Immunology 1978 35 933
Migration of human lymphocytes II.
VARIATION OF LYMPHOCYTE DISTRIBUTION
K. MORGAN & P. J. L. HOLT Department of Rheumatology, University of Manchester Medical School, Stopford Building, Oxford Road, Manchester
Received 30 September 1977; accepted for publication 23 March 1978
different donors' lymphocytes and between one donor's lymphocytes must be assessed, therefore this variation has been determined in this paper. Trypsin treatment reduces lymphocyte migration across the endothelium of post-capillary venules (Woodruff & Gesner, 1968; Woodruff, 1974) suggesting that the surface of the lymphocyte is important. Thus we have looked at the effect of trypsin on lymph node localization of human lymphocytes and of natural alteration of the lymphocyte as with leukaemic lymphocytes. To eliminate the possibility of host factors being involved we have looked at localization after treatment of the lymphocytes with formalin and sodium azide and after antilymphocyte globulin which could mimic either antibody-induced complement lysis, immune adherence
Summary. Variations in the distribution of 5lCrlabelled human lymphocytes from several donors and from one donor on several occasions have been determined after their injection into carbon-treated mice and reasons for the variation discussed. Natural alteration of the lymphocyte surface (leukaemic lymphocytes) or induced change (trypsin-treatment) reduced lymph node localisation as did treatment with formalin, sodium azide and antilymphocyte globulin. We have concluded that the presence of radioactivity in the lymph nodes is due to the presence of viable, metabolically active lymphocytes with normal surfaces capable of interacting with the endothelium of the post-capillary venules.
or opsonization.
INTRODUCTION
MATERIALS AND METHODS Animals 8-12 weeks old, female Manchester Closed Colony mice were used for all experiments.
After the injection of 51Cr-labelled human lymphocytes into carbon-treated mice the radioactivity recovered from the spleen and lymph nodes represents the presence of viable, human lymphocytes (Morgan & Holt, 1978). For changes in lymphocyte migration to be measured intrinsic variation between
Lymphocyte preparation and injection Lymphocyte preparation, labelling with [65Cr]sodium chromate and injection into mice were performed as described previously (Morgan & Holt, 1978). All mice in these experiments were carbon-treated.
Correspondence: Dr P. J. L. Holt, Department of Rheumatology, University of Manchester Medical School, Stopford Building, Oxford Road, Manchester M13 9PT. 0019-2805/78/1200-0933$02.00 ©) 1978 Blackwell Scientific Publications
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K. Morgan and P. J. L. Holt
Treatment oflymphocytes All treatments were performed after 5"Cr-labelling of lymphocytes and washing them twice in Medium 199. Control lymphocytes were incubated under the same conditions but without the active agent. After treatment the lymphocytes were washed twice in Medium 199, unless otherwise stated, before injection. Lymphocyte viability was estimated by eosin dye exclusion and was greater than 85 % in each control.
(1) Trypsin. Lymphocytes were incubated in Medium 199 (1-2 x 106/ml) which contained 0025 % w/v trypsin (Wellcome Labs) for 10 min at 37°. For overnight incubation in vitro at 370 in an atmosphere of 95 % air/5 % CO2 control and trypsintreated lymphocytes were resuspended at a concentiation of 0 5-1-0 x 106 cells/ml in Medium 199 with 20 % v/v foetal calf serum. For the final 30 min 30Ci of [5'Cr]-sodium chromate was added. The lymphocytes were washed four times in Medium 199 before injection into mice.
(2) Formalin. Formalin-treated lymphocytes were incubated for 30 min at 40 in phosphate buffered saline containing 1 % (v/v) formalin.
(1) Fluorescence. 3 x 106 lymphocytes were resuspended in four drops of FITC-labelled sheep antihuman whole immunoglobulin antisera (Wellcome-Reagents Limited) diluted 1:10 in 0 9% (w/v) sodium chloride solution and incubated at 40 for 30 min. The lymphocytes were then washed three times in Medium 199 and resuspended in a drop of glycerol saline (9:1). The proportion of lymphocytes with surface fluorescence was counted. (2) Sheep red blood cell rosetting. A 0X2 ml aliquot of lymphocytes (3 x 106/ml) was added to 0-2 ml of a 1% suspension of sheep red blood cells in Medium 199. The cells were centrifuged for 5-7 minutes at 35 g and incubated at 00 for 60 min. The cells were then gently resuspended and a drop carefully placed in a counting chamber. Lymphocytes with five or more sheep red blood cells adhering to them were counted as rosetted lymphocytes.
RESULTS
Tissue sampling and radioactive counting The mice were killed at various times after injection of lymphocytes and organs removed for radioactive counting as previously described (Morgan & Holt, 1978). Results were expressed as a percentage of the injected radioactivity recovered per whole organ or as a percentage of the injected radioactivity recovered per gram of tissue for lymph nodes and blood for ease of comparison.
Intrinsic variation in the distribution of normal lymphocytes Taking data from the previous experiments (Morgan & Holt, 1978) and adding further results Table 1 was compiled. These experiments were carried out over a period of 544 days and are unselected results on a 28 year old male. The main variation in lymphocyte recovery was seen in the lymph nodes and spleen. To confirm that the recovery of radioactivity in each tissue followed an approximately normal distribution, the results from these 37 mice were assumed to represent 37 independent measurements and a normal distribution fitted. For each tissue the chi-squared value was found to be not statistically significant. Therefore a normal distribution could be assumed and the Student's t test used for comparison of means even though the sample sizes were small. When lymphocytes from different individuals were used (Table 2) variation in recovery was seen in the lymph nodes and spleen but there was increased variation in the recovery from the liver and lungs as well.
Immunofluorescence and rosetting procedures The procedures were performed on the lymphocytes after labelling with [5"Cr]-chromate.
Lymphocytic leukaemia lymphocytes Lymphocytes from four lymphocytic leukaemia patients were used (Table 3).
(3) Anti-human lymphocyte globulin (ALG). Lymphocytes were incubated at 40 for 30 min with ALG (Searle Diagnostics Ltd) diluted 1/10 and 1/1000 with Medium 199.
(4) Sodium azide. Lymphocytes were incubated in Medium 199 containing sodium azide (0 3 M or 0-03 M) for 30 min at 370 in an atmosphere of 95 % air/5 % CO,.
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Variation of lymphocyte distributioi9
Table 1. Distribution in carbon-treated mice of radioactivity at 17-21 h after the injection of one individual's lymphocytes on different occasions Mean % of injected radioactivity recovered
Mean % of injected radioactivity recovered per whole organ
per gram of tissue
Days between tests
4 5 3 4 4 3 2 3 3 3
37 71 52 13 36 138 38 12 44
Lymph node
Blood
20-1 3-7 6-1 ±04 6-6±1-2 12-3 ±25 8-4 1-8 8-3± 30 10-3 0-2 59 ± 16 4-3 ±07 124±2-2 9-7 ±09 9-6 0-9
0- 3 0.10 03 ±004 05±0-07 0-4 ±007 04±0-04 04 ±0-06 03 ± 001 05 ±004 04±009 04±0-04 0-8 0A44 0 4 ±003
Bone
No. of mice
Liver
Lungs
Spleen
Kidney
marrow
H 11-7 1.1* 22-3 ±1-2
11-8±1-5
3-1 i 2-1 0-8 0-2 2*5± 1-
±2.3
1.5 ±07
17-3 ± 2-6 9-4± 2-3 13-1 2-4 10-6 ± 1-2 15-2 ±22 65 ±05 10-8 ± 1-9 9-6 ± 1 2 140± 1-2 13.1 3-2
2-3 ± 0 1 2-1 ±01 2-6±03 2 1 0-3 21 ±0-2 39 ±0-9 2-4 0-02 1-6 +0.1 1.9±0-2 2-2 ±03
79 ±2-0 11-7 0-8
3 9 ±03 2-4 0-1
49 ± 08 3-5 ±05 49±04 3-7 ±04 70 +1-3 3-3 ±06 6-8 +0-6 40 ±0-6 6-1 ±09 6-5 03 5 0 ±09 4-9 ±03
10.5 97 10-5 9-5 126 11-3
±1-2
1-4 ±06
0-9 + 1-4 ± 1-1 1-5
0.5 +01 1.4 ±0 3 05 ±0-2
9-1±0-5 10-7 2-0
103 3 Mean values for 37 mice
12-3
0-8
4*0±09 1-9±0-3 17 ±0-7 1-7 0-3
*Mean i standard error of the mean
Table 2. Distribution in carbon-treated mice of radioactivity at 17-21 h after the injection of lymphocytes of healthy individuals Mean % of injected radioactivity recovered per gram of tissue
Mean % of injected radioactivity recovered per whole organ
Lymphocytes Sex of Age group No. of donor of donor mice Liver injected SC (1) SC (2) JH (I) JH (2) EK AT RB RC PW JD JR
PH(1) PH(2) JG
F F F F F F F M M M M M M M
2 2 2 2 2 2 4 2 2 2 3 4 4 4
2 4 2 2 3 4 5 4 3 1 1 2 3 3
Lungs
17-2 ± 2-5 13 6 X04 8-6 i21 10-3 ±01 10.9 ±09 9-0 0-9 9-7 1.2 6-9 1-8 8-7 ±11 8-3 10-7 9-3 ±30 115 ±0-8 10.9 ±05
Age Group of Donor: 2
=
9 0 ± 3-0 0 9 ±03 1.9 ±06 15 ±0-6 1.9 ±07 4-1 ± 1-4 4-7 ±09 1-7 ±03 3-8 ±09 1-4 25 1-7 0-3 4-4 1-3 5-0 ±11
Spleen
13-8 11.9 11-4 11-2 13-9 19-2 15-7 8-4 10-3 9-8
9*0 9-9 10-6 16-3
20-30 years; 3
=
Kidney
± 13 2-3 ± 0 01 ±05 3 5 ±03 ±50 1-7 ±02 4-7 3-3 0-7 ±45 2-7 ±04 ± 31 2-3 0-1 ±19 1-7 0l1 ±25 1 1 0-2 3-4 1-8 ±01 2-4 2-8 0-2 2-4 0-4 i19 2-3 ±01 ±3-1 2-4 ±04
Bone marrow
54 ± 01 5 4 ± 0-8 5 2 ± 0-7 5-4 ± 1-3 5 2 +11 7-2 0-2 57 ±1-2 3-7 1-0 51 ±05 65 6-9 70 ±04 4-2 ±04 4-4 0-2
30-40 years; 4 = 40-50 years.
Lymph node
Blood
7-8 ± 35 0-3 ± 0-05 0 4 ± 0-06 0 3 ± 0-01 0 4 ± 0 05 0-6 ±021 15-4 ± 1-4 0 4 ± 003 7-0±16 0-8 +053 123 ±3-7 0 4 i004 9-3 2-0 0 7 ±007 7-9 03 4-8 05 3-6 ±09 0-2 +002 3-4±01 10±044 4-1 ±1-3 07 ±0-12
117-7 15 13-0 ±09 6-4 ±26 179 ±6-1
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K. Morgan and P. J. L. Holt
Table 3. Distribution in carbon-treated mice of radioactivity after the injection of lymphocytes from lymphocytic leukaemia patients Mean % of injected radioactivity recovered per whole organ
Injection A B
C D
Time (h) tissues No. of sampled mice 2-3 18-19 2-3 18-19 19-20 1 2 3 15-16 17-5-18-5
3 4 5 8 7 2 2 1 4 4
Liver 17-7 9-8 26-5 13-5 24.7 24-2 15-5 18-7 13-5 12-3
±27 ±05 ± 48
±2-2 ±47 0-8
±14 1-2 1-4
Lungs
±19
Spleen
16-5 ±10 26-4 +52 ± 1-8 18-7 ± 2-9 ±0-03 18-3 ±4-4 1-5± 05 30-6 4-4 14-4 3-3 3-5 1-4 15-6 1-2 3-1 ±11 19-8 6-8 1-7 0-3 8-9 ±09 1-0 ±0-2 9-6 ±2-0 10-3 05 6-5 0-2
±01
Patient A, untreated chronic lymphocytic leukaemia (CLL). Lymphocytes were obtained from a blood sample. When tested with FITC-labelled antihuman immunoglobulin antiserum 94% had surface fluorescence and 3 % rosetted with sheep erythrocytes. Normal healthy individuals had 30-50% of their lymphocytes forming rosettes and 15-20 % giving surface fluorescence by the methods used in this laboratory. Patient B, CLL. Lymphocytes were obtained from a blood sample, 95 % had surface fluorescence and 2 % rosetted with sheep erythrocytes.
Patient C, CLL. Lymphocytes were obtained from leukocytes separated by leukophoresis. 1 % rosetted with sheep erythrocytes. The surface fluorescence
was too weak to count. Patient D. Lymphocytes were obtained from leukocytes separated by leukophoresis. Most of the lymphocytes were non-dividing cells with internal immunoglobulin but little endoplasmic reticulum. The patient had no lymph node enlargement and had a diagnosis of possible prolymphocytic leukaemia. With all four patients lymph node localisation at 20 h post-injection was less than after the injection of normal lymphocytes. The reduced localiza-
Mean % of injected radioactivity recovered per gram of tissue
Kidney
Bone marrow
Lymph nodes
Blood
1-5 ±01 2-1 ±01 2-3 ± 0-3 2-2 ±0-3 2-1 ±0-2 1-4 ±01 1-7 ±0-2 2-4 1-7 0-2 2-1 ±03
1 9 ±03 3-8 +0-6 2-1 ± 03 2-5 ±03 1-6 0-2 25 ±04 3-6 0-6 56 4-2 0-5 39 0-4
07 ±01 0-8 +0-2 0-7 i 005 05 0-06 04 ±01 0-8 ±01 19 ±01 2-2 1-4 0-3 1-4 0-4
1-2 +0-2 04 +01 0 9 + 0.1 0-3 ±0-02 04 +01 1P8 0-2 2-6 0-2 NT 0-4 0-1 0-3 0-02
tion of lymphocytes in lymph nodes was also apparent in the first few hours after injection. The overall pattern for the other tissues sampled was similar to that found with normal lymphocytes. Effect of treatments on lymphocyte distribution In Table 4 the results are expressed as a percentage of the value found for that tissue when the control lymphocytes were injected. Thus less than 100% indicates a reduction in the migration of lymphocytes to a tissue. The mean value was used for each group of four or five mice. All treatments to lymphocytes reduced lymph node localization. The three treatments which altered the lymphocyte surface also decreased localization in the spleen. Tables 5 and 6 show the effect of trypsin treatment on localization of lymphocytes in more detail. The main reduction in localization after trypsination was found in the lymph nodes and was apparent 2-3 h after injection. By 20 h (Table 5) the lymph node localization was still decreased as compared with normal lymphocytes. The increase in splenic localization between 2-3 h and 20 h shown by normal lymphocytes was not seen with trypsin-treated lymphocytes. The characteristic reduction of radioactivity recovered from the liver and lungs with time occurred in both trypsin-treated and in control lymphocytes.
Variation of lymphocyte distribution
937
Table 4. Effect of lymphocyte treatments on the recovery of radioactivity from various tissues in carbon-treated mice at 20 h Treatment of lymphocytes before injection
Liver
Lungs
Spleen
Kidney
Trypsin
ND
ND
58 (0 05)
162 (0-05)
ND
17
ND
Formalin
32 (0-01)
ND
5 (0 002)
122 (0 05)
25 (0 05)
8 (0-01)
ND
Anti-lymphocyte
ND
ND
19 (0 025)
ND
34 (005)
1 (001)
ND
globulin 1/10 dilution 1/1 000 dilution
ND
ND
38 (0 05)
ND
ND
11 (0-01)
ND
ND
ND
ND
ND
ND
ND
164 (0 05)
ND
ND
ND
ND
20 (0 01) 57 (0 05)
Sodium Azide 0*3M 0*03M
Bone marrow Lymph nodes
Blood
ND
The localization of radioactivity is expressed as a percentage of the localization of the equivalent viable control lymphocytes in each experiment using the mean value for each group. Values shown only for tissues where differences between control and experimental group was statistically significantly different by Student's t test. P values are shown in paranethsis. ND = no difference. Three to five mice used in all groups.
Table 5. Effect of trypsin-treatment on recovery of lymphocytes-early and late distribution Mean % of injected radioactivity recovered per whole organ
Mean % of injected radioactivity recovered per gram of tissue
Lympho- Time (h) No. of tissues mice cytes injected sampled
Liver
Lungs
Spleen
Kidney
Bone marrow
Lymph nodes
Blood
0-8 + 0-3
1-0 ± 0 1
KM4
19-7
2-9
9 3 ± 1-3
4-5 ± 1-3
2-8 ± 06
1-7 ± 03
4
31-6
1-4
7-7 ± 1-6
8-9 ± 1-3
1-2
0l1
25±0-3
15-5
2-7
09±0 1
4
11 4 ± 1 8
09±03
75±07
3-6 ±01
3-7
0-8
09
0-2
0-2 ± 0-1
3
10-6 + 2-3
2-3 + 1-5
17-5 + 1-3
2-0 ± 02
4-2
0-6
16-5
1-0
0-4 ± 0-1
2-2-5 h
0-01
NS
NS
NS
NS
0-02
NS
18-5-19-5 h
NS
NS
0 001
0 001
NS
0 01
NS
trypsinized 2-2-5 KMcontrol 2-2-5 KMtrypsinized 18 5-19 5 KMcontrol 18-5-19-5 P values