Michael F. Tosi

Abnormalities of polymorphonuclear leukocyte function associated with a heritable deficiency of high molecular weight surface glycoproteins (GP138): common relationship to diminished cell adherence.

Investigations of polymorphonuclear leukocyte (PMN) function were performed in a 5-yr-old white female with delayed umbilical cord separation, impaired pus formation, and a severe defect of PMN chemotaxis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated an almost total deficiency of a high molecular weight glycoprotein(s) (GP138) in the granule and membrane fractions of the patients cells, and NaB3H4-galactose oxidase labeling demonstrated the absence of a major glycoprotein complex on the surface of her PMNs. Monoclonal antibodies (MAb) were employed in flow cytometry experiments to demonstrate that two previously characterized glycoproteins (Mo1 and LFA1) were undetectable on the surface of the patients PMNs and monocytes. Immunoprecipitation of 125I-labeled patient cells with subunit specific MAbs confirmed that the alpha-subunits of Mo1 (155 kD) and LFA1 (177 kD) and their common beta-subunit (94 kD) were totally deficient. Functional analyses of patient PMNs demonstrated severe impairment of adherence- and adhesion-dependent cell functions including spreading, aggregation, orientation in chemotactic gradients, antibody-dependent cellular cytotoxicity, and phagocytosis of particles (Oil-Red-0-paraffin, zymosan) selectively opsonized with C3-derived ligands. Patient PMNs demonstrated a normal capacity to rosette with IgG or C3b-coated sheep erythrocytes, but rosette formation with C3bi-coated erythrocytes was profoundly diminished. Adhesion-independent functions including shape change, N-formyl-methionyl-leucyl-3H-phenylalanine binding, and O-2 generation or secretion elicited by soluble stimuli were normal. Membrane fluidity, surface charge, and microtubule assembly were also normal. These findings provide new evidence that critical PMN surface glycoproteins are required to facilitate multiple adhesion-dependent cellular functions of the inflammatory response.

Treatment of childhood angiomatous diseases with recombinant interferon alfa-2a.

A heterogeneous group of five patients with progressive, invasive angiomatous diseases including pulmonary hemangiomatosis, angiosarcoma, or massive hemangioma with associated consumptive coagulopathy were treated with interferon alfa-2a for periods of 17 to 33 months. One patient with a large thoracic hemangioma, cardiorespiratory failure, and consumptive coagulopathy died after less than 2 months of treatment. The remaining four patients have shown beneficial responses, including (1) regression of abnormal vessels on pulmonary angiogram and improved exercise tolerance in pulmonary hemangiomatosis (two patients), (2) decreased corticosteroid and/or platelet transfusion requirements in consumptive coagulopathy (two patients), and (3) decreased size and number of tumor nodules in the one patient with angiosarcoma arising in preexisting angiomatous lesions. Responses occurred during periods of 2 to 20 months of treatment. There was no measurable progression of angiomatous lesions in any patient receiving interferon at the therapeutic dose, except possibly in the one who died. Each of the four surviving patients had improved linear growth and weight gain during interferon treatment.

Detection of enhanced neutrophil adhesion to parainfluenza-infected airway epithelial cells using a modified myeloperoxidase assay in a microtiter format

Despite growing evidence that respiratory virus infections precipitate episodes of airway obstruction and airway hyper-responsiveness in young children and in asthma, little information is available on the mechanisms by which virus infections alter the airway physiology. Airway inflammatory changes (including influx of inflammatory cells such as neutrophils) have been described during episodes of airway hyper-responsiveness in both animal models and human subjects. Neutrophil damage to several cell types has been shown to require adhesion as a primary step. In order to examine the potential interactions between virus-infected airway epithelial cells and neutrophils, we have studied the ability of neutrophils to adhere to virus-infected airway epithelial cell cultures. Neutrophil adherence was determined indirectly, using myeloperoxidase as a marker for adherent neutrophils in an assay system described here. Airway epithelial cell cultures (both primary human tracheal epithelial cells, and two permanent cell lines, A549 and BEAS-2B) were grown in 96-well tissue culture plates and infected with human parainfluenza virus type 2. Infected airway epithelial cell cultures supported significantly enhanced levels of neutrophil adherence (up to 50-75% of neutrophils added to the wells) compared to uninfected control cultures. Moreover, this adherence occurred in a virus dose-dependent fashion, with increasing levels of adherence noted at increasing viral multiplicities of infection. The assay system described allows the detection of small numbers of adherent neutrophils (as few as 1000 neutrophils) in a 96-well format.

Tissue-specific fc γ and complement receptor expression by alveolar macrophages determines relative importance of igg and complement in promoting phagocytosis of pseudomonas aeruginosa

ABSTRACT: Because the expression of IgG Fc receptors and complement receptors on macrophages may vary in a tissue-specific manner, we used monoclonal antibodies and flow cytometry to define the expression and function of opsonin receptors on fresh normal and cystic fibrosis (CF) bronchoalveolar lavage (BAL) macrophages. Using flow cytometry to separately analyze individual types of cells, we then determined the relative contributions of IgG and complement to phagocytosis of Pseudomonas aeruginosa by fresh BAL cells, avoiding alterations in receptor expression due to in vitro purification or culturing techniques. Neither normal nor CF BAL macrophages express appreciable amounts of the complement receptors CRI, CR2, or CR3. These results were confirmed by immunohistochemical staining of fixed lung sections. BAL macrophages express a high-affinity IgG receptor, Fc γRI, that is not found on neutrophils (PMN). In contrast, chemoattractant-stimulated blood PMN express large amounts of CR1 and CR3 but do not express Fc γRI. These results correlate with phagocytosis assays, which show that phagocytosis by macrophages is enhanced by relatively low concentrations of IgG but that the addition of complement does not further increase their phagocytosis. In contrast, low concentrations of IgG alone do not promote phagocytosis by PMN, whereas addition of complement markedly enhances phagocytosis by PMN. These results may explain the previously reported sensitivity of macrophages rather than PMN to the “blocking” effects of anti-Pseudomonas antibodies from CF patients, and emphasize the pathologic significance of interference with IgG and complement mediated opsonization in the lung in CF.

Voluntary modulation of neutrophil adhesiveness using a cyberphysiologic strategy.

In a study of voluntary immunomodulation, 45 subjects were assigned either to a control group or one of two experimental groups. All groups had blood and saliva samples collected before and after either a 30 minute rest condition (Control group) or a 30 minute cyberphysiologic strategy (Experimental groups) to increase neutrophil adherence. These samples were analyzed on a range of immunologic measurements including neutrophil adherence. The second experimental group practiced a cyberphysiologic strategy two weeks prior to the experimental session. Subjects in each group returned to repeat their exercise in a second session the following week. Analysis of all immune measurements revealed statistical significance for changes in neutrophil adherence. These studies suggest that such strategies may be used to effect changes in immune cell functions. Analysis further revealed that those subjects with prior cyberphysiologic training were able, by the second session, to induce a significant increase in neutrophil adherence.

Postnatal maturation of total cell content and up-regulated surface expression of Mac-1 (CD11b/CD18) in polymorphonuclear leukocytes of human infants

Markedly deficient expression of membrane‐activated complex 1 (Mac‐1; CD11b/CD18) by polymorphonuclear neutrophils (PMN) of human neonates compared with adults is well documented. To define postnatal maturation of Mac‐1 expression of PMN, lysates of PMN from 21 infants, aged 1–14 months, and concurrent adult controls were assayed by ELISA for total cell content of Mac‐1 and LFA‐1 (CD11a/CD18), and LFA‐1 content was within the normal adult range at all ages tested. Mac‐1 content was ∼50% of adult levels for infants 1–2 months of age and steadily increased to reach normal adult levels by 11–12 months of age. For a separate group of 25 infants, aged 0.5–11 months, measurement of surface expression of Mac‐1 and LFA‐1 on activated PMN by immunofluorescence flow cytometry yielded results that were similar to those obtained by ELISA.

Directional Changes in Neutrophil Adherence Following Passive Resting Versus Active Imagery

This study was designed to determine whether increases or decreases in neutrophil adherence could be achieved following a self-regulation (relaxation/imagery) intervention. Fifteen subjects were randomly assigned to one of three conditions. Two experimental groups employed imagery focussed on either increasing or decreasing neutrophil adherence. Subjects had two weeks of self-regulation practice (4 total training sessions) prior to blood drawings. A third group of control subjects had the same number of resting sessions without imagery training. All subjects had blood samples collected before and after either 30 minutes of self-regulation or resting practice for two sessions. Pulse and peripheral finger temperature measures were taken before and after the blood samples. Both experimental groups demonstrated decreases in neutrophil adherence, and the control showed a tendency toward increases in this measure. The psychophysiologic data for the control group was suggestive of a relaxation response. The experimental group that attempted to increase neutrophil adherence demonstrated psychophysiologic responses that were contrary to relaxation. We concluded that an active cognitive exercise or process is associated with decreases in neutrophil adherence irrespective of the exercise. In contrast, relaxation without an active imagery exercise was associated with increases in neutrophil adherence. The results of this study are discussed in terms of behavioral engineering of directional immune changes.

Pneumocystic carinii pneumonia in a term newborn infant with a transiently depressed T lymphocyte count, primarily of cells carrying the CD4 antigen

A term infant without infection by human immunodeficiency virus had pneumocystis pneumonia at 17 days of life. Initial counts of T lymphocytes carrying the CD4 antigen were approximately 50% of the lower limits of normal; later the counts of T lymphocytes carrying the CD3 and CD8 antigens decreased as well. By 7 weeks after resolution of the pneumonia, CD3+, CD4+ and CD8+ cell counts had returned to normal. These observations suggest that a primary transient deficiency of T cell production or maturation, especially involving CD4+ cells, may occur in otherwise normal newborn infants.

CHARACTERIZATION OF THE MOLECULAR BASIS FOR PMN DYSFUNCTION & RECURRENT INFECTION IN THE GP-138 DEFICIENCY SYNDROME

A deficiency of a high MW glycoprotein present on the surface of normal PMNs, was found in unrelated patients (3 F, I M) with leukocytosis, periodontitis, & recurrent infection. Multiple adhesion-dependent functions including random and directed migration of affected PMNs were profoundly diminished. SDS-PAGE of NP-40 lysates of these PMNs and controls indicated that the deficient protein had a MW of 138,000 daltons. Further characterization showed the protein had a large carbohydrate content and was a major surface glycoprotein with a pI of 5.2-5.4. Galactose oxidase - NaB 3H4 surface labeling demonstrated the three F were totally deficient in surface GP-138, but the M patient had 5-10% of normal on the PMN surface. Immunoprecipitation experiments with polyclonal anti-GP-138 on normal PMNs suggested that the functional protein consisted of two or more polypeptide chains. To further test this hypothesis, monoclonal antibodies against known leukocyte cell surface proteins were tested using fluorescent activated cell sorting (FACS). LFA-1, p150.95, and OKM1 as well as the beta subunit these polypeptides share, were absent in the two F tested. The M patient was identical except he had ∼30% of normal OKM1 by FACS. This data suggests GP-138 and OKM1 may be similar molecular species. Thus, PMN dysfunction in the GP-138 deficiency syndrome is related to impaired expression of at least three related high MW glycoproteins (OKM1, LFA-1, & p150.95) necessary for adhesion dependent cell function.