Int. Archs Allergy appl. Immun. 49: 782-788 (1975)
Leukocyte Migration Inhibition in Nickel Dermatitis1 A. M. M irza, M. G. P erera , C. A. M accia, O. G. D ziubynskyj and I. L. Bernstein Childrens’ Hospital and Division of Immunology (Allergy), Department of Internal Medicine of the General Hospital, University of Cincinnati Medical Center, Cincinnati, Ohio
Abstract. Leukocyte migration inhibitory factor assay was employed as an in vi tro diagnostic aid in nickel dermatitis, the second most common contact dermatitis in North America. 15 patch test-positive and 5 patch test-negative patients, all giv ing a past history suggestive of nickel dermatitis, were investigated. Significant inhi bition of leukocyte migration in both groups of patients was obtained only with nickel sulfate-albumin conjugate and not with unconjugated nickel sulfate. Specifici ty of this system was tested by utilizing an unrelated metallic albumin complex, and no inhibition was found. When patch testing is equivocal or contraindicated, this in vitro technique may be a practical alternative.
Several groups of investigators [5, 6] have demonstrated that cell mi gration inhibition could be employed as an in vitro correlate of delayed hypersensitivity. These methods involved the migration inhibition of guinea pig macrophages after these cells were incubated with antigen in presence of sensitized lymphocytes or soluble factors secreted by antigenstimulated lymphocytes (MIF). A direct MIF assay method [13] demon strated that migration of human peripheral leukocytes was inhibited in the presence of specific antigen. This technique has been applied as a para meter of delayed hypersensitivity induced by a variety of tuberculin-like antigens [14] and chemical contactants [9, 15]. A recent report by R ock lin [12] differentiated leukocyte inhibitory factor (LIF) from MIF.
Received: February 7, 1975.
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1 This investigation was supported in part by NIH Training Grant AM05509-08. Presented in part at the Annual Meeting FASEB, April 7-12, 1974, At lantic City, N.J.
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Allergic eczematous contact dermatitis can usually be diagnosed by patch testing, but occasionally chronic persistence of the lesion or the risk of increased sensitization may contraindicate such a procedure. The oc currence of false positive or negative patch tests is not infrequent. In such instances an in vitro method would obviously be advantageous in estab lishing the diagnosis. Since preliminary trials of LIF in clinical cases of hexavalent chromium [15] and neomycin sensitivity [9] were reported to be successful, it appeared worthwhile to investigate the diagnostic poten tial of LIF assay in other contact dermatitides. Thus, nickel dermatitis, the second most common type of contact dermatitis in North America [10], was chosen for further evaluation of this technique.
Material and Methods
2 To insure uniformity of cell density within capillary tubes, only those with packed cells of similar length (1.5-3 mm) were selected for the experimental proce dure.
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Two categories of patients were compared to a group of nonsensitive volunteers. Group I consisted of 15 patients with a past history compatible with nickel dermati tis and a positive patch test of a 5°/o nickel sulfate solution applied 48-72 h pre viously. There were 5 patients in group II with suggestive past history of contact dermatitis to nickel, but whose patch tests were completely negative. History and patch testing were negative in all 13 normal volunteers. Table I summarizes the types and amounts of metallic antigens used for both clinical patch testing and LIF assay. The patch test solution contained 50 mg/ml of nickel sulfate and for LIF, a limited dose response assay (25, 50, 100 «g/ml of nick el sulfate) was employed. Nickel albumin complex was prepared by incubating 40 mg of nickel sulfate with 2 g of bovine albumin according to the method of T hu lin and Z achariae [15]. After dialysis for 3 days, the dialysate contained 304 ug/ml of nickel sulfate complexed with albumin, equivalent to 1 mol of nickel sulfate per 1.4 mol of bovine albumin [11]. Aliquots of this complex (15.2, 30.4, 45.6 ,ug/ml) were used in the LIF procedure. Human albumin nickel complexes were also pre pared in a similar manner. To test the specificity of this system a potassium dichro mate albumin complex was prepared by the same method and the doses employed were 1.9, 3.9, and 4.9 ¿ig/ml, respectively. Peripheral leukocytes were obtained by sedimentation of 25 ml of blood in a sol ution containing 250 U heparin/ml and 6°/o dextran [2], After leukocytes were washed initially with 0.83% ammonium chloride and twice more with Hanks’ bal anced salt solution (HBSS), they were suspended in TC199 containing 10% calf ser um. This suspension was aspirated into capillary tubes which were plugged, centri fuged at 2,000 g, cut at cell fluid interfaces2 and mounted in tissue culture chambers filled with suspending medium. These chambers were then incubated either with or
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Table I. Types and amount of antigens LIF
Patch
NiSO.»
NiSCh-albumin complex
K2Cr2Û7 K2Cr207-albumin complex
wt/vol mg/ml
molarity1
wt/vol /ig/ml
molarity1
50
3.2 x 10-1
25 50 100
1.6 x 10-4 3.2 x 10-4 6.5 x 10-4
0.304
5 ND
2.0 x 10-3
1.7 x 10-2 ND
15.2 30.4 45.6 ND 1.9 3.9 4.9
9.8 x 10-5 2 x 10-4 2.9 x 10-4 ND 6.9 x 10-5 1.4 x 10-5 1.7 x IQ-5
ND = Not done. 1 Nickel and chromium molarity determined by an atomic absorption technique [11].
without antigens at 37 °C in a 5°/o C 02 incubator for 18-24 h [7], The migration areas were measured planimetrically and migration indices were calculated by the following formula: Mx (migration of cells in presence of antigen) __ Migration index = ------------------------------------ —---------- :----- -— x 100. Mo (migration of cells without antigens) All experiments were done in triplicate and a mean migration index was calculated. Because soluble antigen-antibody complexes may induce positive LIF responses [12], the effect of sera of nickel-sensitive subjects upon normal leukocyte migration was also investigated. 1 ml of serum was incubated with washed normal leukocytes (buffy coat) for 1 h at 37 °C in a 5°/o C 0 2 atmosphere before these cells were processed through the procedure described above.
LIF results in normal volunteers and sensitive patients tested with var ious concentrations of unconjugated nickel sulfate are shown in figure 1. It is evident that there are no differences between these patients at con centrations of nickel sulfate ranging from 25 to 50 ^g/ml. However, defi nite inhibition of migration occurs at lOO/tg/ml in both normal volun-
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Results
Leukocyte Migration Inhibition
785
MIGRATION INDEX, NiS04 140" n«4
120à?
100-
n -6
■ ■-
•*
§
■ ■
•
80-
n -1 0
*
••
• ••
n«10
n »7
■ •
CONTROL GROUP
I
■
•
• s
■
■
■ •
1
n«6
■
•
• I
60>
Cfc
■
$5
40-
20-
■ M-101 SE*
M*91 SE *12 NS
25 pg/ml
M -9 4 SE • 7
M *8 6 SE‘ 5 NS
50 pg/ml
M «61 S E *8
M*61 SE 4 2 NS
100 pg/ml
CONCENTRATION OF N iS 04
Fig. 1. Comparison of migration indices using unconjugated nickel sulfate in patient groups and normal subjects, n = Number of study subjects; M = mean; SE = standard error; NS = not significant by Student’s t test. Enclosed bars indicate 1 SD from the mean. ■ = Control; • = group I.
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teers and patients, suggesting that this concentration of nickel sulfate in hibits migration nonspecifically. Therefore, in experiments in which nick el albumin conjugates were utilized, it was essential that the final concen trations of nickel sulfate contained in these complexes were in the nontox ic range (table I). Figure 2 demonstrates LIF results at several concentra tion levels of nickel sulfate-albumin complexes in the two patient groups and age-matched normal volunteers. Even at the lowest concentration (15.2 /(g/ml of nickel sulfate-albumin complex) LIF results are significant in both groups of patients as compared to normal subjects. Furthermore, it is clear that significant differences in the LIF assay exist between pa tient groups and normal subjects at higher concentrations of the nickel sulfate-albumin conjugate (30.4 and 45.6 jUg/ml). More clear-cut results were obtained at the highest concentration, suggesting a dose-dependent effect. In parallel experiments an additional pulse of 25 /ig/ml of unconju gated nickel sulfate to various concentrations of the nickel sulfate-albu-
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786
MIGRATION INDEX, N iS04 -ALBUM IN COMPLEX 160 -1
140-
*
S
120
100 ■
s
-
80-
1 60-
*î*ï
40
20
P< 0 .0 0 2 M* 106 SE * 16
M * 86
SE *14
pcO.OOl
p< 0.01 M-82 SE*5
15.2pg/ml
M «9 6
SE *19
M«73 SE*13
30.4 pg/ml
p< 0.0001
p