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/microg total RNA in the ventilator group and 61,854 +/- 22,433 copies/microg 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/microg total RNA compared to < 1,000 copies/microg 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/microg total RNA for the AVCO2R group and 5,854 +/- 2,070 copies/microg 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.

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 (gammac) 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) Vbeta-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 gamma-mediated pathways in XCIDL-->Q271.

Blood gammadelta T cells and gammadelta TCR V gene specificities in a single missense mutation (L-->Q271) in the common gamma chain gene.

The numbers of blood CD4+, CD8+, and CD4-CD8- T cells bearing alphabeta T-cell receptor (TCR) or gammadelta TCR molecules in males with a single missense mutation (L-->Q271) in the common gamma chain gene (gamma(c)) were investigated by flow cytometry. Virtually all XCIDL-->Q271 blood T cells that were CD4+ or CD8+ displayed alphabeta TCR but no gammadelta TCR. In contrast, CD4-CD8- T cells from affected males usually displayed gammadelta TCR, but no alphabeta TCR. The gammadelta TCR specificities were also studied. Except for the oldest subject, there was a direct relationship between blood CD3+ T cells that displayed gammadelta TCR and Vgamma9 and Vdelta2a specificities. Relative frequencies of CD3+ blood T cells that were Vgamma9+ or Vdelta2a+ were inversely related to age. In the oldest patient, the only detected gammadelta TCR specificity was Vdelta1. Thus, in contrast to mice with no gamma(c), XCIDL-Q271 blood T cells that bear gammadelta TCR with Vgamma9/Vdelta2a specificities develop but then decline in late childhood and thereafter. TCR with the Vdelta1 specificity then appear in older survivors with XCIDL-->Q271.

Immunodeficiency due to a unique protracted developmental delay in the B-cell lineage.

A unique immune deficiency in a 24-month-old male characterized by a transient but protracted developmental delay in the B-cell lineage is reported. Significant deficiencies in the number of B cells in the blood, the concentrations of immunoglobulins in the serum, and the titers of antibodies to T-dependent and T-independent antigens resolved spontaneously by the age of 39 months in a sequence that duplicated the normal development of the B-cell lineage: blood B cells followed by immunoglobulin M (IgM), IgG, IgA, and specific IgG antibodies to T-independent antigens (pneumococcal polysaccharides). Because of the sequence of recovery, the disorder could have been confused with other defects in humoral immunity, depending on when in the course of disease immunologic studies were conducted. Investigations of X-chromosome polymorphisms suggested that the disorder was not X linked in that the mother appeared to have identical X chromosomes. An autosomal recessive disorder involving a gene that controls B-cell development and maturation seems more likely. In summary, this case appears to be a novel protracted delay in the development of the B-cell lineage, possibly due to an autosomal recessive genetic defect.

Interleukin-10 in human milk.

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 degrees 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.

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.

Oxygen-induced lung injury in the guinea pig proceeds through CD18-independent mechanisms.

The pathogenesis of pulmonary oxygen toxicity is postulated to be related in part to neutrophil-mediated injury. This study examined the effect of a monoclonal antibody directed against the CD11a,b,c/CD18 glycoprotein complex (beta 2 leukocyte integrins) on oxygen-induced lung injury. M8, a monoclonal antibody that binds to the beta chain of the guinea pig leukocyte integrins that facilitate neutrophil adherence to vascular endothelium, was injected into adult guinea pigs prior to and during exposure to > 98% oxygen. Control oxygen-exposed animals were injected with a noninhibitory antibody to the CD18 complex or with saline. Survival in oxygen was similar for animals treated with M8 when compared with those treated with saline (102 versus 105 h, respectively, NS). Pulmonary edema as assessed by protein in the supernatant of bronchoalveolar lavage fluid (BALF) was higher in the three groups of oxygen-exposed animals than in the air-exposed groups (p < 0.01), but it did not differ between the M8 antibody treatment group and the other oxygen-exposed groups. M8 antibody treatment did not decrease hyperoxia-induced neutrophil accumulation into the lung as assessed by myeloperoxidase activity (MPO) in lung homogenates or by neutrophil counts in histologic specimens. M8 antibody also did not decrease neutrophil counts or MPO in alveolar lavage fluid, both of which were significantly elevated in all oxygen-exposed groups. These results suggest that hyperoxia-induced neutrophil migration into the lung and acute lung injury occurs by CD18-independent processes in the guinea pig model of pulmonary oxygen toxicity.

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.

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 CD11b 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 CD11b was increased and L-selectin was decreased. After blood neutrophils were incubated in acellular milk, their expression of CD11b 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.

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.

Repertoire of V alpha and V beta regions of T cell antigen receptors on CD4+ and CD8+ peripheral blood T cells in a novel X-linked combined immunodeficiency disease.

The repertoire of V regions of alpha/beta+ T cell receptor (TcR) on CD4+ and CD8+ T cells from the peripheral blood of six patients with a novel X-linked combined immunodeficiency was investigated by flow cytometry. The relative frequencies of V region subfamilies V alpha 2a on CD4+ T lymphocytes and V beta 5b and V beta 12a on CD8+ T lymphocytes from the peripheral blood from the affected males were decreased significantly. Also, the relative frequencies of certain other V region subfamilies on CD4+ or CD8+ T cells from the peripheral blood of some patients were either considerably below or above the ranges found in normal subjects. Although there may be other explanations including an extrathymic event, we suggest that the abnormalities in the TcR repertoire of peripheral blood T cells are consistent with a dysregulation of the intrathymic maturation/selection of T cells that leads to deficiencies in T cell populations in the peripheral blood of males with this disease.

Tumor necrosis factor-alpha in human milk.

We previously demonstrated that certain biologic activities in human milk were partially blocked by antibodies directed against human tumor necrosis factor-alpha (TNF-alpha). In this study, immunochemical methods were used to verify the presence of TNF-alpha in human milk obtained during the first few days of lactation. Gel filtration revealed the presence of TNF-alpha by RIA in molecular weight fractions between 80 and 195 kD. TNF-alpha 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-alpha-positive fractions from gel filtration, those bands proved to be nonspecific. TNF-alpha in milk was reliably quantified by the competitive RIA. Those studies revealed that the concentrations of TNF-alpha in milk were 620 +/- 183 pg/mL. Although RNA to TNF-alpha was detected in milk leukocytes by Northern blotting, little TNF-alpha was found in those cells before or after stimulation with N-formyl-l-methionyl-l-leucyl-l-phenylalanine or 4 beta-phorbol-12 beta-myristate-13 alpha-acetate. The origin of this cytokine in human milk remains unclear. Nevertheless, this study suggests that TNF-alpha 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.

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.

Chemokinetic agents for monocytes in human milk: possible role of tumor necrosis factor-alpha.

Human milk was found to contain chemokinetic agents for human blood monocytes. The chemokinetic agents were whey proteins that were inactivated by heating at 56 degrees C for 20 min or treatment with trypsin. Three peaks of chemokinetic activity less than 60 kD in size were found by gel filtration chromatography. The chemokinetic activity of each peak obtained by gel filtration was partially blocked by polyclonal rabbit antibodies to recombinant human tumor necrosis factor-alpha (TNF-alpha). TNF-alpha, or a protein that immunologically cross-reacted with it, was also detected in human milk by blockage of the cytotoxicity of human milk by anti-TNF-alpha. Such proteins or others that elicit the release of TNF-alpha from the target cells may be responsible for the enhanced motility of human milk macrophages, and it is possible that they may alter the immunologic or metabolic activities of the alimentary tract of the recipient infant.

Chemokinetic effects of exogenous and endogenous tumor necrosis factor alpha on human blood monocytes.

The possible chemokinetic effect of human tumor necrosis factor alpha (TNF-alpha) upon human monocytes was investigated by using a type 1 collagen gel system. Human recombinant TNF-alpha was found to be chemokinetic, and concentrations of that cytokine as low as 0.7 pg/microliters enhanced the movement of those leukocytes. Furthermore, we explored whether endogenous TNF-alpha stimulates monocyte movement by incubating blood monocytes with a TNF-alpha-stimulating agent, human recombinant interleukin 1, with or without antibodies to human TNF-alpha. Human recombinant interleukin 1 enhanced the motility of blood monocytes, and that effect was blocked by rabbit polyclonal antibodies to human TNF-alpha. Furthermore, the movement of blood monocytes no exposed to exogenous cytokines was decreased after incubation with anti-TNF-alpha. Thus, endogenous as well as exogenous TNF-alpha is a chemokinetic agent for human monocytes.

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 beta [1.10 gm/ml; vitamin B12 binding protein (vit B12 B.P.)-rich] or pre-gamma (1.07 gm/ml; vit B12 B.P.-poor) granular fractions, respectively, whereas 24% was associated with the plasma membrane-rich gamma (1.06 gm/ml) fractions. N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation (10(-8) M, 15 min, 37 degrees C) significantly diminished Mac-1 in pre-gamma (-18% of total, P less than 0.05) but not beta fractions (+6% of total). Under these conditions, the content of Mac-1 in gamma 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.

Recognition of an endothelial determinant for CD 18-dependent human neutrophil adherence and transendothelial migration.

Human neutrophil (PMN) attachment to human umbilical vein endothelial cells (HUVEC) was evaluated in vitro using two MAbs, R6-5-D6 and RR1/1, that recognize intercellular adhesion molecule-1 (ICAM-1), and one MAb, TS1/18, that recognizes CD18. Pretreatment of the HUVEC with anti-ICAM-1 MAbs produced greater than 50% inhibition of attachment to HUVEC, and IL-1 (0.5 U/ml)- or lipopolysaccharide (LPS) (10 ng/ml)-stimulated HUVEC, and greater than 99% inhibition of f-Met-Leu-Phe (0.5 nM) enhanced adherence. Anti-ICAM-1 MAbs also inhibited by greater than 85% the transendothelial migration induced by 4-h IL-1 (0.5 U/ml) and LPS (10 ng/ml) activation of the HUVEC. That these effects involved a CD18-dependent mechanism is supported by the following results: pretreatment of PMN with TS1/18 produced the same degree of inhibition of attachment and migration as seen with R6-5-D6. In addition, the use of both MAbs together did not further increase the inhibition of cell attachment to stimulated HUVEC. The attachment of PMN from patients with CD18 deficiency to stimulated HUVEC was not reduced by R6-5-D6, and both R6-5-D6 and TS1/18 revealed the same time course for appearance and disappearance of an adherence component on stimulated HUVEC not blocked by either MAb. These results demonstrate that attachment and transendothelial migration of PMN in vitro depend substantially on both CD18 on the PMN and ICAM-1 on the endothelial cell.

Two-dimensional and three-dimensional movement of human polymorphonuclear leukocytes: two fundamentally different mechanisms of locomotion [corrected].

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/p150,95-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.

Decreased response of human milk leukocytes to chemoattractant peptides.

To test the hypothesis that neutrophils and macrophages in human milk may not defend by classical inflammatory mechanisms, experiments were conducted to ascertain whether adherence, orientation, and directed motility of these leukocytes would be enhanced by exposure to chemoattractant peptides including N-formyl-L-methionyl-L-phenylalanine and N-formyl-L-methionyl-L-leucyl-L-phenylalanine and C5a generated from zymosan activated human serum. Adherence and spatial orientation were tested on coverglasses and in Zigmond chambers, and chemotaxis was examined by Boyden chambers and a subagarose technique. Whereas, the adherence, orientation, and directed movement of adult peripheral blood neutrophils and monocytes were significantly enhanced by those chemotactic agents, human milk leukocytes failed to respond. The failure of the response of human milk leukocytes was not due to alterations in maternal peripheral blood leukocytes but appeared to be due partially to inhibitors in human milk. The experiments suggest that human milk leukocytes may be modified in the mammary gland to protect by noninflammatory mechanisms.