|Year : 1980 | Volume
| Issue : 4 | Page : 207-209
Complement in aqueous humour and serum of rabbits and monkeys
Shiv Charan, Arvind Rat, LP Agarwal, VM Mahajan
Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India
V M Mahajan
Dr. Rajendra Prasad Centre for Opthhalmic Sciences, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Charan S, Rat A, Agarwal L P, Mahajan V M. Complement in aqueous humour and serum of rabbits and monkeys. Indian J Ophthalmol 1980;28:207-9
The complement (C') system is the primary humoral mediator of antigen-antibody reaction. The interaction between various C' components (CI to C9) leads to the generation of biological activity ranging from lysis of different kind of cells, neutralisation of bacteria and viruses to direct mediation of inflammatory processes through increase in capillary permeability (anaphylatoxin activity), directed migration (chemotaxis) and enhancement of phagocytic activity by polymorphonuclear leucocytes. Also, the level of serum C' in various conditions is known to alter significantly from those in health. The C' system plays a defensive role for the host against infection.
With the demonstration of the presence of both C' activation pathways (i.e. classical and alternative), in tears, a higher order of complexity to defense of ocular surface structure could now be appreciated. However, the role of complement-mediated immunological pathways in ocular tissue has not yet been evaluated. To best of our knowledge, there appears to be no recorded attempt to establish the C' sequence in aqueous humour in normal and disease conditions in laboratory animals.
| Materials and methods|| |
Aqueous humour from 8 rabbits and 4 monkeys was collected under/general anaesthesia (Pentobarbitone sodium 15 gm/lb. body weight), by inserting a 27 gauge needle into the anterior chamber at the limbus. Approximately, 0.1 ml of aqueous humour from both the eyes was pooled separately from each individual and stored at-70°C. Serum samples from these animals and three healthy persons were collected and stored at -70°C.
Three strains of newcastle disease virus (NDV) namely F (lentogenic), R2B (mesogenic) and a local isolate (velogenic) propagated into allantoic cavity of 9-11 days embryos, having titers between 10 8, ELD50/ml to 10 10
ELD501ml in lyophilized form were used after reconstitution in chilled phosphate buffered saline, immediately before use. With each strain of NDV, two eyes of a monkey were instilled (0.1 ml) after abrading the conjunctiva. Three rabbits were inoculated with F strain of NDV by instillation over intact and abraded conjunctiva and by inoculation directly into anterior chamber. Aqueous humour and serum were collected on 7th day of inoculation from these animals.
A microtitration procedure was adapted to assay total hemolytic C'. To sensitise sheep red blood cells (SRBC), equal volume of one percent SBRC and hemolysin diluted to contain 4 MHD50 (1:4000) per unit volume in veronal buffered saline (pH. 7.4) were mixed thoroughly and incubated for 30 min at 37°C in water bath. In 50 p.1 dilutions of aqueous humour and sera, thawed immediately before, an equal amount of sensitised SRBC were added. The plates were gently agitated and allowed to react for one hour at 37°C. Standard human sera and appropriate controls were included in the test. The dilutions causing 50% hernolysis of the sensitised SRBC (indicated by the size of the button) were considered the end points. The aqueous humor and serum samples were also tested for loss of C' activity by heat inactivity by the heat inactivation at 56o for 30 min.
| Observations|| |
We failed to detect hemolytic C' activity in aqueous humour via the classical pathway in any of the samples at a dilution above 1:2 out of 8 samples from rabbits and 4 samples from monkeys (Two aqueous humour from the two eyes of an animal have been pooled into one). The level of C' in aqueous humour at one week postinfection via intraocular routes into rabbits and monkeys remained undetectable by the procedure adopted. Even after introducing NDV directly into anterior chamber the level of C' was below measurable amount. Human sera (Standard) were active in this system up to 1:32 dilution. The C' titers in the monkey sera were also detectable up to 1:32 dilution, while in rabbit sera the C' activity was there in dilutions up to 1:4 only. [Table - 1]. There was no change in serum C' level after intraocular inoculation of NDV. Various controls included in the test system behaved properly. After heat inactivation at 56°C for 30 minutes, C' activity of human, monkey and rabbit sera disappeared totally.
| Discussion|| |
The level of hemolytic C' in aqueous humour from rabbits and monkeys appears to be low enough to be detected in a dilution of 1:2 or above by the method followed, even though the concentration of sensitized SRBC was decreased to twofold to enhance cell lysis at reduced concentration of C' activity. However in rabbit and monkey sera the C, activity could be observed upto a dilution of 4 and 32 respectively. In the standard human sera, the C' activity measured in the same assay system indicated activity at dilutions of 1:16 and 1:32 which were in agreement with Gilbert and associates. Also the disappearance of C' activity on heat inactivation at 56° C for 30 minutes supported that the hemolytic activity of the monkey and rabbit sera was due to C' only. The state of ocular infection with NDV did not appear to enhance the level of C' aqueous humor, even after introducing the virus directly into the anterior chamber.
In a maiden attempt by Yamamota and associates dilutions up to 1:4 of tear samples have been claimed to contain C' activity in classical pathway. On the other hand some of the components of C, (CI and C3) have been reported to be absent from tears as well as aqueous humor of human beings, both of which are essential for classifiable pathways of C' activation. The absence of C' in aqueous humour of monkeys and rabbits in our studies goes in agreement with the findings of Chandler and coworkers. The presence of C' in serum but its absence in aqueous humor means that C' is prevented being poured into aqueous humor at the capillary bed under the ciliary epithelium, as nearly 80% of the plasma. proteins are held back by the capillary wall and C' being a high molecular weight protein are retained into plasma so the possibility of some immune mechanism mediated through C' under operation in aqueous humour cannot be favourably supported. However, the consistently demonstration of C4 component of C' in aqueous humor may propose some function yet unknown,, other than the typical C' activity.
| Summary|| |
Twelve aqueous humour samples (8 from rabbits and 4 from monkeys) and 15 serum samples (8 from rabbits, 4 from monkeys and 3 from human) assayed for complement activity, revealed its activity in human and monkey sera up to a dilution 32 and in rabbit sera up to a dilution 4. In aqueous humor from either species complement activity could not be demonstrated even upon ocular infection with Newcastle disease virus indicating little role of complement mediated immune defense in aqueous humor of eye.
| References|| |
Fundenberg, H.H, Stites, D.P., Caldwell, J.L, and Wells, J.V., 1978, Basic and clinical immunology. Lange Medical Publications, Los Altos, California, 67.
Ruddy, S., Gigli, I and Austen, K.F., 1972, N. Engl. J. Med. 287, 489, 545, 592, 642.
Yamamoto, G.K. and Allansmith, M.R., 1979, Amer J. Ophthalmol, 88: 758.
Chandler, J.W., Leder, R., Kaufman, ME and Caldwell, J.R,, 1974, Invest. Ophthalmol., 13:151.
Fatt, 1., 1978, Physiology of the eye An introduction to the vegetative functions, Butterworth, London, 16.
[Table - 1]