of one animal. The increase in nitrate/nitrite plasma levels in injured controls (12 hrs: 17 ± 2 vs. 6 ± 1 μM in sham animals; p < .001) was linked to increases in inducible NOS messenger RNA and 3-nitrotyrosine formation in lung tissue (48 hrs: 22 ± 1 vs. 0.8 ± 0.3 nM in sham animals; p < .001). 7-Nitroindazole treatment prevented the injury-associated changes in inducible NOS messenger RNA, nitrate/nitrite, and 3-nitrotyrosine, thereby attenuating the loss of hypoxic pulmonary vasoconstriction and improving gas exchange. In addition, 7-nitroindazole decreased lung tissue concentrations of hemoxygenase-1 and ameliorated myocardial depression, airway obstruction, pulmonary edema, ventilatory pressures, and histopathologic changes seen in injured controls. Conclusions:The present study provides evidence that neuronal NOS–derived nitric oxide plays a pivotal role in the pathogenesis of acute respiratory distress syndrome resulting from combined burn and smoke inhalation injury.

Two-Dimensional and Three-Dimensional Movement of Human Polymorphonuclear Leukocytes:Two Fundamentally Different Mechanisms of Location

Patients with an inherited deficiency of the adherence glycoproteins LFA‐1, Mac‐1, and p150,95 are unable to mobilize polymorphonuclear leukocytes (PMNLs) to peripheral sites of inflammation. LFA‐1/Mac‐1/p150,95‐deficient PMNL exhibited profoundly impaired movement stimulated by chemotactic factors when the cells were required to move over two‐dimensional surfaces. Less impairment of movement was demonstrated in three‐dimensional movement through cellulose filters. A possible explanation for this difference in cell translational mobility is that movement in cellulose filters is less adherence dependent than movement over a two‐dimensional plastic surface. Movement of PMNL in collagen gels is known to be relatively independent of adherence. No deficiency of translational mobility of PMNL from LFA‐1/Mac‐1/p 150,95‐deficient patients was observed in collagen gels. Antibodies against the common beta subunit effectively blocked two‐dimensional movement but had little effect on three‐dimensional movement through cellulose filters or collagen gel matrices. HL‐60 cells were employed as a model to investigate the effects of adherence on cell movement. Treatment of HL‐60 cells with phorbol myristate acetate resulted in the appearance of Mac‐1 and p150,95 on the cell surface. Concurrently, the cells exhibited increased adherence to glass and plastic. In spite of increased adherence, HL‐60 cells showed no translational movement, indicating factors other than the ability to adhere were important in cell motility. These experiments implied that PMNLs undergo two fundamentally different kinds of motion, one adherence dependent (two‐dimensional movement) and the other largely adherence independent (three‐dimensional movement). These findings are consistent with the view that egress of PMNLs from the vascular space is adherence dependent. Movement through extravascular tissues may be adherence independent.

Combined burn and smoke inhalation injury impairs ovine hypoxic pulmonary vasoconstriction

Objective:To examine the effects of combined burn and smoke inhalation injury on hypoxic pulmonary vasoconstriction, 3-nitrotyrosine formation, and respiratory function in adult sheep. Design:Prospective, placebo-controlled, randomized, single-blinded trial. Setting:University research laboratory. Subjects:Twelve chronically instrumented ewes. Interventions:Following a baseline measurement, sheep were randomly allocated to either healthy controls (sham) or the injury group, subjected to a 40%, third-degree body surface area burn and 48 breaths of cotton smoke according to an established protocol (n = 6 each). Hypoxic pulmonary vasoconstriction was assessed as changes in pulmonary arterial blood flow (corrected for changes in cardiac index) in response to left lung hypoxic challenges performed at baseline and at 24 and 48 hrs postinjury. Measurements and Main Results:Combined burn and smoke inhalation was associated with increased expression of inducible nitric oxide (NO) synthase, elevated NO2/NO3 (NOx) plasma levels (12 hrs, sham, 6.2 ± 0.6; injury, 16 ± 1.6 &mgr;mol·L−1; p < .01) and increased peroxynitrite formation, as indicated by augmented lung tissue 3-nitrotyrosine content (30 ± 3 vs. 216 ± 8 nM; p < .001). These biochemical changes occurred in parallel with pulmonary shunting, progressive decreases in Pao2/Fio2 ratio, and a loss of hypoxic pulmonary vasoconstriction (48 hrs, −90.5% vs. baseline; p < .001). Histopathology revealed pulmonary edema and airway obstruction as the morphologic correlates of the deterioration in gas exchange and the increases in airway pressures. Conclusions:This study provides evidence for a severe impairment of hypoxic pulmonary vasoconstriction following combined burn and smoke inhalation injury. In addition to airway obstruction, the loss of hypoxic pulmonary vasoconstriction may help to explain why blood gases are within physiologic ranges for a certain time postinjury and then suddenly deteriorate.