Quantitation of microbicidal activity of mononuclear phagocytes: an in vitro technique.NN Rege, SA Dahanukar
Dept of Pharmacology, Seth GS Medical College and KEM Hospital, Parel, Bombay, Maharashtra.
An in vitro assay technique was set up to determine the phagocytic and microbicidal activity of a monocyte-macrophage cell line using Candida species as test organisms. The norms were determined for the activity of peritoneal macrophages of rats (24.69 +/- 2.6% phagocytosis and 35.4 +/- 5.22% ICK) and human (27.89 +/- 3.63% phagocytosis and 50.91 +/- 6.3% ICK). The assay technique was used to test the degree of activation of macrophages induced by metronidazole, Tinospora cordifolia and Asparaqus racemousus and to compare their effects with a standard immunomodulator muramyl-dipeptide. All the three test agents increased the phagocytic and killing capacity of macrophages in a dose dependent manner upto a certain dose, beyond which either these activities were found to have plateaued or decreased. The optimal doses for MDP, Metronidazole, Asparagus racemosus and Tinospora cordifolia were found to be 100 micrograms, 300 mg/kg, 200 mg/kg and 100 mg/kg respectively. Patients with cirrhosis were screened for defects in monocyte function. The depressed monocyte function (20.58 +/- 5% phago and 41.24 +/- 12.19% ICK; P < 0.05) was observed indicating a compromised host defense. The utility of this candidicidal assay in experimental and clinical studies is discussed.
Keywords: Adult, Animal, Candida, physiology,Human, Macrophages, Peritoneal, drug effects,physiology,Metronidazole, pharmacology,Monocytes, physiology,Phagocytosis, drug effects,Plant Extracts, pharmacology,Rats, Rats, Sprague-Dawley,
Many techniques are available to assess the killing activity of the monocyte-macrophage system. These involve incubation of microbes with either monocytes or macrophages. The numbers of organisms killed are determined either by studying the oxpen dependent phase of phagocytosis and killing,,,, quantitation of released lysosomal enzymes and inhibition of labelled nucleotide uptake by ingested organisms using radioautography or directly by colony counting. All of them however pose a few inherent problems viz. necessity of sophisticated instruments, need of costly chemicals and supervision by expert. In addition, sluggish phagocytosis by monocyte-macrophages necessitates the use of antibiotics to eliminate the extracellular organisms, which themselves may effect the intracellular microbes as well as interfere with assay system. During the search for a simpler rt reliable technique the differential staining procedure appeared suitable. This assay is based on the principle of Giemsa staining characteristics of live and killed Candida organisms. The present paper describes the methodology, inclusive of modifications carried out while setting up the candidicidal assay for assessment of microbicidal activity of macrophages obtained from rat peritoneal cavity and monocytes derived from human blood. It also presents the data for the range of normal values in rats and humans. Applicability of this assay in experimental and clinical studies is also demonstrated.
The work was carried out in two phases: Phase A: Animal study. The study was conducted on Sprague-Dawley rats of either sex weighing between 100-120 gms.
Part 1: Candidicidal assay was set up which involves following steps:
i) Preparation of macrophage suspension: Peritoneal exudate was collected from the peritoneal cavity of anaesthetised rats. The fluid was transferred to a sterile siliconized test tube and centrifuged at 500 rpm for 10 minutes. After removing the supernatant, the pellet was resuspended in 1 ml of cold distilled water along with 1 ml of minimum essential medium (MEM; Hi-media) to destroy the neutrophils. This suspension was centrifuged again for 10 min, the supernatant was removed and the pellet was resuspended in 2 ml of MEM. Total and differential counts of the peritoneal exudate cells were carried out. Recovery of macrophages was approximately 85-90%. Viability of cells was determined using a dye exclusion technique and was found to be more than 96%. The final concentration was adjusted to 10 cells per ml.
2) Preparation of yeast cell suspension: Candida pseudotropicalis NCIM 3522 (supplied by NCL, Pune) was selected as the test organism. This strain was grown in the medium for growth of the yeast phase (MGYP). This medium is composed of 0.3% malt extract, 1 % glucose, 0.3% yeast extract and 0.5% peptone and has a pH 6.4 - 6.8. After incubation in this medium for 24 hrs at room temperature (32?C - 34?C), the organisms entered the yeast phase. The organisms were washed twice in 0.9% saline. The yeast characteristics of candida and their viability were checked while counting the organisms in the haemocytometer. For this purpose, 0.5 ml of 0.3% trypan blue and 0.1 % eosin (3:1) were added to 0.5 ml of the washed culture, mixed well for 10 min and the hemocytometer chamber was filled with this mixture. The yeast cells looked as single spherical cells or attached doublets; the filamentous forms were absent and 98% of yeast cells were viable, as shown by their ability to exclude the dye. The concentration was then adjusted to 10 yeast cells/ml.
3) Assay system: The assay system consisted of 0.1 ml of C. pseudotropicalis suspension, 0.3 ml of macro phage suspension, 0.3 ml of MEM and 0.1 ml of pooled serum obtained from normal volunteers with blood group AB. These quantities were selected to facilitate optimal pelleting of organisms with macrophages. The tubes containing the assay system were incubated at 37?C in a carbon dioxide environment for one and a half hour. Smears were prepared using the cytocentrifuge, stained with Giemsa and examined by light microscopy under oil-immersion.
Two hundred macrophages were scanned. The cells with ingested candidae were counted. This was expressed as percentage phagocytosis. While counting these macrophages with phagocytosed candidae, a record of all intracellular forms of candida was maintained. Usually 3 forms were observed, (1) yeast cells with homogenous blue cytoplasm; (2) yeast cells with partially or completely decolourised cytoplasm (ghost cells) and (3) filamentous forms which consist of blue staining yeast cell with one (rarely two) bud, denoting germination within the macrophage. With the given incubation length, the filamentous forms constituted less than 8% of total intracellular forms. The "ghost cells" were considered as the killed and digested organisms by macrophages. The intracellular killing capacity (ICK) was expressed as the number of ghost cells present per 100 phagocytosed candidae.
To compare the results obtained with this technique, another method i.e. methylene blue dye exclusion tests was used. Intracellular killing of peritoneal macrophages obtained from 12 rats was determined using both the techniques.
Part II: After setting up the technique, effects of different dosages of various agents on macrophage functions were evaluated. For this purpose, a known immunomodulator - murarnyl dipeptide (MDP) was chosen as a standard along with three test agents viz. metronidazole, Asparagus racemosus and Tinospora cordifolia, the last two are indigenous plant products. Roots of Asparagus racemosus and, stems of Tinospora cordifolia in the form of a decoction in water were used.
Rats (n=120) were divided into 4 groups and received either of the agents. Each group was further subdivided depending on the dosages selected. [Table - 1] illustrates the dosages, route of administration and duration of therapy. At the end of the treatment period, rats were sacrificed for collection of peritoneal macrophages. Candidicidal assay was performed as mentioned earlier.
Phase B: Human study. Heparinized venous blood was collected from 50 healthy adult volunteers (age ranging from 25-35 years). Monocytes were isolated by buoyant density centrifugation in Ficoll Hypaque as previously described in detail. Candidicidal assay was performed using Candida albicans as test organisms.
Screening of monocyte activity was carried out in 16 patients diagnosed as cirrhosis of liver with portal hypertension. The same assay technique was used.
Phase A: Animal Study. The macrophages obtained from fifty normal rats exhibited 24.69 + 2.6% phagocytosis. These macrophages killed 35.4 + 5.22% of ingested yeasts (See [Figure] on next page). The results were consistent when tested on different occasions.
When the present technique was compared with the methylene blue dye exclusion test, it was observed that there is no significant difference in the results obtained. The ICK of peritoneal macrophages determined by both the techniques is illustrated in [Table - 2].
MIDP produced a dose dependent increase in phagocytic activity of macrophages. The ICK was significantly increased with 50 and 100 ?g doses compared with basal values (p < 0.05); however, a higher dose (200 ug) failed to elicit any change in ICK [Figure:1].
Metronidazole given in increasing doses did not produce any significant increase in phagocytic activity. However, the candidicidal activity increased with higher doses and achieved peak with a dose of 300 mg/kg (50.66 + 3.07% and p < 0.05) and then plateaued. [Figure:2].
On the other hand, dose-dependent increase in both was observed. But again at these higher doses macrophage functions were significantly higher than the normal (p < 0.05).
percentage phagocytosis and killing activity of macrophages was observed in groups receiving Asparaqus racemosus. The doses higher than 200 mg/kg, however, produced decline in these functions, particularly in killing capacity. Nevertheless, both the activities remained significantly more compared with normal values (p < 0.05) [Figure:2].
Tinospora cordifolia in doses ranging from 25 to 100 mg/kg also produced a significant dose dependent rise in both phagocytosis and candidicidal activity of macrophages [Figure:2]. Further increase in the dose (200 mg/kg) led to decrease in phagocytic activity, but no noteworthy change was observed in killing capacity. At a dose of 300 mg/kg decreased candidicidal activity
Phase 8: Human study. Monocytes isolated from normal volunteers had 27.89 t 3.63% phagocytosis. Intracellular killing capacity was 50.91 + 6.3%. The phagocytic (20.58 + 5%) and cidal (41.24 + 12.19%) activity of monocytes collected from cirrhotic patients was found to be significantly decreased (p < 0.05).
As evident from the methodology, the candidicidal assay is not only simple to perform, but is also rapid and less cumbersome as compared to the standard technique of colony counting. Our results prove that the number of degraded organisms 'Ghost Cells' correlate closely with the percentage of non-viable candida determined by methylene blue dye exclusion test. The main advantage of this assay system lies in direct and simultaneous assessment of phagocytic and intracellular killing capacities of monocyte - macrophage system.
Using this assay, range was established for phagocytosis and ICK of normal rats. Rats, the commonly available laboratory animals, are often selected for innumerable research experiments. Standardization of assay system with peritoneal macrophages of rats helped us in screening immunomodulators. Metronidazole and the other two indigenous agents (Asparagus racemosus and Tinospora cordifolia) have been found to increase phagocytic activity of peritoneal macrophages against Staphylococcus aureus in our previous studies,. With the help of candidicidal assay dose-response curves of all the 3 agents were studied and compared with that of a known immunomodulator - murarnyl dipeptide. We observed that though these drugs showed a dose dependent rise in phagocytic and intraceilular killing activities, beyond certain doses these activities either failed to rise or even showed a decline. The optimal dose for MDP appears to be 100 ?g, for metronidazole 300 mg/kg, for Asparaqus racemosus 200 mg/kg and Tinospora cordifolia 100 mg/kg. Thus the assay can be used to determine degree of activation of macrophages and to select appropriate dose for further immunological studies.
The function of the circulating monocytes, precursor of tissue macrophages, was also assessed, as they are the most readily available cells to monitor host defense status 17 and judge the defects at tissue level. To demonstrate this, we selected the patients with cirrhosis. Depression of Kupffer cell function (macrophage system of liver) is a well-recognized feature of cirrhosis,,. Monocyte functions (both phagocytosis and IcK) of these patients were studied using our candidicidal assay. Significant depression of these functions was found as compared to those of normal individuals. This illustrates that monocyte activity itself sheds light on the tissue macrophage status. Thus for screening purposes this assay can be employed routinely and appears to be an asset for the physician.
We are grateful for the valuable suggestions given by Dr (Mrs) UD Wagle, Reader in Microbiology, during standardization of this assay technique. We also acknowledge the technical help rendered by Miss Sanjeevani P Malagi and Miss Mala Kulkarni.
[Table - 1], [Table - 2]