Leucocyte function in patients with rheumatoid arthritis: quantitative in-vivo leucocyte mobilisation and in-vitro functions of blood and exudate leucocytes.

Quantitative leucocyte mobilisation in vivo and the in-vitro random migration, chemotaxis, phagocytosis, and oxidative metabolic activity were studied in 15 patients with rheumatoid arthritis (RA). Patients mobilised leucocytes to chambers covering skin windows to the same degree as control subjects, and the mobilisation correlated with the blood leucocyte numbers and serum concentration of alpha-l-antitrypsin. Peripheral blood leucocytes showed slightly reduced migration in Boyden chambers but increased phagocytosis and increased unstimulated reduction of nitroblue tetrazolium. Exudate leucocytes from patients with RA showed migratory and phagocytic activity which did not differ from that of control subjects, but unstimulated exudate leucocytes reduced nitroblue tetrazolium more actively than leucocytes from control subjects. The observations indicate that leucocyte accumulation at an experimental inflammatory lesion and the function of these exudate leucocytes are not impaired in patients with rheumatoid arthritis.     

Leucocyte function in Crohn's disease. Studies on mobilisation using a quantitative skin window technique and on the function of circulating polymorphonuclear leucocytes in vitro.

Leucocyte function was evaluated by mobilisation to skin windows with chambers and by the chemotactic, phagocytic, and nitro blue tetrazolium (NBT) reducing activity of circulating leucocytes in vitro in 20 patients with Crohn's disease, 21 healthy volunteers, and nine patients with sarcoidosis or tuberculosis. Leucocytes had been mobilised in significantly reduced numbers at 12, 24, 36, and 48 hours in Crohn's disease compared with healthy volunteers (P less than 0.01) and patients with sarcoidosis/tuberculosis (P less than 0.01). The leucocyte migration rate showed that mobilisation in Crohn's disease begins after a prolonged lag phase and is reduced compared with healthy volunteers (P less than 0.01) and patients with sarcoidosis/tuberculosis (P less than 0.02). The reduced mobilisation was not correlated with disease activity. In vitro random migration by leucocytes was slightly lower in Crohn's disease (P less than 0.05) than in healthy volunteers, but there was no difference after removal of the autologous plasma. Chemotactic response to casein did not differ between the groups studied. Serum independent and dependent phagocytosis did not differ from control groups. Serum independent phagocytosis was positively and significantly correlated to the disease activity (rho 0.4812, P less than 0.05). Resting leucocyte NBT reduction was increased in Crohn's disease and sarcoidosis/tuberculosis (P less than 0.01), but during phagocytosis a lower NBT reduction was found in Crohn's disease than in healthy volunteers (P less than 0.02). The inflammatory response in Crohn's disease, with reduced leucocyte accumulation, differs from patients with other granulomatous reactions and is independent of the disease activity. Our data suggest that the defect is not cellular. They support the hypothesis that a pathogenic factor in Crohn's disease may be foreign material that is normally eliminated remaining in the tissue and eliciting a chronic inflammatory response.     

Leucocyte scintigraphy to localize inflammatory activity in ulcerative colitis and Crohn's disease.

The validity of using autologous leucocytes labelled with technetium -99m hexamethyl-propyleneamine-oxine (Tc-HMPAO) for scintigraphy in inflammatory bowel disease was evaluated in 12 patients with clinically active ulcerative colitis (UC) and 10 with Crohn's disease (CD). Colonoscopy and biopsy were used as reference. Scintigrams taken 1 h and 3 h after leucocyte reinjection were evaluated blindly by two independent observer groups. Full agreement was found in 11 of 12 UC patients when compared with colonoscopy but in only 3 of 10 CD patients. Segments with agreement in CD patients often showed neutrophilic granulocyte infiltration at biopsy. The judgements of clinicians and physiologists differed for only 2 of totally 70 UC segments but for 13 of 59 CD segments (kappa, 0.94 and 0.52). It is concluded that Tc-HMPAO scintigraphy might be an alternative to colonoscopy in the control of disease extent in UC. In CD patients the technique might warn about infections complications.     

Skin reactivity and phagocytic function of neutrophil leucocytes in Crohn's disease and ulcerative colitis

A decreased capacity of the leucocytes to phagocyte yeast particles was found in patients with Crohn's disease of the ileum. Patients with lesions only in the colon showed normal phagocytic activity as did patients with ulcerative colitis (U.C.). Intradermal injection of killed Strep. pyogenes produced an increased erythrmatous reaction after 48 hours in patients with U.C., and the reaction was pustular in half of them. None of the controls or the patients with Crohn's disease of the ileum had such a reaction. An increased erythematous reaction to streptokinase-streptodornase was also more common in patients with U.C. than in those with Crohn's disease and a control group. No such difference was seen to other bacterial antigens. The reactivity to kallikrein, prostaglandin E1, histamine or bradykinin did not differ from that of normal subjects.     

Inhibition of leucocyte motility by drugs used in ulcerative colitis.

The effects on leucocyte motility of sulphasalazine (Salazopyrin) and its metabolites sulphapyridine and 5 amino-salicylic acid have been compared with those of prednisolone and indomethacin. Sulphasalazine, its active metabolite 5 amino-salicylic acid, and prednisolone are all potent inhibitors of leucocyte motility. Sulphapyridine and indomethacin are non-inhibitory. Inhibition of leucocyte motility may explain why sulphasalazine and 5 amino-salicylic acid are effective in ulcerative colitis while sulphapyridine is not. The lack of effect of indomethacin suggests that this action of sulphasalazine does not involve inhibition of prostaglandin synthesis.     

Human leucocyte antigens in Crohn's disease and ulcerative colitis

The frequency of occurrence of 15 of the commoner human leucocyte antigens was determined in 18 patients with Crohn's disease and in 16 patients with ulcerative colitis, using an in-vitro lymphocyte cytotoxicity test (Harris, Wentzel, Cocking, Dodsworth, and Ukaejiofo, 1970). The overall results showed that, with the exception of human leucocyte antigen 3 in patients with Crohn's disease, in neither disease was there any major difference in any of the other antigen frequencies compared with a panel of 50 healthy controls. Nor was there an excess frequency of any particular antigen occurring in both Crohn's disease and colitis.     

Reticuloendothelial function in coeliac disease and ulcerative colitis.

Patients with ulcerative colitis and coeliac disease who had been shown by impaired clearance of heat damaged red cells to have diminished splenic phagocytosis, were examined for evidence of more generalised reticuloendothelial malfunction by measuring their circulatory clearance of micro-aggregated albumin. Although in animals micro-aggregated albumin is largely removed by Kupffer cells, we found impaired clearance in otherwise normal subjects who had previously had surgical splenectomy. In patients with hyposplenism because of bowel disease there was no additional impairment of micro-aggregated albumin clearance, indicating that their hyposplenism is an isolated phenomenon and not part of a generalised reticuloendothelial atrophy. Patients with coeliac disease and normal splenic function had increased reticuloendothelial catabolic activity; this was not present in patients with coeliac disease and abnormal splenic function.     

Processed blood volume impacts clinical efficacy in patients with ulcerative colitis undergoing adsorptive depletion of myeloid lineage leucocytes

Background  

Hitherto, therapeutic depletion of granulocytes and monocytes by adsorption (GMA) has been associated with significant and insignificant efficacy in patients with ulcerative colitis (UC). Further, the processed blood volume in one GMA session has been fixed at 30 mL/min × 60 min, regardless of patients’ body weight (BW). We were interested to see the efficacy and safety of GMA when administered in relation to patients’ BW.     

Leucocyte and platelet-derived bioactive substances in stored blood: effect of prestorage leucocyte filtration

Abstract: Adverse reactions to transfusion of allogeneic blood may depend on content of leucocytes and platelets and on storage-time of the erythrocyte suspensions. Therefore, we studied the efficacy of prestorage leucocyte reduction by filtration on total content and extracellular accumulation of histamine, eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO), plasminogen activator inhibitor type-1 (PAI-1) and interleukin-6 (IL-6) in samples obtained from 5 units of SAGM blood, 7 units of plasma-reduced whole-blood and 6 units of whole-blood before and after filtration, respectively. In addition, we analysed supernatants from the same units after storage at +4°C for 0, 21 and 35 d, respectively. The filtration was performed at room temperature within 2–4 h after donation. The substances were analysed by ELISA and RIA methods and we also analysed the donor plasma levels of the same bioactive substances. The total content of histamine, ECP, EPX, and MPO were 10–70-fold higher in all unfiltered erythrocyte products compared to donor plasma concentrations, while PAI-1 content was 15–20-fold higher only in plasma-reduced whole-blood and whole-blood. Prestorage leucocyte filtration significantly reduced the total histamine, ECP, EPX, MPO and PAI-1 content to levels similar to donor plasma levels in plasma-reduced whole-blood and whole-blood, while PAI-1 was still low in filtered SAGM blood. In addition, the levels of extracellular bioactive substances at d 0 after donation and filtration were within the range of concentrations in donor plasma, and there was no time-dependent accumulation during storage for 35 d at +4°C. IL-6 was not detected in either plasma or samples obtained from the blood bags. These results suggest prestorage leucocyte filtration to deplete leucocyte contents to levels, which prevent the previously shown time-dependent accumulation of leucocyte derived bioactive substances in various erythrocyte suspensions. In addition, the PAI-1 results suggest leucocyte filters to reduce the obligatory platelet content in whole-blood products.     

Leucocyte integrin and CR1 expression on peripheral blood leucocytes of patients with rheumatoid arthritis.

Expression of the leucocyte integrins (CD11a, b, c/CD18) and of CD35 (CR1) on leucocytes from the peripheral blood of patients with rheumatoid arthritis (RA) (n = 14) and control subjects (n = 12) was measured by flow cytometry using a rapid fixation and leucocyte preparation procedure. The mean (SE) percentages of lymphocytes expressing CD11a (RA 93.4 (1.7)%; controls 97.2 (1.8)%) and CD18 (RA 91.3 (2.3)%; controls 97.0 (2.6)%) were lower and the percentage of monocytes expressing CD11b (RA 86.9 (11.4)%; controls 78.4 (11.9)%) and CR1 (RA 62.6 (15.5)%; controls 36.6 (17.6)%) were higher in patients with RA than in controls. In addition, the mean fluorescence intensity of CD18 (RA 22.1 (2.3); controls 30.7 (2.5)) on lymphocytes was decreased and that of CD11b (RA 4.5 (0.8); controls 2.9 (0.9)) and CR1 (RA 2.4 (0.4); controls 1.5 (0.5)) on monocytes was increased in patients with RA compared with controls. The functional importance (if any) of the altered expression of the antigens on lymphocytes is not yet known. Altered expression on monocytes is consistent with activation within the circulation.     

Effect of ulcerative colitis and smoking on rectal blood flow.

Rectal blood flow was measured by laser doppler flowmetry over 60 minutes in eight patients with colitis in remission and eight healthy male non-smokers. Ten smokers were also examined on two occasions, one of which included smoking a cigarette. Plasma nicotine concentrations were measured in smokers. All subjects showed a pronounced fall in rectal blood flow in the first 30 minutes and patients with colitis had significantly higher values compared with smokers (p less than 0.002; p less than 0.04) and non-smokers (p less than 0.007; p less than 0.002) during the first and second 30 minutes respectively. Values in smokers and non-smokers were similar, but smoking a cigarette was associated with a significant fall in blood flow (p less than 0.04) and this change was inversely related to the rise in plasma nicotine concentration (r = -0.63; p less than 0.05). The findings may be relevant to the association between colitis and the smoking history.     

The chemotactic function of neutrophils in ulcerative colitis

Neutrophil chemotactic function was investigated in 30 patients with ulcerative colitis (UC) in an active (no. = 15) or an inactive (no. = 15) stage. Peripheral blood neutrophils from active cases had a significantly decreased stimulated migration in modified Boyden chambers. Simultaneously done investigations of neutrophil migration into skin window chambers showed normal results in both active and inactive cases of UC. Thus, the defective chemotaxis during in vitro conditions was not reflected in a quantifiable neutrophil dysfunction in experimental acute inflammation.     

Polymorphonuclear leukocyte function in ulcerative colitis and Crohn's disease

The aim of this study was to determine whether polymorphonuclear leukocyte chemotaxis, adhesion, and electrophoretic mobility were altered in inflammatory bowel disease and whether such alterations could be related to prior ingestion of immune complexes. Polymorphonuclear leukocytes from patients with ulcerative colitis and Crohn's disease showed significantly impaired stimulated migration (P<0.05), increased adhesiveness (P<0.01 in ulcerative colitis,P<0.001 in Crohn's disease), and reduced electrophoretic mobility (P<0.02 in ulcerative colitis,P<0.001 in Crohn's disease) compared with healthy controls. The disease control of patients with rheumatoid arthritis demonstrated reduced stimulated migration (P<0.025) but normal adhesion. Preincubating normal cells in inflammatory bowel disease sera suggested that the altered migration and adhesion were due to circulating serum factors. Circulating immune complexes, detected by the C1q PEG binding assay, were present in 12.5% of patients with ulcerative colitis and 30% with Crohn's disease. Direct immunofluorescence of polymorphonuclear leukocytes suggested binding and/or ingestion of complexes in 57% of patients with ulcerative colitis, and 67% with Crohn's disease. There was a dorect correlation between positive immunofluorescence and impaired cell migration in ulcerative colitis (P<0.05), but no such relationship was found in the other parameters of polymorph function.     

Lymphoblastic response to autologous colon epithelial cells in ulcerative colitis in vitro

The blastic transformation in vitro of peripheral blood lymphocytes was measured by the 72-hour uptake of tritiated thymidine (³H-6-thymidine) in 23 patients with mucosal ulcerative colitis, three patients with acute Crohn's colitis with rectal involvement, and seven normal subjects. The 23 patients with ulcerative colitis were subdivided into three groups, graded according to severity into seven with acute, severe, nine with active, chronic, and seven with quiescent disease.

In the control cultures of lymphocytes without any added potential stimulant the uptake of ³H-6-thymidine in the clinical subgroup of seven patients with acute, severe ulcerative colitis was significantly greater than in seven normal subjects (p<0·01). This contrasted with a reduced uptake of ³H-6-thymidine by lymphocytes from seven patients with acute severe colitis when compared with seven normal subjects after stimulation with phytohaemagglutinin-P (PHA-P) (p<0·01).

In further duplicate cultures of lymphocytes specifically stimulated by an equal number of viable autologous rectal epithelial cells, the uptake of ³H-6-thymidine was significantly greater in seven patients with acute severe colitis when compared with seven normal subjects (p<0·01). The results in three patients with acute Crohn's colitis with rectal involvement showed no such evidence of lymphocyte sensitivity to autologous rectal epithelial cells and their uptake of ³H-6-thymidine lay within the normal range.

Evidence that the degree of lymphoblastic transformation was related to the clinical severity of ulcerative colitis was provided by the results obtained in the unstimulated and epithelial cell stimulant cultures. The uptake of ³H-6-thymidine was directly related to the clinical severity of ulcerative colitis in the three subgroups studied.

In addition, four of the seven patients with acute severe colitis were studied later in clinical remission. They were then found to have a significantly reduced uptake of ³H-6-thymidine in response to autologous rectal epithelial cells (p < 0·01).

     

Predicting therapeutic outcome in severe ulcerative colitis by measuring in vitro steroid sensitivity of proliferating peripheral blood lymphocytes.

BACKGROUND: Up to 29% of patients with severe ulcerative colitis (UC) fail to respond to steroid treatment and require surgery. Previous studies have failed to show a clear correlation between failure of steroid treatment in severe UC and measures of disease severity. The reasons for treatment failure therefore remain unknown. AIM: To investigate the hypothesis that patients with severe UC who fail to respond to steroid treatment have steroid resistant T lymphocytes. METHODS: Eighteen patients with severe UC were studied. After seven days' treatment with high dose intravenous steroids they were classified as complete responders (CR), incomplete responders (IR), or treatment failures (TF). Within 48 hours of admission blood was taken and the antiproliferative effect of dexamethasone on phytohaemagglutinin stimulated peripheral blood T lymphocytes was measured. Maximum dexamethasone induced inhibition of proliferation (I(max)) was measured. RESULTS: In vitro T lymphocyte steroid sensitivity of TF and IR patients was significantly less than that of CR patients. Both TF and 3/5 IR patients had an I(max) of less than 60%; all CR patients had an I(max) of greater than 60%. No significant correlation was seen between response to treatment and disease severity on admission. When in vitro T lymphocyte steroid sensitivity was remeasured three months later, there was no difference between the groups. CONCLUSIONS: Results suggest that T lymphocyte steroid resistance is an important factor in determining response to steroid treatment in patients with severe UC and may be more predictive of outcome than disease severity.