A comparison of the inhibition of leucocyte migration and monocyte spreading as in vitro assays for tuberculin hypersensitivity in man.

The ability of leucocyte migration inhibition and monocyte spreading inhibition test to detect tuberculin hypersensitivity was compared in the same twelve Mantoux-negative and fifteen Mantoux-positive persons. Tuberculin hypersensitivity expressed in vitro as migration or spreading inhibition, induced by 100 mug of PPD/ml, was assessed after 2 and 24, or 4 and 20 hr of incubation. A significant difference was found between negative and positive persons by migration inhibition at the early interval and by spreading inhibition at both intervals. When the two tests were compared on the basis of individual results, monocyte spreading inhibition appeared more discriminating (fewer results in the group of positive persons overlapped with those found among negative persons). Results of the monocyte spreading inhibition test correlated well with cutaneous reactions at both incubation intervals, while with migration inhibition the correlation was not so well expressed at either interval. Furthermore, a given change in skin reactivity of tuberculin-positive persons was reflected better in spreading inhibition than in migration inhibition indices. We conclude that the method of monocyte spreading inhibition compares favourably with the method of leucocyte migration inhibition, and it seems to be a suitable in vitro test for detection of tuberculin hypersensitivity in man.     

In vitro delayed hypersensitivity in normal and hyporeactive patients

The in vitro macrophage migration inhibition test can be used to evaluate human delayed hypersensitivity. Using purified protein derivative of tuberculin (PPD) as the antigen, twenty of twenty-seven in vitro tests in non-anergic persons with negative PPD skin tests were negative and fifteen of sixteen in vitro tests in persons with positive skin tests were positive. In patients with drug or disease-induced cutaneous hyporeactivity, twelve of twenty-eight tests were positive despite negative skin tests. In two anergic patients with mucocutaneous candidiasis positive in vitro tests were obtained with Candida albicans antigen as well. Measurable levels of IgG were seldom detected in the test media.

The results indicate that the macrophage migration inhibition test measures delayed hypersensitivity in man and is sometimes positive in cases of reduced skin reactivity.

     

Production of Monocyte Chemoattractant Protein 1 in Tuberculosis Patients

To investigate the role of monocyte chemoattractant protein 1 (MCP-1) in the immune response to Mycobacterium tuberculosis, we studied MCP-1 production in tuberculosis patients. CD14⁺ blood monocytes from tuberculosis patients spontaneously expressed higher levels of MCP-1 mRNA and protein than CD14⁺ monocytes from healthy tuberculin reactors. MCP-1 production in lymph nodes from tuberculosis patients was also markedly increased. These findings suggest that MCP-1 can contribute to the antimycobacterial inflammatory response by attracting monocytes and T lymphocytes.     

An inhibitory factor against monocyte spreading in the sera of patients with systemic lupus erythematosus

The effect of the sera of patients with systemic lupus erythematosus (SLE) on monocyte function was studied using cell spreading as an indicator. Monocyte spreading induced by exogenous stimuli was shown to be inhibited by SLE sera. Gel filtration of SLE sera on Sephadex G-200 revealed that the factor responsible for this inhibition had a molecular weight of about 50,000. Pretreatment of monocytes with the inhibitory factor led to suppression of cell spreading induced by subsequent stimulation, but this hyporeactivity was reversible. Spreading of monocytes was rapidly aborted by the addition of this inhibitory factor. Thus, the inhibitory factor appeared to affect monocyte itself, but its effect seemed to be transient.     

In vitro studies of delayed hypersensitivity: inhibition of macrophage spreading in rats sensitive to tuberculin and diphtheria toxoid

Wistar rats were sensitized by footpad injection of BCG in adjuvant, or Mycobacterium butyricum in adjuvant, or diphtheria toxoid in Freund's incomplete adjuvant. It was found that the cell population of the peritoneal washings contained approximately 57 per cent macrophages, 22 per cent lymphocytes, 11 per cent granulocytes and 8 per cent mast cells. The lymphocyte count was significantly reduced and the granulocyte count increased after sensitization. The animals sensitized to M. butyricum exhibited delayed skin reactivity to tuberculin and the spreading of macrophages in vitro was significantly inhibited with the same antigen. On the contrary, the spreading of macrophages obtained from animals sensitized to BCG was not inhibited by tuberculin and there was no cutaneous reactivity. Spreading of macrophages obtained from rats sensitized by diphtheria toxoid was significantly inhibited in the presence of diphtheria toxoid, but not in the presence of tuberculin. These animals displayed delayed cutaneous hypersensitivity to diphtheria toxoid. Spreading of macrophages from normal rats was unaffected by serum antibodies. This was true either when the peritoneal cells were treated with antiserum prior to contact with antigen, or when the antigen—antibody reaction took place in the chamber containing the macrophages ready to spread. These results indicate that the technique of macrophage spreading inhibition is able to detect specifically hypersensitivity of delayed type and offers a convenient method for the in vitro study of delayed hypersensitivity.     

Development of sensitivity to dinitrochlorobenzene in patients with sarcoidosis

(1) The ability to develop delayed hypersensitivity to dinitrochlorobenzene (DNCB), a potent chemical antigen, has been studied in twenty-two patients with sarcoidosis.

(2) In three patients under treatment with prednisone the Mantoux was negative, and DNCB testing was negative.

(3) In fourteen untreated patients with active sarcoidosis DNCB testing was negative. In eight of these patients the Mantoux was also negative and in six it was positive.

(4) In one patient with sarcoidosis of doubtful activity, DNCB testing was positive, and the Mantoux was negative.

(5) Five patients with healed sarcoidosis developed delayed hypersensitivity to DNCB in the normal manner. In three the Mantoux was positive, and in two it was negative.

(6) It is suggested that these results can best be interpreted in terms of the afferent, central and efferent aspects of the immune response. The results are related to previous work on the association of sarcoidosis with abnormalities of delayed hypersensitivity.

     

In vitro studies of delayed-type hypersensitivity: The time course of the primary immunological reaction in individual rats determined by macrophage spreading inhibition

The aim of this work was to develop a method for the continuous follow-up of the onset and evolution of delayed-type hypersensitivity in individual rats. This hypersensitivity was determined by an in vitro macrophage spreading inhibition test in Wistar rats sensitive to tuberculin and diphtheria toxoid. Circulating antibodies to diphtheria toxoid were evaluated by passive haemagglutination. Samples of peritoneal cells and blood were taken 2 days and a few hours before sensitization, and on days 3, 4, 5, 6, 7, 11, 20, 27 and 40 after sensitization. It was found that: (1) Multiple consecutive washings of the peritoneal cavity, repeated bleedings and sensitization alone produced no change in the percentage of peritoneal macrophages spreading in medium alone. (2) Similarly, the percentage of spread macrophages of non-sensitized rats in the presence of tuberculin remained unaltered during the course of daily peritoneal washings. (3) In sensitized rats, sensitization was followed by a significant inhibition in macrophage spreading in the presence of sensitizing antigens. Thereafter, it was possible to trace individual curves reflecting the onset and evolution of delayed-type hypersensitivity to tuberculin and diphtheria toxoid. (4) The mean macrophage spreading inhibition corresponding to the delayed-type hypersensitivity was found to be maximal for both antigens on day 7 following sensitization, and decreased thereafter. (5) On the other hand, high titres of circulating antibodies to diphtheria toxoid did not appear earlier than 20 days after sensitization and continued to increase towards the end of the observation period.     

Diminished monocyte function in microfilaremic patients with lymphatic filariasis and its relationship to altered lymphoproliferative responses

Antigen-specific hyporesponsiveness to filarial antigens is a phenomenon observed in patent infection with lymph-dwelling filarial parasites of humans. This phenomenon has been attributed to a multitude of factors, one of which is altered monocyte function. To examine the role played by monocytes in filarial infection, we assessed the responses of monocytes obtained from normal and filarial parasite-infected individuals to both crude filarial antigen and purified recombinant filarial antigen WbSXP-1 and attempted to relate these to the altered lymphoproliferative responses seen in filarial infection. Monocytes from microfilaremic (MF) patients demonstrated an inability to respond to lipopolysaccharide compared to monocytes from endemic normal persons or from lymphedema patients. Indeed, interleukin 1beta (IL-1beta) production was severely limited, a finding that did not extend to monocyte responses to filarial antigens. Serum from MF patients reduced adherence and spreading of normal monocytes, a finding not seen with serum from the other clinical groups. Interestingly, there was a significant correlation between the production of IL-1beta and adherence. Moreover, the levels of spontaneous production of IL-1beta correlated with high levels of spontaneous secretion of IL-10. The effects observed were not a result of diminished viability or alteration in the expression of the cell surface markers CD14 and HLA-DR. These data suggest that monocyte function is dampened in MF patients, a finding which could alter lymphoproliferative responses during patent infection.     

Assessment of chemokine receptor function on monocytes in whole blood: In vitro and ex vivo evaluations of a CCR2 antagonist

Inhibition of monocyte and macrophage function by targeting chemokine receptors represents an attractive strategy for therapeutic intervention in inflammatory diseases. We describe an assay to assess chemokine receptor function on whole blood monocytes by measuring chemokine stimulated change in cell shape as measured by flow cytometry. The relative potential of the chemokine receptors CCR1, CCR2, CCR5, CX₃CR1, and CXCR4 to activate monocytes in whole blood was evaluated and compared. Analysis of MCP-1 response for monocytes in blood from numerous donors revealed that the assay method had excellent intra-donor reproducibility and sensitivity. Further, the utility of this assay to determine target engagement by chemokine receptor antagonists was demonstrated using a CCR2 antagonist in rhesus monkeys. Blockade of CCR2 on whole blood monocytes was demonstrated ex vivo on blood samples collected from rhesus monkeys administered a small molecule CCR2 antagonist (MK-0812). Using a delayed-type hypersensitivity reaction to elicit monocyte recruitment to the skin of rhesus monkeys, we also evaluated the ability of MK-0812 to block monocyte migration in vivo. Blockade of CCR2 stimulation of whole blood monocytes was correlated with the inhibition of monocyte recruitment to the skin, validating the potential to use this approach in the evaluation of dose selection for chemokine receptor antagonists clinically.     

Tuberculin anergy in clinically normal individuals. I. Lymphokine and lymphocyte transformation studies

Tuberculin anergy was demonstrated in a number of clinically normal individuals who, on being immunized with Bacillus Calmette-Guérin (BCG), failed to demonstrate a positive Mantoux skin test. Lymphoid cells from these anergic subjects when stimulated with purified protein derivative (PPD) in vitro did not produce macrophage migration inhibition factor (MIF). Lymphocyte transformation studies demonstrated that while Mantoux-positive, non-BCG immunized Mantoux-negative and anergic individuals could all undergo blast cell transformation in the presence of PHA, only lymphoid cells from Mantoux-positive and a proportion of anergic people were capable of blast cell transformation when cultured with PPD. These results suggest that more than one mechanism may account for tuberculin anergy in clinically normal people operating at the level of lymphokine production and in some cases lymphocyte transformation.     

Histometric studies on cellular infiltrates of tuberculin tests in patients with sarcoidosis.

The density and microanatomical location of CD4 and CD8 lymphocytes and of monocytes/macrophages at the site of a tuberculin test were measured in 13 patients with sarcoidosis, and the results were compared with those seen in a group of healthy controls. The cellular infiltrate was significantly reduced in the sarcoid subjects compared with the controls for all cell phenotypes studied; the ratio of CD4 positive:CD8 positive lymphocytes was significantly increased in the sarcoid group. Clinically negative reactions showed substantial numbers of infiltrating mononuclear cells, although not as great as in clinically apparent reactions. A clinically negative tuberculin reaction does not necessarily imply anergy to the test substance and should not be termed "negative".     

Migration inhibition experiments with mixtures of human peripheral blood lymphocytes and guinea pig peritoneal exudate cells

Migration inhibition using mixtures of human peripheral blood lymphocytes and non-sensitized guinea pig peritoneal exudate cells was demonstrable with purified protein derivative of tuberculin (PPD) and Candida albicans extract (CAE) when lymphocytes were obtained from individuals with a delayed type skin reaction to these antigens. No migration inhibition occurred when lymphocytes were used from skin test negative individuals. Furthermore it was found that lymphocytes from some skin test negative persons, which did not induce migration inhibition, showed, however, an increased DNA synthesis when cultured in vitro with PPD. This indicates that a dissociation between the results of migration inhibition, skin test and lymphocyte transformation can occur not only in patients with immune disorders, as has been described by other investigators, but also in healthy individuals.     

Antigen specific lymphocyte activity in vitro by peripheral blood leucocytes from Mantoux positive and negative human beings. I. Comparison of quantitative and qualitative differences in the PPD-specific lymphoproliferative response of lymphocytes from the two kinds of donors.

Lymphocytes from some PPD (purified protein derivative from tubercle bacillus) skin test negative (Mantoux negative=Mx--) human beings reacted against PPD in the lymphoproliferative assay with a time course and dose response very similar to those of lymphocytes from Mantoux positive (Mx+) individuals. Other Mx-- persons were PPD non-responsive in the lymphoproliferative assay. The PPD response of (immunoglobulin=Ig) Ig anti-Ig column passed lymphocytes (T-cells) from Mx--/LP+ (LP+=lymphoproliferative) persons was significantly reduced whereas the in vitro PPD response of T-lymphocytes from Mx+/LP+ was the same or increased. Purified B-lymphocytes from all kinds of tested individuals did not respond in vitro against PPD. Serological investigations indicated that one of the reasons for the negative skin reaction of individuals whose lymphocytes gave a positive lymphoproliferative response against PPD in vitro, is that such individuals had recirculating PPD of high molecular weight (greater than 900,000) and/or PPD anti-PPD antibody complexes in the serum. These substances could block the PPD-specific T-lymphocytes.     

Linkage Between Monokine Production and Regulation of the Negative Surface Charge Density of Human Monocytes

The regulation of the negative surface charge density of human monocytes was investigated with the help of the synthetic glycolipid analogue BAY R 1005. This compound is incorporated into the outer membrane of isolated monocytes during 24 hours of incubation. After this time the electrophoretic mobility (EM) of monocytes is unchanged at 0.95 × 10^-4 (cm² V^-1 s^-1) and remains unchanged even under conditions where non-treated monocytes increase their EM up to 1.1 × 10^-4 (cm² V^-1 s^-1). In addition BAY R 1005 stops differentiation of monocytes to macrophages, it triggers monokine production and abolishes monocyte suppressor activity and spreading capability. The results show that BAY R 1005 affects intracellular features. In connection with earlier investigations of the regulation of the negative surface charge density of human monocytes (1,2) the study suggests that monokine production and maintenance of the EM of monocytes are linked.     

A micro-method for peripheral leucocyte migration in tuberculin sensitivity

Inhibition of buffy layer peripheral leucocyte migration by tuberculin purified protein derivative (PPD) from micro-capillaries mounted in small tissue culture chambers correlates in all cases with a positive Mantoux test. This quick and reproducible test of cellular hypersensitivity in man requires only 5-15 ml of blood and is applicable for study in children and in clinical situations where repeated monitoring of cellular immunity may be required.     

Linkage Between Monokine Production and Regulation of the Negative Surface Charge Density of Human Monocytes

The regulation of the negative surface charge density of human monocytes was investigated with the help of the synthetic glycolipid analogue BAY R 1005. This compound is incorporated into the outer membrane of isolated monocytes during 24 hours of incubation. After this time the electrophoretic mobility (EM) of monocytes is unchanged at 0.95 × 10^?4 (cm² V^?1 s^?1) and remains unchanged even under conditions where non-treated monocytes increase their EM up to 1.1 × 10^?4 (cm² V^?1 s^?1). In addition BAY R 1005 stops differentiation of monocytes to macrophages, it triggers monokine production and abolishes monocyte suppressor activity and spreading capability. The results show that BAY R 1005 affects intracellular features. In connection with earlier investigations of the regulation of the negative surface charge density of human monocytes (1,2) the study suggests that monokine production and maintenance of the EM of monocytes are linked.     

Tuberculin Skin Reactivity in Italian Military Recruits Tested in 1996–1997

 In 1996–1997 data was collected and a Mantoux tuberculin test performed in 2882 Italian military recruits aged 18–23 years in order to establish the prevalence of tuberculin reactivity. In addition, the annual risk of infection, defined as the probability that a non-infected individual would be infected during the following year, was calculated. Of the 2882 recruits, 513 had received a BCG vaccination, the remaining 2369 had not. The overall prevalence of subjects with a tuberculin skin reaction size >5 mm (the cut-off point for positivity corresponding to the antimode in the reaction size frequency curve) was 6.1% (144/2369). The prevalence of skin reactivity increased with age but remained similar when related to area of residence, duration of father's school education and family size. The same general trend was observed if the standard pre-established cut-off point of 10 mm was used. In this case the overall prevalence of a positive skin reaction was 4% (95/2369). The annual risk of infection was 0.3% for a prevalence of tuberculin skin reactivity of 6.1% (cut-off point 5 mm) and 0.19% for a prevalence of 4% (cut-off point 10 mm). Analysis of the population sample vaccinated with BCG showed a lack of correlation between the positive reaction after vaccination reported retrospectively by the subject and the current skin reaction observed by the physician in this study (K=0.254). Moreover, a significant difference was observed between the skin reaction in subjects vaccinated with BCG in 1993–1994 (average size 12.5 mm) and that of subjects vaccinated in 1995–1996 (average size 10.1 mm, P<0.01), probably as a consequence of mycobacteria circulating in the general population which act as a natural booster in people already vaccinated with BCG. A booster effect of tuberculin in Mantoux assays also cannot be excluded.     

Appraisal of the total blood lymphocyte proliferation assay as a diagnostic tool in screening for tuberculosis.

The total blood lymphocyte proliferation assay (TLP) was evaluated as a screening test for infection with Mycobacterium tuberculosis and was compared with the tuberculin (Mantoux) skin test. The results of TLP assays performed on 33 patients with tuberculosis and 37 non-tuberculous subjects were compared with results of skin tests performed in the previous year. There was a high correlation between skin test responses and TLP responses to PPD which was statistically significant. The sensitivity, specificity and the predictive value of a positive test were also similar for the skin test and TLP test. These findings suggest that the TLP test is as effective in screening for M. tuberculosis infection as tuberculin skin testing. Future research leading to further simplification of the TLP method may lead to it replacing intradermal skin testing.     

Role of in vitro and in vivo tests of hypersensitivity in beryllium workers.

The value of the beryllium macrophage migration inhibition (Be MIF) and Mantoux tests in the diagnosis of chronic beryllium disease and in the detection of hypersensitivity in healthy beryllium workers is demonstrated. In the absence of steroid treatment the Be MIF test is positive in chronic beryllium disease patients. Seven of 50 (14%) helathy beryllium workers were Be MIF positive, while all the control subjects, normal and sarcoidosis patients, were negative. Healthy beryllium workers tend to be more often Mantoux negative than a comparable group of non-exposed workers, and although not conclusive this finding correlates with a positive Be MIF test. Although the detection of hypersensitivity is not diagnostic of disease, the Be MIF test can be used as an additional method for monitoring the health of beryllium workers. The full significance of our results should be assessed by a long-term study.