Optimal timing for collection of PBPC after glycosylated G-CSF administration.

Daily administration of granulocyte colony-stimulating Factor (G-CSF) results in progenitor cell mobilization with maximum blood levels achieved after 4-7 days. In this study the short-term effects of glycosylated G-CSF at a dose of 5 microg/kg s.c. were determined so as to allow optimization of the timing of progenitor cell collection. In the first study involving 20 normal volunteers, a significant fall in neutrophil count and G-CSF levels was observed 2 h after the G-CSF injection. To investigate this phenomenon serial measurements were made in a further six volunteers after the 6th daily injection of G-CSF. A fall in the neutrophil count occurred which was maximal at 1 h and recovered to baseline within 3 h. There was also a fall in CD34+ cells (P = 0.034), GM-CFC (P = 0.025) and BFU-E (P = 0.066) and recovery to baseline levels took 4-12 h. We conclude that glycosylated G-CSF should not be given immediately prior to stem cell collection.

Crossover study of the haematological effects and pharmacokinetics of glycosylated and non-glycosylated G-CSF in healthy volunteers.

A cross-over study of glycosylated and non-glycosylated G-CSF was performed in 20 healthy male volunteers to compare the effects of the different forms of G-CSF, the extent of inter-individual progenitor cell mobilization and to determine whether any differences observed were related to the serum concentrations of G-CSF attained. The peak WBC achieved during 6 d of G-CSF administration at a dose of 5 microg/kg/d was significantly higher with the glycosylated than the non-glycosylated product (P = 0.02) as was the peak level of granulocyte-monocyte colony forming cells (GM-CFC) (P=0.03). The average GM-CFC count on days 5, 6 and 7 was 28% higher with the glycosylated product (P=0.003). Serum concentrations of G-CSF achieved were significantly higher with the non-glycosylated G-CSF, however, suggesting that the difference in bio-efficacy was not due to a difference in G-CSF stability. Marked inter-individual variation in progenitor mobilization was observed, but this was not related to serum G-CSF levels. The G-CSF concentrations on day 6 were approximately one third of those on day 1, with both forms of G-CSF.

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.

Effects of chlorhexidine solution on neutrophil locomotion in vitro.

The aim of this study was to ascertain the effect of low concentrations of chlorhexidine on the locomotor behaviour of neutrophils. A special feature was the use of a direct test of chemotaxis. Neutrophils recovered from peripheral blood of healthy adult volunteers were tested for chemokinesis and chemotaxis in the presence of chlorhexidine. A moderate dose-related direct response was observed in respect of both these parameters. It was also apparent that increasing concentrations of the drug inhibited a greater proportion of cells, so that there was total immobility at 0.2 per cent and many cells appeared lysed.

Analysis of autoantibody reactivity and common idiotype PR4 expression of myeloma proteins.

Sera from 75 patients with monoclonal gammopathies and with no clinical evidence of autoimmune disease have been screened for a wide range of autoreactivity including binding to DNA, cardiolipin, extractable nuclear antigen (ENA), rheumatoid factor activity and the presence of the common anti-DNA antibody idiotype PR4. The sera of 17/75 (23%) patients possessed autoreactivity: six were positive for anti-DNA activity, two had anticardiolipin activity and the PR4 ID was found in two sera (both of which possessed anti-DNA activity). Antibodies to ENA were found in one serum (anti-Ro) and anti-organ-specific antibodies in five. Using iso-electric focusing and immunoblotting we have shown that the PR4 ID and DNA binding activity are carried on the paraprotein and not on some other serum constituent. The IgG subclass distribution of 55 IgG paraproteins has also been investigated. The majority of IgG paraproteins belong to IgG1 subclass (55%), with the others, being IgG2 (4%), IgG3 (9%) and IgG4 (27%). In this study we have shown that sera from myeloma patients frequently possess autoreactivity, and that in many cases this can be attributed to the paraprotein.

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.

Does rheumatoid factor protect lupus patients from the development of nephritis?

It has been suggested again recently that the presence of rheumatoid factor (RF) in the serum of patients with systemic lupus erythematosus (SLE) protects them from the development of nephritis. In this study the RF is measured by standard latex test and by radioimmunoassay to detect IgM, IgA, and IgG isotypes, in patients with SLE with (26 patients) and without (25 patients) renal involvement, and in a control group of 21 patients with idiopathic renal disease. In addition, patients with SLE and nephritis were tested during active and inactive phases of their disease. No significant protective effect was observed from the presence of RF.

A comparison of autoantibodies and common DNA antibody idiotypes in SLE patients and their spouses.

In order to determine whether environmental influence per se might influence autoantibody production, sera from the spouses of 20 SLE patients were examined. No antibodies to cardiolipin, poly (ADP-ribose), or ENA were detected and none had detectable rheumatoid factor. One weakly positive ANA reaction was noted, one had anti-DNA antibodies (by RIA and ELISA) and in two sera the common DNA antibody idiotype 16/6 was found. The idiotype was not, however, present on either anti-DNA or anti-K30 antibodies. Although long-term analyses are required, it is evident that sharing the same environment with patients who commonly express a wide range of autoantibodies and common idiotypes rarely leads to their expression in non-autoimmune subjects.

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.

Measurement of anti-DNA antibodies: a reappraisal using five different methods.

One hundred and thirty coded sera, 60 from patients with systemic lupus erythematosus (SLE) and 70 from patients with other autoimmune rheumatic diseases were tested for deoxyribonucleic acid (DNA) binding activity by five different types of assay. These were enzyme linked immunosorbent assay (ELISA) (distinguishing IgG and IgM anti-ssDNA and anti-dsDNA), Crithidia luciliae, a nitrocellulose filter assay, the Amersham kit, and another modified Farr assay, the radioimmunoassay (RIA) (UK). The Crithidia test was the most specific, none of the controls was positive, but the least sensitive (13% positive only). The RIA (UK) was the most sensitive (57% positive). In most of the assays 3-9% of the controls were positive. When the SLE sera were analysed according to disease activity the IgG anti-dsDNA ELISA, all three RIA values, and the Crithidia test values were raised in all the patients with severely active disease. Some patients with inactive disease, however, were positive in each of the tests. The best interassay correlations (r less than 0.49) were found between RIA (UK), and ss IgG and the Amersham kit; and between ds IgG and ss IgG. In the main, however, it was clear that different assays are dependent upon distinctive properties of DNA antibodies. It seems inevitable that most major rheumatology units will require more than one anti-DNA antibody assay.