Neutrophil activation in preeclampsia. Are defensins and lactoferrin elevated in preeclamptic patients?

OBJECTIVE: To determine if human defensins and lactoferrin, both markers of neutrophil activation, are elevated in preeclamptic plasma. STUDY DESIGN: Blood samples were obtained from 18 preeclamptic and 29 normal pregnant women in the third trimester. Demographic and clinical data were obtained from the medical record. No patient had evidence of labor and/or infection when blood was drawn. Preeclamptic patients were defined by elevated blood pressure, 140/90 mm Hg, proteinuria of > 300 mg in a 24-hour collection and hyperuricemia. Human defensins and lactoferrin were measured by enzyme immunoassay of plasma samples diluted 1:100 and 1:10, respectively. Standard curve values ranged from 0.25 to 16 ng/mL. Data for human defensins and lactoferrin are presented as median values +/-SE. Statistical analysis included Student's t test for comparison of clinical data, Mann-Whitney U test for comparison of absolute values between groups and Fisher's exact test, when appropriate. RESULTS: There was no difference in age or estimated gestational age between the two groups. There were more nulliparous patients in the preeclamptic group. Human defensin levels were significantly elevated (P = .005) in plasma of preeclamptic patients (25.1 ng/mL +/- 16.2) as compared to normal controls (9.0 ng/mL +/- 8.9). Nine of 18 (50%) preeclamptic patients and 2 of 29 (7%) normal controls had defensin levels above the low point on the standard curve (P = .001). There was no difference in lactoferrin levels between the two groups. CONCLUSION: Our results suggest that preeclampsia is associated with neutrophil activation. The biologic effect of elevated human defensins in preeclamptic plasma remains to be determined.

Human neutrophil defensin and serpins form complexes and inactivate each other.

Defensins, antimicrobial and cytotoxic peptides of neutrophils, bind to and are inactivated by blood proteins. We identified defensin interactions with alpha 1-proteinase inhibitor (alpha 1-PI), alpha 1-antichymotrypsin (alpha 1-ACT), alpha 2-antiplasmin (alpha 2-AP), and antithrombin III (AT III) and examined defensin binding to alpha 1-PI and alpha 1-ACT in more detail. Defensin interactions with either alpha 1-PI or alpha 1-ACT were not affected by iodoacetamide or high salt concentration. Preincubation of alpha 1-ACT or alpha 1-PI with increasing concentrations of defensin resulted in a progressive decrease of antiprotease activity of both inhibitors against cathepsin G and antiprotease activity of alpha 1-PI against human neutrophil elastase. At higher concentrations, defensin also ablated the inhibitory effect of normal human serum on cathepsin G and human neutrophil elastase. Both alpha 1-PI and alpha 1-ACT inhibited defensin cytotoxicity toward the human lung carcinoma cell line A549, whereas the elastase inhibitor antileukoprotease did not. Complex interactions between serpins and defensin may have a role in regulating inflammatory processes.

Identification of defensin binding to C1 complement.

In human serum we found strong defensin binding to the complexes of activated C1 complement (C1) and C1 inhibitor (C1i). Purified C1q, activated C1 tetramer (r2s2) and C1i did not bind defensin. When r2s2 was dissociated by EDTA, only the activated C1s (C1s) bound defensin. Binding of defensins to C1 complement represents a newly recognized bridge between the complement- and phagocyte-mediated host defenses, and a potential mechanism for protecting infected tissue from cytotoxic injury by defensin.

Plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis.

We measured concentrations of defensins (human neutrophil peptides) in the plasma of healthy volunteers and patients with sepsis and meningitis. When a sensitive enzyme immunoassay was used, defensins were detected in plasma samples from 13 of 24 healthy blood donors, with a mean +/- SD of 42 +/- 53 ng/ml. Defensin levels in plasma samples from seven patients with sepsis at the onset of disease ranged from 900 ng/ml to 170,000 ng/ml. In 10 patients with meningitis in the initial phase of disease, plasma defensin concentrations ranged from 120 ng/ml to 910 ng/ml. Defensin concentrations in the plasma of both patient groups were significantly higher than those in healthy blood donors (p << 0.01), and patients with sepsis had higher defensin levels than patients with meningitis (p < 0.01). Defensin levels were significantly (p < 0.01) lower after the beginning of specific antibiotic therapy. Defensin concentrations in the plasma of patients with sepsis and meningitis correlated only weakly (r = 0.38) with blood neutrophil count. In vitro studies of defensin added to plasma indicated that all defensin was bound to plasma proteins. At high concentrations (1000 micrograms/ml), defensins caused precipitation of plasma proteins. Because plasma defensin levels may reflect neutrophil activation at sites of infection and inflammation, studies of the clinical utility of defensin ELISA are indicated.

The effect of biotinylation on the antigenic specificity of anti-defensin monoclonal antibodies.

We studied the effects of biotinylation on three monoclonal antibodies (Mabs) that were raised against carrier protein conjugates of human defensin HNP-1, and of rabbit defensins NP-2 and NP-5 respectively. Before biotinylation, each Mab specifically bound to its peptide hapten. Biotinylation of these Mabs by the N-hydroxysuccinimide-biotin (NHS-biotin) resulted in crossreactivity of each Mab with the two irrelevant defensin peptides. In contrast, Mab specificity was preserved after biotinylation with biotin hydrazide, which links biotin to the glycan moiety of antibodies. The effects of NHS-biotinylation were in part mimicked by 2,4-dinitrofluorobenzene, another agent that modified primary amine groups of proteins, suggesting that this modification contributed to the change in antibody specificity. When a high degree of antigenic specificity against peptide immunogens is required, biotinylation on the glycan moiety of Mabs may be preferable.

Mononuclear cells from HIV-infected patients produce factors which enhance functional activity of polymorphonuclear neutrophils from healthy subjects.

The influence of mononuclear cell supernatants (MNCS) from nine healthy donors and 35 HIV-infected patients (17 with lymphoadenopathy syndrome (LAS), 15 with ARC and three with AIDS) on functional activity of polymorphonuclear neutrophils (PMN) from healthy donors was investigated. MNC after short-term cultivation (24 h) produced factors which enhanced chemiluminescence (CL) and chemotaxis of PMN. This augmentation did not depend on stimulation of MNC by mitogens (lipopolysaccharide Escherichia coli (LPS) and concanavalin A (Con A)) or on activation of PMN by FMLP. After 48 h of cultivation only MNC stimulated by LPS produced these factors. MNCS from HIV-infected patients provoked a more pronounced augmentation of PMN CL compared with MNCS from healthy subjects. This enhancement was observed in patients at all stages of infection, but was more pronounced in patients with LAS. MNCS impact on PMN CL was not connected with proliferative activity of MNC but was correlated with the level of CD4 cells. It was shown that removal of adherent cells from MNC fraction resulted in decreased MNCS impact. Treatment of MNCS by antibody to IL-1 beta, IL-8, interferon-alpha (IFN-alpha) and tumour necrosis factor-alpha (TNF-alpha) did not decrease MNCS impact on PMN CL.

An enzyme immunoassay for human defensins.

We developed and optimized an enzyme immunoassay for human neutrophil defensins, cationic cysteine-rich peptides that participate in host defense and inflammation. The assay utilizes a sandwich design with a monoclonal capture antibody and a biotinylated monoclonal detecting antibody. Cetrimonium bromide is employed to obviate non-specific binding of defensins to surfaces. The assay has a sensitivity of 0.04-0.05 ng/ml and a working range of 0.05-10 ng/ml.

Tumor necrosis factor alpha induction in human monocytes.

The present study was undertaken to assess the presence of tumor necrosis factor (TNF)-alpha mRNA and protein in circulating human blood monocytes and to study the TNF-alpha gene expression in human monocytes isolated by continuous Percoll gradient fractionation. The technique of RNA isolation directly from the blood samples was used to study TNF-alpha mRNA expression in circulating human blood leukocytes. It was shown that human blood leukocytes of healthy donors contained no presynthesized pool of TNF-alpha mRNA as well as no TNF-alpha protein. It was found that early pretreatment with cycloheximide interferes with TNF-alpha mRNA induction by Staphylococcus aureus.

The use of syngeneic cells producing human somatotropic hormone (hSTH) for immunization of mice and development of hybridomas.

We studied the possibility of syngeneic cells expressing heterologous protein being used for sensitization of mice and production of hybridomas. Recombinant retroviral vector containing cloned human somatotropic hormone (hSTH) gene was used to express hSTH in BALB/3T3 cells. BALB/c mice were injected intrasplenically (i/s) or combination of intraperitoneally (i/p) and intrasplenically with hSTH-producing cells. Sensitized splenocytes were fused with myeloma cell P3X63-AgB.653. Screening for anti-hSTH hybridomas was performed by enzyme-linked immunoassay. Both single i/s injection of producer cells as well as combined i/s and i/p injections were effective for sensitization of splenocytes. Combined injection was effective for production of IgG and IgM secreting hybridomas. Single i/s injection led to generation of only IgM producing hybridomas. The results proved that syngeneic cells expressing genes of heterologous proteins can be used for splenocyte sensitization and hybridoma preparation.

Induction of tumor necrosis factor synthesis in human monocytes treated by transcriptional inhibitors.

Human blood monocytes and lymphocytes were separated by Percoll gradient fractionation. The synthesis of RNA was inhibited by actinomycin D (AcD) or alpha-amanitin (Amn). Monocytes were stimulated with LPS, lymphocytes were stimulated with phytohaemagglutinin (PHA). The activity of tumor necrosis factor (TNF-alpha) and lymphotoxin (LT) was measured by L-929 cell assay. It was shown that induction of TNF-alpha synthesis by LPS was not blocked by AcD and Amn. In contrast, the production of LT was blocked in cultures of lymphocytes treated by the inhibitors. Moreover, TNF-alpha synthesis was induced in resting monocyte cultures by AcD. Cycloheximide (Cy) inhibited the production of TNF-alpha. The data imply that TNF-alpha synthesis by human blood monocytes can be induced by posttranscriptional regulation of TNF-alpha mRNA presynthesized in vivo.