The effects of human macrophage inflammatory protein-1 alpha and its genetically modified variant, BB10010, on phagocyte function.

BB-10010 is a genetically modified form of human macrophage inflammatory protein-1 alpha (MIP-1 alpha) with a single amino acid substitution of Asp26 by alanine, which inhibits aggregation of the native molecule. BB-10010 has stem cell protective properties, and has entered clinical trials for this purpose. The aim of this study was to determine the effects of BB-10010 on human phagocyte function and compare them with the native molecule. MIP-1 alpha and BB-10010 had identical dose-response curves in assays of calcium mobilization; however, neutrophils were less sensitive than monocytes to either MIP-1 alpha form, suggesting differences in receptor expression. Neither MIP-1 alpha type directly stimulated phagocyte superoxide production, nor did it have any priming effect on agonist-induced superoxide production. Both MIP-1 alpha and BB-10010 enhanced monocyte migration; however, cells were more sensitive to the native molecule, with optimal effects seen at 1 ng/ml compared with 100 ng/ml BB-10010. Concomitant alteration of CD11b, CD18, and CD49d (VLA-alpha 4) cell adhesion molecule expression was not seen with either MIP-1 alpha type. With the exception of the difference in monocyte sensitivity in chemotaxis assays, BB-10010 reproduces the effects of the native molecule on phagocytes. BB-10010 does not have proinflammatory effects on neutrophil activation, and this bodes well for its clinical use.

Leukocyte migration in the leg in response to experimental venous hypertension.

PURPOSE: Leukocyte trapping and activation in the microcirculation of the legs may play an important role in causing skin damage in venous disease. Leukocyte emigration from the microcirculation and subsequent locomotion in response to venous hypertension was studied in a group of 12 normal volunteers using a "skin window" technique. METHODS: Two 0.5-cm square dermal abrasions were made with a dental stone over the gaiter area of the leg and the flexor aspect of the forearm (control), which were covered with moist micropore membranes. The volunteers lay supine for 30 minutes, and then stood supported for 30 minutes to raise the venous pressure in the leg, and then lay supine again for 30 minutes. The experiment was repeated in six volunteers who lay supine for the whole period. The membranes were changed and collected every 15 minutes, fixed in formal saline solution, and dual-stained for monocytes and polymorphonuclear leukocytes. The type and numbers of cells that emigrated and the furthest distance traveled (leading front) by the cells through the membrane were measured. RESULTS: Both in arms and legs, the vast majority of cells that emigrated were neutrophils, with very few monocytes (arm, 93% neutrophils and 7% monocytes; leg, 97% neutrophils and 3% monocytes). In the 30 minutes after venous hypertension, leukocyte migration significantly decreased in the leg (median leukocyte locomotion: basal, 75.3 microns; standing, 73.5 microns; after hypertension, 62.9 microns; p = 0.012, Wilcoxon matched pairs signed rank test), but not in the arm (basal, 86.2 microns; standing, 84.4 microns; after hypertension, 85.5 microns; p = NS) or when the experiment was repeated with the volunteers lying supine for the entire period (basal, 91.5 microns; standing, 89.4 microns; after hypertension, 92.6 microns; p = NS). CONCLUSIONS: Leukocyte migration is decreased immediately after experimental venous hypertension, which may be a result of the release of factors that inhibit migration.

Interleukin-3 administration enhances human monocyte function in vivo.

In addition to its haemopoietic effects, interleukin-3 (IL-3) enhances leucocyte function in vitro. In this study we examined the effects on haematological variables and monocyte function of a single IL-3 infusion in five haematologically normal individuals. There was a rapid fall in circulating monocyte (to 24 +/- 6% of pre-infusion value) and eosinophil numbers (to 3 +/- 2%) with a nadir at 30 min and gradual return to baseline over 6 h. No significant changes in monocyte expression of the adhesion molecules CD11b or L-selectin or of monocyte respiratory burst activity were detected. There was a significant increase in monocyte phagocytosis and killing of Candida after IL-3 infusion: the percentage of monocytes which had ingested Candida increased from 39 +/- 10% to 62 +/- 12% and the total number of Candida killed per 100 monocytes increased from 63 +/- 34 to 210 +/- 59 (P < 0.05 and P < 0.01 respectively). There was no inhibition of neutrophil migration into a 'skin window' site and monocyte migration was moderately enhanced (peak increase of 260 +/- 47%). These results show that IL-3 has significant effects on monocyte function in vivo and could be of use in augmenting host defence mechanisms in immunocompromised patients.

A skin window study of neutrophil migration in subjects with localized juvenile periodontitis.

This study investigated the effect of a chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (FMLP), upon skin window migration of neutrophils into filters in 5 patients with a history of localized juvenile periodontitis (LJP) and 8 controls. On 2 occasions, each subject had 2 superficial skin abrasions made on the inner aspect of the forearm. Initial periodontal treatment was carried out on the disease group between the visits. On one skin window filters were placed that were soaked in physiological saline, and on the other filters soaked with FMLP. Leading fronts and cell densities were measured in each filter. At visit 1, LJP subjects had significantly lower leading fronts and cell densities. At visit 2, the differences were insignificant. The leading fronts for the LJP group were significantly improved on the second visit. No difference was observed between saline and FMLP. The findings of this study indicate that neutrophil migration is reduced in LJP patients where treatment is not involved, and that FMLP has no effect on neutrophil migration from the skin windows under the conditions of this study.

Pentoxifylline at clinically achievable levels inhibits FMLP-induced neutrophil responses, but not priming, upregulation of cell-adhesion molecules, or migration induced by GM-CSF.

Pentoxifylline (PTX) administered after bone-marrow transplantation reduces procedure-related organ damage mediated by TNF alpha. GM-CSF is also given post-transplant to stimulate earlier neutrophil recovery. Because PTX has been shown to inhibit neutrophil function, we sought to determine whether it also inhibited the effects of GM-CSF on neutrophil activity. The study confirmed that PTX at clinically achievable concentration (5-10 mumol/l) attenuated the responses of human neutrophils to chemotactic peptide, whereas it did not inhibit the effect of GM-CSF on neutrophil function even at high concentrations. In experiments with human neutrophils, neither the direct effects of GM-CSF such as stimulation of migration and increased expression of CD11b, nor the priming effects of GM-CSF on the respiratory burst, were inhibited by PTX. In experiments with monkeys, intravenous administration of PTX did not block subsequent GM-CSF-induced neutrophil CD11b upregulation or phagocyte margination, even when near millimolar plasma levels of pentoxifylline were obtained. The retention of cytokine-stimulated activities suggests that PTX will not compromise the response of neutrophils to stimuli from infectious foci.

Role of leucocyte integrins in phagocyte responses to granulocyte-macrophage colony stimulating factor (GM-CSF): in vitro and in vivo studies on leucocyte adhesion deficiency neutrophils.

The role of leucocyte integrins in phagocyte function has been studied by comparing normal neutrophils with those from a patient with partial leucocyte adhesion deficiency (LAD), in whom the levels of CD11b and CD11c were 10% of controls, whereas CD11a levels were normal. Unstimulated LAD neutrophils exhibited defective adhesion to plastic (4.4 +/- 1.5% cf. 14.4 +/- 3.8% in controls), but not to human umbilical vein endothelial cells (HUVECs). The adhesion to HUVECs could be further upregulated by granulocyte-macrophage colony stimulating factor (GM-CSF), but not by 12-O-tetradecanoylphorbol 13-acetate (TPA) which, in normal cells, is a more potent 'pro-adhesive agonist'. The normal neutrophil-endothelial interaction induced by GM-CSF in LAD neutrophils was confirmed in vivo when administration of GM-CSF resulted in rapid phagocyte margination. Neutrophil migration and phagocytosis/killing were defective in LAD neutrophils, and some improvement in phagocytosis/killing was seen following in vivo administration of GM-CSF. These studies illustrate that the degree to which the leucocyte integrins mediate adherence-related phagocyte functions varies not only with the particular function, but also with the conditions of stimulation. High levels of CD11b and CD11c expression appear not to be required for unstimulated or GM-CSF-stimulated neutrophil-endothelial interactions, either in vitro or in vivo. Other neutrophil functions, on the other hand, such as migration and phagocytosis/killing are much more dependent on the leucocyte integrins.

In vivo effects of macrophage colony-stimulating factor on human monocyte function.

Macrophage colony-stimulating factor (M-CSF) is reported to enhance a variety of functions of mature monocyte/macrophages in vitro. We have examined the effects of a 2 h intravenous infusion of M-CSF obtained from human urine (hM-CSF) on haematological parameters and selected monocyte functions. There was a rapid, small, but consistent reduction in Hb concentration (mean 6.5 +/- 2.3%, P less than 0.0005 by paired t test) by the completion of the hM-CSF infusion and small, transient falls in platelet, monocyte and neutrophil counts were noted in the 2 h following the end of the infusion. No effect on monocyte or neutrophil CD11b cellular adhesion molecule expression was detected. Exposure to hM-CSF in vivo did not directly stimulate the monocyte respiratory burst, but increased the percentage of monocytes responding to f-met-leu-phe from 9.8 +/- 2.5 to 16.6 +/- 4.2 (P less than 0.01). The number of candida ingested and degraded per 100 monocytes increased from 101 +/- 14 pre-infusion to 160 +/- 22 post-infusion (P less than 0.01). There was a rapid increase in the numbers of monocytes entering a skin window membrane from a mean of 226 +/- 71 pre-infusion to 1064 +/- 404 at the end of the infusion, with no effect on neutrophil migration. These data show that the administration of hM-CSF enhances several of the functions of peripheral blood monocytes in vivo, and this may be of benefit in the treatment of selected infections.

An in vivo study of neutrophil locomotion in relation to periodontal disease status and local chlorhexidine.

Neutrophil migration from skin window abrasions was studied in 10 patients with no history of periodontitis, 10 with localised juvenile or post-juvenile periodontitis, and 10 with chronic adult periodontitis. Filters contained either saline or chlorhexidine (0.002% or 0.02%). The leading front was measured in filters placed for 30 min after cell migration had been established for 2 h. Subjects in the juvenile/post juvenile group showed a reduced range of migration distances, but were still within the normal range when compared with the other 2 groups. Chlorhexidine at 0.002% tended to increase leading front distances, and 0.02% to decrease them. We conclude that: 1) migrating neutrophils in vivo may move less far in patients with a history of juvenile periodontitis; 2) chlorhexidine may inhibit cell migration, possibly decreasing the host response in vivo if applied at current therapeutic concentrations.

Granulocyte-macrophage colony-stimulating factor may inhibit neutrophil migration in vivo.

Recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) was administered by constant intravenous infusion to eight patients with malignant disease prior to chemotherapy. rh GM-CSF was given at a dose of 15 to 25 micrograms m-2 h-1 for 1 or 2 h. Neutrophil migration into an inflammatory zone was monitored throughout this period using a micropore skin window technique. Neutrophil migration into the micropore membrane prior to the commencement of the rh GM-CSF infusion was almost identical to that in eight normal control individuals (leading front distance of migrating neutrophils in 20-min period 81.3 +/- 7 microns in the patients compared to 79.4 +/- 4 microns in the control individuals). During the rh GM-CSF infusions there was a significant fall compared to controls in the number of neutrophils entering the membrane and the leading front distance (P less than 0.05). In four out of nine patient studies (eight patients) there was a greater than 30% fall from the pre-infusion level. In the control individuals the largest fall recorded was less than 10% and overall there was a progressive rise in the number of neutrophils entering the membrane throughout the period of study. This study suggests that intravenous infusion of rh GM-CSF may impair the ability of neutrophils to infiltrate an inflammatory focus.

In vivo leucocyte migration in Behçet's syndrome.

Serial studies of leucocyte migration in vivo were carried out in 15 patients with Behçet's syndrome using a skin window technique. Where possible, patients with and without active disease were studied during and in the absence of treatment. In patients with active disease neutrophil migration was frequently greater than normal, particularly with respect to numbers of cells migrating. There was also an increased frequency of emigrating neutrophils with less or more nuclear lobes than normal. In three patients in whom function of skin window neutrophils was studied nitroblue tetrazolium reduction and phagocytosis and killing of Candida guilliermondiae were normal. The monocyte component of the skin window was more often reduced in patients than in normal controls. Corticosteroid treatment did not exert a major effect on leucocyte migration, though the doses involved were relatively small. Neutrophil abnormalities were common in patients and particularly those with active disease. These results suggest that neutrophil hyperactivity may have an important role in the pathogenesis of Behçet's syndrome.

The effects of recombinant human granulocyte-macrophage colony-stimulating factor on phagocyte kinetics in man.

Four patients with advanced resistant malignant disease received recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) for 10 days. All developed a moderate neutrophilia and monocytosis over this period. A transient phagocytopenia was observed during the first hour of administration. Radionuclide labelling studies showed that this cytopenia was due to sequestration predominantly within the lungs and that the recovery was due to re-entry of the same cells into the circulation. Studies of neutrophil lobularity during this time showed no reduction in lobe count suggesting that there had been little if any release of immature cells from bone marrow reserves. Skin window responses were present in 2 out of 3 patients during the period of neutropenia showing that cells were also present in the marginated pool of the skin at this time.

A human skin window technique using micropore membranes.

A skin window technique using micropore membranes is described. Eight micrometre pore-size membranes are placed on abrasions made with a dental stone and covered with moist filter paper and impermeable film: adhesive tape is placed overall. The assembly is unobtrusive and stable and may be worn during normal activities. For clinical studies, a series of membranes used over 4-5 h gives information on distribution of leucocyte emigration sites, numbers and types of emigrant cells, their rate of locomotion in the membrane (solely under the influence of endogenous factors) and any difference in form, surface phenotype or function between cells at the dermal surface and those which have traversed the membrane. This system, using a 3-dimensional labyrinthine structured membrane, avoids errors inherent in previous techniques, e.g., differential adhesiveness, competition for space, no quantification of locomotion after emergence or indication of the productive area of the abrasion. Incidental findings include no major change in the nature of the emigrating population over 24 h, the presence of monocytoid cells from the earliest phase in normal subjects, the origin of emigrating cells in discrete foci, and an even distribution of cells in a fully infiltrated membrane. The skin window technique is capable of greater clinical precision and experimental elaboration than hitherto realised.

Gingival crevice neutrophil function in periodontosis.

This study examined gingival crevicular polymorphonuclear leucocyte function in periodontosis patients. Cells were examined for viability, function and ultrastructure. Eighty percent or more of the cells in each sample were viable as assessed by the fluorescein diacetate technique, but the test organism, Candida guillermondiae, was not phagocytosed. Gingival crevicular fluid contained many lysing neutrophils and nonphagocytosed organisms. Recognizable polymorphs contained Gram-negative and Gram-positive organisms. On the basis of this and previous studies it is concluded that gingival crevice neutrophils from periodontosis sites show reduced phagocytic function compared with cells from normal or periodontitis-affected gingival crevices. It is possible that the behavior of neutrophils from gingival crevices may be irrelevant. Original changes by that stage may have obscured their capabilities.

Neutrophil function in systemic lupus erythematosus and other collagen diseases.

Using a whole blood technique we assessed neutrophil migration, phagocytosis, and killing in a group of 20 patients with systemic lupus erythematosus (SLE) and in 8 patients with other connective tissue disorders. In the untreated cases of SLE neutrophil migration was significantly depressed, but it was usually normal in the treated group. This may be attributable either to an intrinsic neutrophil abnormality or to a humoral factor. Although isolated abnormalities of phagocytosis and killing were observed in SLE, these functions were normal when the patients were considered as a group. The treated patients with other collagen diseases showed enhanced migration in both autologous and control plasma, normal phagocytosis, and enhanced killing in autologous plasma only. The small group of untreated, non-SLE patients showed some depression of all 3 functions. There was no correlation between neutrophil function and clinical activity of disease. In the SLE patients there was no correlation between neutrophil function and circulating immune complexes.

Neutrophil function in advanced Hodgkin's disease: effect of therapy.

Whole-blood techniques were used to study neutrophil migration and uptake and killing of Candida in patients with advanced (Stages IIIB and IV) Hodgkin's disease (HD). In order to assess the effect of therapy on neutrophil function, 17 patients were studied either before treatment or on days 1, 8 and 14 of successive cycles of chemotherapy. Neutrophil migration was reduced in 20% of patients, and was largely corrected by normal control plasma. Phagocytosis was normal but killing was reduced in most patients; this reduced killing was not corrected by normal control plasma. Neither chemotherapy nor previous splenectomy had any effect on neutrophil function. It is concluded that defective neutrophil killing, attributable to an intrinsic defect of the cell, is a consistent feature of advanced HD and that a similar defect is present before and after therapy.

Neutrophil function during chemotherapy for Hodgkin's disease.

Simultaneous measurement of neutrophil migration, phagocytic activity, candidacidal and bactericidal activity were made during quadruple chemotherapy of advanced Hodgkin's disease (HD). Measurements were also made in normal individuals, hospital patients not on chemotherapy, untreated patients with advanced HD and patients off chemotherapy for over a year. Neutrophil migratory activity was usually normal in untreated HD patients and those on chemotherapy, but less than 20% of all tests showed depressed values, some of which were corrected by plasma. Similar results were found with neutrophil phagocytosis. Abnormalities in these functions were found in both early and late cycles, but there was a tendency for migration to deteriorate during later chemotherapy cycles. Neutrophil candidacidal and bactericidal activity were frequently depressed in patients on treatment and there was deterioration in candidacidal activity during the chemotherapy cycle. These abnormalities of killing activity were frequently corrected in control plasma. Neutrophil function is normal in most patients with advanced HD and in patients in remission. In a minority of patients on treatment there are marked functional defects, especially in killing activity. These defects are partly cell-associated and partly plasma-related. Susceptibility to infection during chemotherapy of HD may be partly due to defective neutrophil function.

Defective neutrophil function in low-birth-weight, premature infants.

A whole-blood technique was used to measure simultaneously neutrophil migration, uptake, and killing of candida in 27 premature infants of low birth weight (less than 1500 g). Neutrophil migration was consistently reduced, especially in the first two weeks of life. Phagocytosis was also reduced, particularly in the first week of life and in sick patients. Killing was usually normal, except in sick patients. The three functions were not altered when the test was performed in normal adult, rather than autologous, plasma, and the reduced migration and uptake are therefore the result of an intrinsic defect of the cell. The results clarify the previous controversy concerning neutrophil function in premature infants and provide an explanation for their increased susceptibility to infection.

Thymus-dependent and thymus-independent antibody responses: contrasting patterns of immunosuppression.

The effects of mitoclomine, an anti-tumour agent, and of cortisone acetate on the antibody response of mice to thymus-dependent and thymus-independent antigens were compared. Both agents yielded patterns of immunosuppression which differed from that seen after X-rays, an agent which is likely to be active equally against T and B cells. Mitoclomine, at low doses, depressed thymus-independent responses the most; this effect can be explained if the drug is more active against B than T cells. Cortisone acetate at low doses, while depressing thymus-dependent responses, increased thymus-independent responses; this can be explained if the drug is more active against T than B cells. Thus three different patterns of immunosuppression (for X-rays, mitoclomine and cortisone acetate) can be interpreted in terms of effects simply on T or B cells.

Use of whole blood in the measurement of neutrophil migration.

A whole-blood method was used for measuring neutrophil migration in micropore membrane. The method is reproducible (coefficient of variation (CV) 10.3% and 9.8% in two normal individuals tested repeatedly) and can be performed on an 0.25 ml lithium heparin blood sample. Migration is independent of leucocyte and erythrocyte counts and is comparable with that obtained for separated cells. As in separated cell techniques, cytochalasin and casein respectively inhibit and stimulate neutrophil migration. The technique is of value when testing large numbers of samples and when only small volumes of blood can be obtained, for example, in neonates.