Neutrophil Mobility Research Articles

CD18-dependent and L-selectin-dependent neutrophil emigration is diminished in neonatal rabbits

Human neonatal neutrophils manifest decreases in mobility, adherence, and emigration compared with adult neutrophils that may contribute to the increased susceptibility of neonates to infection. In a developmental rabbit model, we show a reduced ability of neutrophils from 1-day-old rabbit pups to emigrate to inflamed peritoneium (3.7 +/- 0.35 x 10(6) neutrophils/mL peritoneal exudate) compared with 14-day- old (8.5 +/- 0.7 x 10(6)/mL) and adult rabbits (9.4 +/- 1.4 x 10(6) mL, P < .05) despite significantly increased blood neutrophil counts. Because the reductions in functional Mac-1 (CD11b/CD18) as well as the amount of surface L-selectin are hypothesized to be primarily responsible for the differences in human neonatal neutrophil mobility, we examined CD11b/CD18 and L-selectin in our model. Using flow cytometric analysis we found that similar to human neonates, neutrophils from 1-day-old rabbit pups had 57% of adult rabbit levels of L-selectin and, in contrast with adults, failed to show significant decreases in L-selectin after chemotactic stimulation. In addition, neutrophils from 1-day-old pups compared with adults showed a significantly diminished capacity to upregulate CD11b/CD18 after chemotactic stimulation in vitro, or after emigration to the inflamed peritoneum. Systemic administration of anti-L-selectin monoclonal antibody (MoAb) resulted in significant reduction in peritoneal neutrophils in adult (47%, P < .05) and 14-day-old rabbits (47%, P < .05), but was without effect in 1-day-old rabbits. Administration of anti-CD18 MoAb resulted in significant reduction in peritoneal neutrophil accumulation in all age groups though less in 1 day and 14 day (58% and 65%, respectively) than in adults (91%, P < .05). Only in the 14-day-old rabbits was there an additive effect of anti-L-selectin and anti-CD18 MoAbs compared with anti-CD18 alone (84% v 65%, P < .05). The findings in this in vivo rabbit model support the hypothesis that the previously described in vitro defects in human neonatal L-selectin and CD11b/CD18 may be major contributors to human neonatal inflammatory deficits.

Improvement of neutropenia and neutrophil dysfunction by granulocyte colony-stimulating factor in a patient with glycogen storage disease type Ib

Patients with glycogen storage disease type Ib (GSD Ib) suffer from recurrent bacterial infections due to neutropenia and neutrophil dysfunction. To improve the quality of life in a 9-year-old boy with GSD Ib, we subcutaneously administered recombinant human granulocyte colony-stimulating factor (G-CSF). Daily injections of 100 micrograms/m2 of G-CSF significantly increased absolute neutrophil counts and augmented neutrophil mobility. The patient was then treated with 70 and 100 micrograms/m2 of G-CSF daily and twice-weekly. The treatment maintained absolute neutrophil counts at significantly higher levels than those without treatment for 22 months and markedly decreased the frequency of infections and the necessity for hospitalisation. Weekly injections of 70 micrograms/m2 of G-CSF were less efficient. No adverse effects were observed during treatment. These findings indicate that daily and twice-weekly treatment with G-CSF of long duration are safe and effective for patients with GSD Ib. G-CSF may be a useful therapeutic agent in patients with neutrophilic impairment as a consequence of a metabolic disorder.

Autocrine motility factor and its receptor: role in cell locomotion and metastasis.

The ability to locomote and migrate is fundamental to the acquisition of invasive and metastatic properties by tumor cells. Autocrine motility factor (AMF) is a 55 kD cytokine produced by various tumor cells which stimulates their in vitro motility and in vivo lung colonizing ability. AMF stimulates cell motility via a receptor-mediated signalling pathway. Signal transduction following binding of AMF to its receptor, a cell surface glycoprotein of 78 kD (gp78) homologous to p53, is mediated by a pertussis toxin sensitive G protein, inositol phosphate production and the phosphorylation of gp78. Cell surface gp78 is localized to the leading and trailing edges of motile cells but following cell permeabilization is found within an extended network of intracellular tubulovesicles. Gp78 tubulovesicles colocalize with microtubules and extension of the tubulovesicular network to the cell periphery is dependent on the presence of intact microtubules. Gp78 labeled vesicles can be induced to translocate between the cell center and periphery by altering intracellular pH as previously described for tubulovesicles labeled by fluid phase uptake. Anti-gp78 mAb added to viable motile cells is localized to large multivesicular bodies which, with time, relocate to the leading edge. Binding of AMF to its receptor induces signal transduction, similar to chemotactic stimulation of neutrophil mobility, as well as the internalization and transport of its receptor to the leading edge stimulating pseudopodial protrusion and cell motility.

Chronic inflammatory bowel disease in glycogen storage disease type 1B

Two children with glycogen storage disease type 1B developed chronic inflammatory bowel disease. The first, a 7-year-old boy, had ileitis and later developed perianal disease. The second developed colitis by the age of 9 years; in both the features were consistent with Crohn disease. The children had neutropenia and neutrophil mobility defects characteristic of GSD-1B. It is suggested that these neutrophil abnormalities are important in the pathogenesis of the bowel inflammation.

Leiner's disease associated with metabolic acidosis

A female infant with failure to thrive, erythroderma and recurrent diarrhoea died at 10 months of age from a Haemophilus influenzae respiratory tract infection. Her clinical course was complicated by a persistent metabolic acidosis. Investigations revealed a low level of the fourth component of complement and reduced neutrophil mobility.

Murine neutrophil migration during the course of an intra-abdominal abscess.

Studies of neutrophil migration in heterogeneous groups of septic patients have reported elevated, normal, and depressed neutrophil mobility. This experiment, conducted in a standardized murine population, measured neutrophil migration under agarose during the course of a chronic intra-abdominal abscess initiated by cecal ligation. Compared with both control (unoperated) and sham celiotomy mice, those mice with an abdominal abscess showed elevations of 22% and 14% of directed and random migration, respectively, at 48 hours. They had 30% to 40% decreases in these functions at days 4 through 10. Murine neutrophil migratory response during the course of an intra-abdominal abscess consisted therefore of an early elevation followed by a prolonged depression. Such a biphasic response could underlie the variable findings of neutrophil studies in septic patients. This model could be further applied to the study of the effects of chemotherapy and immunologic adjuvants on abscess course and neutrophil function, as well as to the study of the mechanisms of neutrophil response to sepsis.

Neutrophil mobility in granuloma annulare and necrobiosis lipoidica.

Summary Neutrophil mobility was investigated in thirteen cases of granuloma annulare and in twelve cases of necrobiosis lipoidica and found to be normal. Plasma from these patients did not inhibit the random or directed migration of neutrophils obtained from normal donors. The nature of the ‘in vivo’ defect of neutrophil migration previously reported in these conditions is at present unclear.

667 THE EFFECT OF TRIPLE DYE ON SHEDDING OF THE UMBILICAL CORD

Triple dye is commonly applied to the umbilical cord of newborns to decrease the incidence of bacterial infection and colonization. We examined the time of umbilical cord separation in 100 full term infants and 52 premature infants whose cords had been treated with a single application of triple dye. The preterm infants' mean birthweight was 1856 gm. and mean gestational age 33.5 wks. The umbilical cords had not been manipulated for a procedure. There were no anomalies of the umbilical cord in this series. In the term infants the mean age of cord separation for males was 12.2 days and for females was 11.9 days. In the premature group the mean age for both males and females was 11.5 days. Several previous studies have shown that in the absence of triple dye use, 80% of the umbilical cords were shed on or before the 8th day of life. Less than 5% of the cords were shed beyond 10 days. 56% of our full term infants and 63% of the preterm infants shed the umbilical cord beyond 10 days. A recent study (Hayward A et al, Lancet 1979 1:1099) reported delayed separation of the umbilical in association with defective neutrophil mobility. This data suggests that triple dye may prevent bacterial colonization and perhaps remove the antigenic stimulation for neutrophil chemotaxis to the umbilical cord.

DELAYED SEPARATION OF THE UMBILICAL CORD, WIDESPREAD INFECTIONS, AND DEFECTIVE NEUTROPHIL MOBILITY

In six infants, from two families, the umbilical cords were still attached at 3 weeks of age. Five of these developed severe local and disseminated bacterial infections from which four died. Two of these children were tested, and both, including the survivor, had defective neutrophil mobility; in the survivor this was improved in vitro and in vivo by ascorbic acid. It is suggested that a primary genetic defect of a contractile protein could explain the association. The sixth child, with delayed cord separation but normal neutrophil mobility and no excess of infections, who has survived without special treatments, also has mastocytosis, apparently inherited independently.

An inherited defect of neutrophil mobility in Shwachman syndrome

Selected immunologic functions were assessed in 14 patients with the Shwachman syndrome. Nine patients were neutropenic and four had low levels of IgA or of IgM. Neutrophil mobility was significantly defective in the group of patients as a whole (in 12 it was below the lower limit of normal) and in their parents. No other consistent abnormality in immunity was found. These results suggest that the defective neutrophil mobility is a feature of Shwachman syndrome which may contribute to the vulnerability of these patients to frequent infections. The defect appears to be a primary genetic one, inherited as an autosomal recessive characteristic consistent with the assumed inheritance of Shwachman syndrome.

Defective Neutrophil Migration in Granuloma Annulare, Necrobiosis Lipoidica, and Sarcoidosis

The neutrophil mobility in patients with granuloma annulare, necrobiosis lipoidica, and sarcoidosis was tested using both in vivo and in vitro techniques. Use of a skin window chamber for measurement of chemotaxis in vivo demonstrated defective neutrophil migration in each group. This contrasted with the finding that, in vitro, chemotaxis toward casein and endotoxin-activated sera was normal. The importance of this dissociation is discussed. Abnormal neutrophil accumulation at sites of inflammation may be of importance in the pathogenesis of these granulomatous disorders.

Clinicopathological findings in patients with primary and secondary defects of neutrophil mobility.

Twenty-eight patients with defective neutrophil mobility were classified, largely on results of parent values, into primary (17)--mainly Shwachman's syndrome--and probable secondary (11). They presented with frequent infections and/or allergy and these symptoms were essentially similar in both groups. Neutropenia was virtually confined. to the patients with Shwachman's syndrome. Diagnoses associated with secondary neutrophil mobility defects included hydrocephalus with shunt, cystic fibrosis, and immunoglobulin deficiency. The neutrophil mobility of nearly nearly all the patients studied increased when levamisole was added in vitro, but there was no clear evidence of clinical benefit when patients were given the drug.

The visualization of receptors for the Fc portion of the IgG molecule on human neutrophil leukocytes

Human neutrophils treated with either Fc or with intact IgG and subsequently with fluorescein-labelled anti-IgG showed binding of the Fc or the IgG to the cell membrane. Neutrophils did not appear to bind F(ab′) 2 fragments. Under suitable conditions, polar capping of fluorescence was seen. The data suggest receptors for Fc on the neutrophil membrane and mobility of these receptors.

QUALITATIVE IMMUNOGLOBULIN (Ig) DEFICIENCY, RECURRENT INFECTIONS, AND INTACT CELLULAR IMMUNITY

A 10 year old female has had repeated episodes of bacterial infections including pneumonia, otitis, adenitis, and urinary tract infections. She is short, has lymphadenopathy and a protuberant abdomen. IgG=1500 mg%, IgM=430 mg%, IgA= 68 mg%, IgD 18.5 mg% and IgE=l4 IU/ml. Ig Subclasses, inmunoelectrophoresis and complement component activity are normal. After isolating Group A streptococci from the throat and an excised node, ASO and other streptococcal antibody titers were low or undetectable. The excised lymph node showed reactive hyperplasia. Lymphocyte counts were normal. Blood is Group O and isoagglutinin titers (A and B) were absent. Responses to diphtheria, typhoid and hexavalent pneumococcal vaccine were minimal or absent. Normal numbers of B lymphocytes were determined by membrane immunofluorescence and rosettes. Spontaneous rosette formation with sheep erythrocytes was normal. In vivo delayed hypersensitivity was normal. Normal lymphocyte responses to mitogens and allogeneic cells occurred. Neutrophil (PMN) chemotaxis, mobility, phagocytosis and bactericidal activity were normal. A serum related defect in PMN opsonic and cidal activity with strep and E. coli was reversed with normal serum. The patient appears to represent a profound abnormality of antibody formation associated with recurrent infections.

Enhancement of random migration and chemotactic response of human leukocytes by ascorbic acid.

Incubation of human leukocytes with ascorbic acid at neutral pH and at concentrations 10-50 times that of normal blood levels augmented both the in vitro random migration and chemotaxis of the cells by 100-300% without influencing their phagocytic capacity. Enhancement of mobility by ascorbate was evident for isolated neutrophils, eosinophils, and mono-nuclear leukocytes and was independent of the specific chemotactic stimulus. Stimulation by ascorbate of the hexose monophosphate shunt of adherent neutrophils and augmentation by ascorbate of neutrophil mobility had comparable dose-response relationships, could be reversed by washing the cells, and were both suppressed by preincubation of the neutrophils with 6-aminonicotinamide, but not with the neutrophil-immobilizing factor. Glutathione, the proposed intermediate for ascorbate action, similarly stimulated hexose monophosphate shunt activity and enhanced migration. The enhancement in vitro of leukocyte mobility by ascorbate at concentrations found in some normal tissues, therefore, appears to be dependent upon stimulation of the leukocyte hexose monophosphate shunt.