Leukocyte Migration Inhibition Assay Research Articles

Prospective analysis of human leukocyte functional tests reveals metal sensitivity in patients with hip implant

BackgroundThe aim of the study was to examine the reactivity of peripheral human leukocytes to various metal ions prior and following hip replacement in order to investigate implant-induced metal sensitivity.MethodsThree patient groups were set up: (1) individuals without implants and no history of metal allergy (7 cases), (2) individuals without implants and known history of metal allergy (7 cases), and (3) patients undergoing cementless hip replacement (40 cases). Blood samples were taken in groups 1 and 2 at three different occasions; in group 3, prior and 3, 6, 12, 24, and 36 months after surgery. Peripheral leukocytes were separated and left either untreated or challenged with Ti, NiCl2, CoCl2, CrCl3, and phytohemagglutinin. Cell proliferation, cytokine release, and leukocyte migration inhibition assays were performed. Metal-induced reactivity was considered when all three assays showed significant change. Skin patch tests were also carried out.ResultsBoth skin patch tests and leukocyte functional tests were negative in group 1, and both were positive in group 2. In group 3, after 6 months, 12% of the patients showed reactivity to the tested metals except for NiCl2. Following the 36-month period, 18% of group three became sensitive to metals (including all the earlier 12%). In contrast, patch tests were negative at each time point in group 3.ConclusionsOrthopedic implant material may induce metal reactivity after implantation in a manner where susceptibility is yet to be elucidated. Leukocyte triple assay technique might be a useful tool to test implant material-related sensitivity.

Macrophage procoagulant activity as an assay of cellular hypersensitivity to gluten peptides in coeliac disease

Gluten and casein digests were tested for their ability to induce a cellular immune response on peripheral blood mononuclear cells (PBM) obtained from coeliac patients as well as healthy volunteers and disease control patients using the macrophage procoagulant assay. PBM from coeliac patients who had been on a gluten-free diet for less than 2 years responded strongly to gluten peptides, while coeliac patients at diagnosis or who had maintained a strict gluten-free diet for longer than 5 years showed weaker responses. PBM from healthy volunteers did not respond to gluten or casein peptides, whereas those from patients with Crohn's disease displayed weak reactivity to gluten and casein peptides. Our study using the macrophage procoagulant assay confirms previous findings that lymphoid cells from patients with coeliac disease exhibit a specific cellular immune response to gluten. This assay represents an alternative measure for cell-mediated immunity and is technically much simpler than the previously described leucocyte migration inhibition assay. Macrophage procoagulant activity is measured using a simple plasma recalcification time assay or spectrophotometrically using commercially available chromogenic substrates.

Determination of the immunotoxic potential of pesticides on functional activity of sheep leukocytes in vitro

The effect of eight pesticides with different chemical structure (atrazine, bentazone, chloridazone, dichlofluanid, endosulfan, MCPA, simazine, triallate) on sheep peripheral blood phagocytes and lymphocytes was examined under in vitro conditions by iodo-nitro-tetrazolium reductase test and leukocyte migration-inhibition assay. The pesticides, dissolved in DMSO, were tested at the concentrations of 10 −1–10 −6 M. The significant suppression of metabolic activity of phagocytic cells was registered after exposure to dichlofluanid (10 −1–10 −3 M), endosulfan, simazine and triallate (10 −1 M). The significant cytotoxic effect (the decrease of spontaneous migration of leukocytes) was registered for bentazone, dichlofluanid, endosulfan and MCPA (10 −1 M); chloridazone (10 −1 M–10 −2 M) and triallate (10 −1–10 −5 M). The significant immunotoxic effect (the decrease of lymphocyte activation with PHA) was observed for atrazine (10 −1–10 −2 M); bentazone (10 −2–10 −4 M); dichlofluanid, endosulfan (10 −2–10 −3 M); MCPA (10 −2–10 −6 M) and simazine (10 −1–10 −4 M). Three of the pesticides tested suppressed both, the metabolic activity of phagocytes and mitogenic activation of lymphocytes (dichlofluanid, endosulfan and simazine). Triallate suppressed the metabolic activity of phagocytes and showed a strong cytotoxic effect. Pesticides atrazine, bentazone and MCPA influenced the mitogenic activation of lymphocytes and chloridazone showed a significant cytotoxic effect. The different chemical structure of pesticides influenced the metabolic activity of phagocytic cells as well as mitogenic activation of lymphocytes to various intensity.

Comparison of immune parameters in cows with normal and prolonged involution time of uterus

Indices of cellular immunity in postpartum Holsteincows with the normal (n = 9) and prolonged (n = 4) uterine involution time were evaluated. Peripheral white blood cells were isolated by lysis from postpartum animals. An indirect immunofluorescence method for staining and flow cytometric analysis was employed to determine the cell subpopulation of lymphocytes. The function assays were also used to examine the activity of lymphocytes and phagocytes. A significant decrease in the lymphocyte absolute number, and subpopulation of T (CD2+, CD4+, CD8+), and B (IgM+) cells in dams with postparturient complications and prolonged uterine involution time was observed. The quantitative changes of immune cells were accompanied by a significant decline of phagocyte functional activity in an iodo-nitro-tetrazolium reductase test and polyclonal lymphocyte activation to phytohemagglutinin in a leukocyte migration-inhibition assay. In conclusion, a significant decrease in the lymphocyte absolute number and subpopulation of T (CD2+, CD4+, CD8+), and B (IgM+) cells was observed and the host defense role of phagocytes and lymphocytes was impaired in cows with prolonged uterine involution, which can increase their susceptibility to infections.

Cell-mediated immunity to the mycelial phase of Malassezia spp. in patients with pityriasis versicolor and controls.

Malassezia is the aetiological agent of pityriasis versicolor. The mycelial phase of the organism predominates in lesions of pityriasis versicolor. To evaluate the cell-mediated immune (CMI) response to the mycelial phase of Malassezia in patients with this disease, which has not previously been studied. The CMI status of 12 patients with pityriasis versicolor and 12 age- and sex-matched controls to mycelial antigen(s) of the organism was examined. The responses to the mycelial form of three strains of the organism were assessed using lymphocyte transformation and leucocyte migration inhibition assays. The transformation responses of the lymphocytes from both patients and controls gave transformation indices < or = 3, although the responses of lymphocytes from patients with pityriasis versicolor to the mycelial form of Malassezia strains were generally higher than those of the controls. In the leucocyte migration inhibition assay, leucocytes from patients with pityriasis versicolor and controls responded to the mycelial antigens of three different Malassezia strains; however, there was no significant difference in leucocyte response between patients with pityriasis versicolor and controls. Patients with pityriasis versicolor do not therefore have a CMI deficiency to Malassezia mycelial antigens but fail to generate a protective CMI response to mycelial antigens over and above that of control individuals during active disease.

Pityrosporum species as a cause of allergy and infection.

Pityrosporum species as a cause of allergy and infection.

Cell-mediated immunity in chickens vaccinated with the V4 strain of Newcastle disease virus

A leukocyte migration inhibition assay was used to demonstrate antigen-specific cell-mediated immunity (CMI) in chickens vaccinated with the V4 strain of Newcastle disease virus. Chickens were vaccinated when 5 weeks old, and again 3 and 7 weeks later. CMI was detected 9 days after initial vaccination by eyedrop, but only after the second dose of vaccine delivered into the crop. In some chickens there was a temporary suppression of CMI to Newcastle disease virus, especially after the initial application of vaccine to the crop. Haemagglutination inhibition antibodies developed to similar levels in chickens after vaccination by either route. There was no obvious correlation between antibody and CMI responsiveness.

Cell‐mediated immune responses to Malassezia furfur serovars A, B and C in patients with pityriasis versicolor, seborrheic dermatitis and controls

It has been postulated that patients with Malassezia furfur-associated dermatoses have a deficient cell-mediated immune response to M. furfur. This study examined the cell-mediated immune responses to M. furfur serovars A, B and C of 10 patients with pityriasis versicolor and 10 age- and sex-matched controls; and 10 patients with seborrheic dermatitis and 10 age- and sex-matched controls. The responses to each serovar of M. furfur were assessed using the lymphocyte transformation assay and the leukocyte migration inhibition assay. The lymphocyte transformation responses of the patients with pityriasis versicolor to M. furfur serovars A, B and C (0/10, 6/10 and 5/10 respectively) were not significantly different from those of controls (0/10, 2/10 and 1/10). However, for patients with seborrheic dermatitis, significantly more patients' lymphocytes responded to serovars B and C (6/10 and 6/10 respectively) than those of controls (1/10 and 1/10). No patient or control responded to serovar A. In the leukocyte migration inhibition assay, the leukocytes from a greater proportion of patients with pityriasis versicolor (5/7) responded to serovar B than controls (2/10); and the leukocytes from a greater proportion of patients with seborrheic dermatitis (4/10) responded to serovar C than controls (0/9). Thus, this data did not indicate the presence of any cell-mediated immune deficiency to M. furfur in patients with pityriasis versicolor or seborrheic dermatitis, as measured by the lymphocyte transformation assay or the leukocyte migration inhibition assay. The greater responsiveness of T lymphocytes from patients may indicate that T lymphocytes might be involved in the pathogenesis of these diseases.

The effect of adjuvant and specific or non-specific vaccination on development of protective immunity of rabbits against Trichostrongylus colubriformis infection

Adult New Zealand rabbits were vaccinated subcutaneously with one dose of 100 μg adult nematode phosphate buffered salinesoluble proteins (PBS-ASP, groups I and II), a detergent-soluble fraction of adult somatic proteins (DS-ASP, group III) or three doses of l mg normal rabbit serum proteins (group IV). Injections of the immunogens in groups II, III and IV were accompanied with beryllium hydroxide, Be(OH) 2 as an adjuvant. Vaccinated rabbits and also those of group V (naive) were challenged orally with 10,000 infective larvae of T. colubriformis 14 days after antigen injection and necropsied 2 weeks later. A single dose of PBS-ASP induced 33.5% protection when the antigen was given alone (group I) and 69.4% when injected with Be(OH) 2 (group II). A detergent-soluble fraction of ASP given with the adjuvant provided 87.2% protection (group III), whilst non-specific vaccination with serum proteins plus Be(OH) 2 elicited 99% protection (group IV). Mesenteric lymph node leukocyte responses were measured using a leukocyte migration inhibition assay. A significant response was observed only in group IV. In ELISA tests IgA antibodies specific to PBS-ASP reached the highest level in the intestinal mucosa of groups I and II and in the bile of groups I and III. Antibody levels of IgG isotype were similar in the intestinal mucosa of all the immunized groups. Nematode antigen was detected using a ‘sandwich’ ELISA method in faecal protein extracts of rabbits of groups II and III on days 2–6 after challenge.

Cell-mediated immune response in rotavirus-infected calves: Leucocyte migration inhibition assay

The cell-mediated immune (CMI) response was determined in rotavirus-infected calves by leucocyte migration inhibition assay with blood, spleen, mesenteric lymph node and intestinal lymphocytes. The inhibition of migration was more prominent in intestinal and mesenteric lymph node lymphocytes than in spleen and blood. In rotavirus-infected calves, the assay indicated the presence of CMI response which was more prominent at the local site of infection.

Stabilization of Salmonella-specific dialyzable leukocyte extracts

Activity of Salmonella-specific dialyzable leukocyte extracts (DLE) prepared from mesenteric lymphatic nodes of calves and stabilized with bovine albumin was studied in this work. The effect of ambient temperature and storage period on the activity of DLE was evaluated. Testing for DLE activity by means of capillary leukocyte migration inhibition (LMI) assay showed that DLE stabilized with albumin retained 60% of its activity for 12 months of storage at 4°C. This level of activity was retained in the native DLE (without albumin) kept at −20°C. DLE stabilized with albumin and stored for 12 months at 4°C inhibited the penetration of salmonellae into the liver and spleen, and their colonization in the gastrointestinal tract was significantly reduced.

An immunological aspect of melanoma and its potential application in adjuvant therapy

In an attempt to understand some of the natural immunological characteristics of cutaneous melanoma so that we can plan justifiable immunotherapeutic approaches, 23 patients were studied. The autologous leukocyte migration inhibition assay was utilized to assess in vitro their tumor-specific cellular immunity. This assay was specifically used because when presensitized lymphocytes are exposed to the same antigen, they release lymphokines which inhibit the natural migration of the leukocytes. All the patients were staged pathologically according to the TNM system. Twelve of them had regional metastasis, i.e., stage III, and underwent regional lymphadenectomy. The other 11 had distant metastases, i.e., stage IV disease, and all their gross tumors were resected. These 23 patients were the source of tumor material and peripheral blood. Fresh autologous leukocytes were obtained for the assays, from each patient, on the day of surgery and prior to the administration of the preoperative medications. These were tested in vitro, on the same day, with freshly prepared autologous tumor extracts as the source of autologous tumor antigens. The results revealed that the preoperative leukocytes of patients with stage III melanoma expressed hypersensitivity to their tumors, with significant inhibition of their leukocyte migration, compared to those with distant metastases who expressed no such sensitivity, P = 0.012. Such hypersensitized lymphocytes may be capable of producing more efficient lymphokine activated killer (LAK) cells for an effective adjuvant adoptive immunotherapy.

Studies on cell-mediated immune response to rabies virus immunization in dogs

Peripheral blood leucocytes (PBL) from unvaccinated dogs and dogs vaccinated subcutaneously with beta-propiolactone (BPL) inactivated sheep brain antirabies vaccine were exposed in vitro to rabies antigen. A direct leucocyte migration inhibition (LMI) assay in agarose was used to study the cell-mediated immune (CMI) response. The CMI response could be detected as early as 7 days postvaccination. It reached a peak by 14 days postvaccination followed by a decline. The PBL from unvaccinated control dogs were not stimulated by rabies virus antigen.

Cellular hypersensitivity to a synthetic dodecapeptide derived from human adenovirus 12 which resembles a sequence of A-gliadin in patients with coeliac disease.

The human intestinal adenovirus serotype 12 (Ad12) may be implicated in the pathogenesis of coeliac disease by virtue of immunological cross reactivity between epitopes shared by its early region E1b protein and A-gliadin. In the present study a synthetic dodecapeptide from the corresponding viral epitope (Ad12E1b, residues 384-395) was tested for its effect on peripheral blood mononuclear cells from 22 treated and eight untreated patients with coeliac disease, 22 healthy subjects, 11 patients with ulcerative colitis, and 11 patients with Crohn's disease by an indirect leucocyte migration inhibition assay. In addition, the effect of both the viral and the gliadin synthetic peptides was studied by proliferation and migration assays simultaneously performed in an unselected subgroup of 12 treated coeliac patients and 12 healthy subjects of the study. Coeliac patients with untreated disease showed no response to the viral peptide compared with treated patients (p greater than 0.1). Treated coeliac patients showed a significantly different response from healthy control subjects and control patients with disease (p less than 0.001) which was dependent on the concentration of the viral peptide. In the subgroup of the treated coeliac patients (n = 12) there was a significant correlation between the responses in the migration and the proliferation assay using either the viral (p less than 0.02) or the gliadin (p less than 0.005) peptide at the highest concentration (33.3 micrograms/ml). Furthermore, the responses obtained using viral peptide correlated significantly with the responses obtained with gliadin peptide in both the migration (p less than 0.001) and the proliferation (p less than 0.001) assays. These results show that in coeliac patients there is pronounced cross reactivity at the level of T cell recognition between synthetic peptides derived from the Ad12 and A-gliadin. This antigenic cross reactivity may be involved in the pathogenesis of coeliac disease.

Epstein-Barr virus (EBV) antigen-specific leukocyte migration inhibition in infectious mononucleosis: II. Kinetics of sensitization against five EBV-encoded nuclear proteins and the latent membrane protein

The T cell-mediated immune response of infectious mononucleosis (IM) patients to five Epstein-Barr virus (EBV)-determined nuclear antigens, EBNAs, and to the membrane antigen associated with growth-transformed cells (latent membrane protein, LMP) was measured by the leukocyte migration inhibition (LMI) assay. Two different antigen sources were used: extracts from cells that only expressed EBNA-1, EBNA-2, or LMP after transfection with the corresponding EBV-DNA fragment, and synthetic peptides deduced from the corresponding genes. Patients in the acute phase of the disease failed to respond to EBNA-1, -5, -6, and LMP, but became responsive during convalescence. The majority of the patients responded to EBNA-2 and/or EBNA-3 in the acute phase ( 9 15 and 12 15 , respectively). The response to EBNA-3 disappeared more often in convalescence than the response to EBNA-2; 6 of 15 patients were negative to EBNA-2 and 12 of 15 to EBNA-3 during recovery. In addition to its value in the assessment of host sensitization to virus EBV antigens, these studies and the derived hypotheses also provide certain predictions about the predominant antigen expression in the EBV-infected host under normal and pathological conditions that can be subjected to direct experimental tests.

Cellular autoimmunity in psoriasis and lichen planus

The objective of this investigation was to determine whether specific cellular recognition of the epidermis is associated with the human skin diseases, psoriasis and lichen planus. Epidermal cells (EC) obtained from biopsies of involved and uninvolved skin of patients with these diseases were used as stimulators and targets for autologous peripheral blood mononuclear cells (PBMC) in assays of three conventional manifestations of cellular immunity: lymphocyte transformation, leukocyte migration-inhibition and cell-mediated cytotoxicity. Parallel tests were conducted with autologous PBMC as stimulators to ascertain the tissue specificity of the reactions evoked by autologous EC. Similar assays were conducted with EC and PBMC from a large group of normal subjects, and the results were compared to those of the dermatology patients by rigorous statistical analyses. No evidence of lymphocyte-mediated cytotoxicity towards autologous EC was obtained with any of the subject groups, but autologous EC, and to a lesser extent PBMC, of the psoriasis patients, but not of the other two groups, evoked significant lymphocyte transformation. These results were obtained only with patients on Goeckerman therapy, raising the possibility that they were a manifestation of the treatment (topical coal-tar and ultraviolet light irradiation) rather than of the disease, although reasons are presented why this is unlikely. Clearer evidence of disease-associated autoimmunity was obtained in the leukocyte migration-inhibition assays, where autologous EC, and to a lesser extent, PBMC, of the psoriasis patients in general, not just those on Goeckerman therapy, and not those of the lichen planus patients or of the normal subjects, stimulated the release of a leukocyte migration-inhibition factor. These results support the concept of a central role for T-cell mediated autoimmunity in the pathogenesis of psoriasis.

Cellular and humoral immune responses to synthetic peptides deduced from the amino‐acid sequences of Epstein‐Barr virus‐encoded proteins in EBV‐transformed cells

Ten synthetic peptides containing 18-22 residues deduced from the amino-acid sequences of the EBV-encoded latent-infection-associated membrane protein (LMP) and the 2 principal nuclear antigens, EBNA-1 and EBNA-2, were tested for their ability to induce lymphokine release from sensitized T-cells of EBV-seropositive donors, as measured by the leukocyte migration inhibition assay (LMI). Only one of the 10 free peptides induced EBV-specific LMI. After Sepharose-coupling, 4 additional peptides were regularly active. In parallel, the sera of the same and other donors were screened for synthetic peptide-binding antibodies, as measured by an ELISA assay. Antibodies to 9 of the 10 peptides were detected in 25-80% of EBV-antibody-positive, but not in EBV-antibody-negative sera. A comparison of the two responses indicates that the humoral immune system tends to react with more epitopes on a given protein than the cellular immune system. Furthermore, the antibody reactivity pattern to different epitopes is more variable from individual to individual than the T-cell response. Also, the epitopes detected by antibodies and sensitized T-cells are often not identical.

Humoral autoimmunity in the pathogenesis of insulin-dependent diabetes mellitus

The aetiology and pathogenesis of insulin-dependent diabetes mellitus (IDDM) remain to be fully clarified, even though numerous observations of the last two decades indicate that humoral and/or cellular autoimmune destruction of the pancreatic β cells is involved (Nerup & Lernmark 1981; Cahill & McDevitt 1981). Several extensive reviews on this subject have been published recently (Irvine (ed) 1980; Martin et al. (eds) 1981; Kolb et al. (eds) 1983; Andreani et al. (eds) 1984), from which only the most significant findings are mentioned here. In 1965, Gepts reported that insulitis, a mono-nuclear cell infiltration in the islets of Langerhans, was common in patients with newly diagnosed IDDM (Gepts 1965). Later Nerup’s group and others showed that peripheral blood mono-nuclear cells obtained from diabetic patients, when tested in a leucocyte migration inhibition assay, reacted against endocrine components of the pancreas (Nerup et al. 1971, 1973a; MacGuish et al. 1974a). These findings suggested that cellular immune reactions could be involved in the development of diabetes. However, whether the reactive cells represented cytotoxic T-lymphocytes or other immune effector cells, was not determined. Others found that the incidence of organspecific autoimmune diseases in diabetic patients was higher than expected (Irvine et al. 1970; MacCuish & Irvine 1975) and that autoantibodies against various endocrine cells, other than pancreatic endocrine cells, were often present in clinically unaffected diabetics (Irvine et al. 1970; Nerup & Binder 1973b). The nature of increased susceptibility to autoimmunity in diabetic patients has yet to be explained.

Epstein-Barr virus (EBV) antigen-specific leukocyte migration inhibition (LMI) in infectious mononucleosis (IM): I. Kinetics and response to a membrane protein on EBV-transformed cells

Cell-mediated immune response of mononucleosis (IM) patients to Epstein-Barr virus (EBV)-determined antigens was measured by the leukocyte migration inhibition (LMI) assay. Patients in the acute phase of the disease failed to respond to partially purified nuclear antigen, EBNA, or to cell extracts that contained EBNA as the predominant EBV antigen. They showed a strong specific response to cell extracts enriched in early antigen (EA) and virus capsid antigen (VCA). The LMI response to EBNA appeared in convalescence in parallel with EBNA-specific antibodies, slightly later in children than in adults. Membrane fractions of EBV-carrying, virus nonproducer Raji cells induced an EBV-specific LMI at approximately the same time. A bacterial fusion protein containing the hydrophilic part of the virus-coded membrane antigen of latently EBV-infected cells also induced an EBV-specific response that parallelled the LMI reaction elicited by the Raji membrane fraction. This is in line with our previous finding (D. Sulitzeanu et al., J. Virol. 58, 230, 1986) that this fusion protein shares an epitope with Raji cell membranes.

The indirect leucocyte migration inhibition assay--an endotoxin-sensitive chemokinetic assay. Part II.

Depletion of agarose for endotoxins resulted in a low spontaneous migration of polymorphnuclear cells (PMNC). Re-addition of endotoxin, in casu lipopolysaccharide from E. coli 026:B6 (LPS), enhanced the spontaneous PMNC migration in a two-phased dose-response pattern, reaching maximum migration with LPS 1 x 10(-7) g/ml. Thus, the migration of PMNC under agarose seems to be a chemokinesis. Leucocyte migration inhibition factor (LIF), induced by PPD 50 micrograms/ml at endotoxin-free conditions, significantly reduced the PMNC migration compared to supernatants from control cultures, however not compared to the conventional limit of significance, MI = 0.80. With increasing PMNC migration there was an insignificant decrease in the MI. Addition of LPS, 1 x 10(-9) g/ml, during LIF induction caused a significant increase in LIF production, an effect which overshadowed the effect of PPD. Thus, the application of the conventional limit of significance, MI = 0.80, may result in false-negative or false-positive conclusions, depending upon the endotoxin contamination. A standardization of the endotoxin content in both steps of the indirect leucocyte migration inhibition assay seems mandatory in order to obtain a reliable and reproducible bioassay.