Immunolocalisation of the kinin Bl receptor

CONTROL

3.7.3 Immunolocalisation of the kinin Bl receptor

Figure 3.46 Confocal images of kinin Bl receptor labeling on control spinal cord

Positive labeling for B 1 kinin receptor 1s shown on the neurons of the substantia gelatinosa of the spinal cord (A). There is an absence of labeling m the negative method control (B). The intensity of the immunofluorescence is shown on the colour strip: red to white: maximum, green to yellow: moderate, light blue = low and purple to blue = zero.

Figure 3.47 Immunovisualisation of the kinin Bl receptors on the neutrophil membrane

Immunolabelling of kinin BI receptors on circulating neutrophils of volunteers (A), and the circulating (B) and synovial fluid neutrophils (C) of RA patients. The intensity of the immunoflourescence is shown on the colour strip: red to white = maximum, green to yellow ⁼ moderate, light blue ⁼ low and purple to blue ⁼ zero.

The corresponding phase contrast images are shown in the second panel.

N -­

e ::t

N 0 �

•

• •

• •

• ^•

-mean

•

^-median

• •

- •

- ow.:...- _______________________ __,

circulating neutrophils circulating neutrophils

Volunteers RA Patients SF neutrophils RA Patients ( patients: n = 8; neutrophils: n =IO neutrophils per patient)

Figure 3.48 Intensity of immunolabelling of kinin Bl receptors on neutrophils

25

20 _lfiJl

15 10

5 0

D Circulating neutrophils healthy volunteers

■ Circulating neutrophils RA patients

■ Synovial Fluid neutropbils

Figure 3.49 Histogram of immunoreactive kinin Bl receptor on neutrophils showing mean values for the intensity of labeling of the kinin Bl receptor (in pixels x 10²/µm²⁾ on the neutrophil membrane

Although there was a clear increase in the intensity of labeling of the kinin B 1 receptor on the SF neutrophils from RA patients ( n⁼8), when compared to the circulating neutrophils from healthy volunteers (n⁼8), the mean values did not reach significance (Kruskal Wallis;

p > 0.05). However, a significant increase in B 1 receptor labeling was observed on both the circulating and SF neutrophils of the RA patients (n⁼80) when compared to circulating neutrophils of the healthy volunteers (n⁼80) (1 Way ANOV A, p < 0.01 and < 0.05 respectively). Details of the intensity of labeling for each of the subjects are shown in Fig.

3.48 and Table 3.12

Table 3.12 Intensity of immunolabelling for the kinio Bl receptor

No (i) Circulating neutrophils (ii) Circulating neutrophils healthy volunteers RA patient

1 7 12

2 ^{4 29.2}

3 2 3.6

4 2 2

5 21 25.9

12 14.5

7 14 18.9

8 3 9.8

Mean 8.1 14.5

Median 5.5 13.3

(iii) SF neutrophils RA patient

0.2 26 0.7 5.8 25.3 2.3 42.4 16.8 36 15.6 Kruskal- (i)vs. (ii) : > 0.05 (i)vs. (iii) : > 0.05 Wallis

Correlation with disease activity

There was a positive correlation between the immunoreactivity for the B 1 kinin receptor on the circulating neutrophils from the RA patients and the local activity index (r = 0.783;

p < 0.05) (Fig 3.50).

35 30 25 20 15

5

• •

0+---

0 10 20

Local activity index

Figure 3.50 Correlation between immunoreactive kinin Bl receptor on circulating oeutrophils from RA patients and the local activity index

3. 7.4 Immunolocalisation of the kinin B2 receptor

Kinin B2 receptors have been localized on the collecting ducts and tubules of normal kidneys (Naidoo et al. 1996a). During each labeling experiment, normal human kidney tissue was used as the positive controls. For the method control, loss of labeling following preadsorption with an excess of the rabbit anti-human B2 receptor antibody and replacement of the primary antibody with PBS was demonstrated (Fig. 3.51 ).

Figure 3.51 Confocal image of control human kidney

Positive labeling for the kinin B2 receptor is present on the collecting ducts and tubules of normal human kidney. The intensity of the immunoflourescence is shown on the colour strip:

red to white = maximum; green to yellow = moderate; light blue⁼ low and purple to blue = zero

The immunolabelling for the kinin B2 receptor on the circulating neutrophils from healthy volunteers and the circulating and SF neutrophils from RA patients is shown in Fig. 3.52.

The mean intensity for the kinin B2 receptor on the circulating neutrophils from the healthy volunteers was 4.6 pixels x 10²/µm² with a median of 5 pixels x 10²/µm² (Fig 3.53

& 3.54 and Table 3.13). For the circulating and SF neutrophils from the RA patients, the mean intensity for B2 receptor was 22.4 pixels x 10²/µm² and 16.1 pixels x 10²/µm² respectively, with corresponding medians of 15.5 pixels x 10²/µm² and 12 pixels x 10²/µm²_

Figure 3.52 Immunovisualisation of the kinin B2 receptor on the neutrophil membrane

Immunolabelling of kinin B2 receptor on circulating neutrophils of (A) volunteers, and (B) circulating neutrophils and (C) synovial fluid neutrophils of RA patients. The intensity of immunofluorescence is shown on the colour strip: red to white: maximum, green to yellow = moderate, light blue = low and purple to blue = zero. The corresponding phase contrast images are illustrated in the second panel.

80

_• - -

^mean

N

e

_::j 60

• •

N

...

40

•

·c

₂₀ ⁰

... ^• - _{.... • •} ^• ... - ^.. _• ^•

circulating neutrophils circulating neutrophils SF neutrophils Volunteers RA Patients RA Patients (patients: n⁼8; neutrophils: n⁼ 10 neutrophils per patient) Figure 3.53 Immunolabelling for kinin B2 receptor on neutrophils

25 22.4

Circulating neutrophils

-

MN

= ^e

^1.0 healthy volunteers

=

^:::1.

·- --

.5� _{� �}

....

_{lo'! 15}

Circulating neutrophils

> ^r,J

■

·- - = ·-

I,,, -CJ lo'! � C.

-

^� 10 RA patients

c:> _I,,,

=

^c:>

= e

^C. � 5

■

Synovial Fluid

e

^CJ

...

^{� I,,,} neutropbils

0

Figure 3.54 Histogram of immunoreactive kinin B2 receptors on neutrophils showing mean values for the intensity of labeling of the kinin B2 receptor (in pixels x 1«>2/µm²⁾ on the neutrophil membrane

Although there was a clear increase of the kinin 82 receptor on the circulating and SF neutrophils from the RA patients compared to circulating neutrophils from healthy volunteers, this was only significant for the circulating neutrophils from the RA patients (p

= 0.05). Details for each patient are shown in Fig. 3.53 and Table 3.13.

There was no correlation between the measures of disease activity and the intensity of labeling for the kinin 82 receptor on the neutrophils from the RA patients.

Table 3.13 Intensity of immuoolabelling for the kinin B2 receptor

No (i) Circulating neutrophils (ii) Circulating neutrophils healthy volunteers RA patients

1 3 7

2 8 14

3 1 17

4 5 10

5 5 2

6 2 18

7 5 63

8 8 48

Mean 4.6 22.4

Median 5 15.5

Kruskal- (i) vs. (ii) : < 0.05 Wallis

(iii) SF neutrophils RA patients

7 48

1 11

l3 1 37 13 16.1

(i) vs. (iii) : > 0.0512

0\ V,

Appendix 3.1

Lab.no

1 191

2 192

3 195

4 193

5 196

6 197

7 200

8 206

9 214

10 216

11 220

12 222

13 226

14 228

15 236

16 236

17 237

18 238

19 237

20 247

Clinical characteristics of the patients with rheumatoid arthritis

Natne Age (yrs) Sex Duration Nodules RF No . ACR criteria DMARD

SM 46 Female 4 Pos 5 No

DB 52 Female 6 Pos 6 3

CA 65 Female 14 Pos 6 5

SM 50 Male 20 present Pos 7 3

MP 53 Male 12 Pos 6 2

KN 40 Female 1.6 Pos 6 2

IB 47 Female 17 Pos 6 4

MN 56 Female 1.6 present Neg 6 No

MT 49 Female 6 Neg 5 2

CD 60 Female 14 Pos 6 2

MS 50 Female 15 Neg 5 No

MM 44 Female 5 Pos 4 2

RS 46 Female 15 Neg 5 2

LP 46 Female 20 Neg 4 3

MG 50 Female 2.5 Pos 5 No

EC 50 Female 15 Pos 6 2

KM 50 Female 10 Neg 5 1

PC 40 Female 11 :Jeg 5 2

GN 60 Female 7 Neg 4 2

ZE 45 Female 11 Pos 6 3

Prednisone dose Co-morbid diseases nil

5 mg HT

7.5 mg HT

10 mg

5 mg DM

7.5 mg 5mg nil

nil Hypothyroid

10 mg HT

nil

7.5 mg

HT

nil

3 mg HT

nil

nil HT

nil HT

4mg

nil HT

nil