Phagocytic and chemotactic function of human polymorphonuclear cells during tetanus infection.
Alexander et al used neutrophil antibacterial function, to assess susceptibility to infection in patients with burns and following transplantation. Alexander and Meakins used this test to show the importance of abnormalities of neutrophilic function in relation to genesis of opportunistic infection in man.
With this background, we took up the study to determine neutrophil function in patients primarily suffering from a lethal disease like tetanus which is a major cause of death in India. Ensuing muscle rigidity and poor chest movements lead to the abolition of protective reflexes such as swallowing and coughing. This predisposes patients to pulmonary infections which threaten life maximally towards the end of the second week, when the severity of tetanus has just passed its peak.8 As the drugs11 and nutritional deficiences3,17 are known to affect host defences adversely, serum protein estimation and in vitro study of drug effect on phagocytosis were also carried out in our study.
Fifty adult cases of tetanus admitted in K.E.M. hospital were studied totally, 40 for phagocytosis; and ten for chemotaxis and phagocytosis both. There were 39 males and 11 females in the age group of 15-72 years. According to the established score system. the patients were subgrouped as 17 mild, 16 moderate and 17 severe cases.
Sampling interval: The first sample was collected on day 1 where day 0 was the day of admission. Subsequent sampling was done on alternate days. The frequency of further collections was determined by the severity of the infection (more often if severe), and the duration of hospitalisation (at wider intervals during recovery phase).
Tests performed: Phagocytosis was done by modification of Alexander et al's method, and chemotaxis by a modified method by Deitch et al as described below.
In phagocytosis, human neutrophils were incubated with Staphylococcus aureus (502 A) in presence of plasma of the same individual for 1 hour at 37°C. Thin smear of the above suspension was made after completion of the incubation period and stained by Giemsa stain. Slides were observed under oil immersion lens on a light microscope for % neutrophils containing intracellular organisms. Internalised bacteria were also counted in 50-100 neutrophils to determine the average number of bacteria/ cell.
Chemotaxis by agarose method, included punching wells in straight line in agarose in a plastic petri dish. The outer two wells were filled with chemoattractant (pooled human serum), the centre well with "minimal essential medium" (MEM) and the other two wells with neutrophil suspension. The plates were incubated for 3 hours and migration was observed under light microscope. The distance travelled was measured with a micrometer and labelled as chemotactic migration when it was in the direction of the chemoattractant and as random when it was towards the well containing MEM. The number of cells migrating in their direction were also measured.
Migration Index or chemotaxis was calculated as follows:
MI = (Cc-Cr) x (Dm-Dr)
Where Dm = Distance of maximum migration,
Dr = Distance of random migration,
Cc = PMN chemotaxis migrated,
Cr = PMN random migrated.
Drugs commonly used in the treatment of tetanus like benzyl penicllin, diazepam, tetanus antitoxin were added to the system of phagocytosis prior to incubation, to study the effect of the same on phagocytosis in vitro. The concentrations chosen were such as to stimulate the peak level achieved in serum after a therapeutic dosage. Two other concentrations, one above and one below, were also studied.
The drug was diluted either in distilled water or saline and a ten times concentrated solution was made. 0.1 ml of this was incubated as a part of the phagocytic system (total quantity 1 ml) with the organisms and leucocytes for 1 hour at 37°C. Percentage phagocytosis and average number of bacteria per cell were determined according to the procedure described above. A control without incorporating the drug was also run simultaneously for comparison.
In addition to these, total and differential count of leucocytes, total count of RBCs, ESR, hemoglobin content, and PCV were estimated routinely by conventional methods everytime, when blood samples were collected. Total protein estimation was done by the Biuret method and fractionation by electrophoretic method was done once or twice during the period of hospitalisation of the patient.
Control subjects: The control values for all the above tests were obtained by performing tests in exactly similar manner on blood samples obtained from healthy voluntary donors from blood bank. Age group was 19-45 years and sex was considered no bar. A total of 50, normals was studied for phagocytosis and 20 control values were obtained for chemotaxis. Protein pattern, haemoglobin, ESR, PCV, total count of RBCs and total and differential count of WBCs were done in all of the above controls.
[Table - 1] gives comparison of the basal values i.e. day 1 values of tetanus patients with that of control. It was observed that haemoglobin content of tetanus patients was less than that of control and ESR markedly raised. Albumin levels were not significantly different from the control values, but percentage phagocytosis and chemotaxis were very low, and this decrease was highly significant.
In [Table - 2] results of phagocytic function along with other parameters in 40 patients are tabulated. These patients were subgrouped as mild, moderate and severe depending upon the severity of the disease. Within each group, the observations of sub sequent days of hospitalisation were compared individually with the basal values, i.e. day 1 values. This comparison revealed no significant changes in any of the above groups in haemoglobin, ESR and phagocytosis form that of the basal values. Haemoglobin and phagocytosis remained low throughout hospitalisation and ESR remained high [Table - 1].
[Table - 3] shows the results of chemotaxis in 10 patients along with phagocytosis. The total number of patients being small, subgrouping as mild, moderate and severe was not done. Comparison of chemotaxis, during hospitalization with that of the basal chemotaxis showed an increase on day 7, whereas it remained comparable with the basal values on days 3, 5, 10 and 15. Only one value for 20,25 and 30 was available as only one patient out of 10 was hospitalised for such a long duration, comparison of this value with the basal showed an increase in chemotaxis.
The absolute count of PMN cells was 6298 ± 967 in the mild group, 7730 ± 997 in the moderate group and 9266 ± 967 in the severe group. Comparison of the mild with moderate and moderate with severe groups did not show any significant difference as studied by Kruskal and Wallis' test.
Incidence of tetanus is high in low socioeconomic group and this is likely to exhibit iron deficiency anemia and protein malnutrition. Hence haemoglobin and serum albumin were chosen to observe correlation if any of iron deficiency anemia and protein malnutrition with PMN function. Serum albumin in absence of other pathology can serve as an index of protein malnutrition. In our study the overall values of serum albumin could not explain predisposition to manifest infection. Similarly significantly low values of haemoglobin reflect on low socio-economic status of patients. The high values of ESR may be the result of fever the patients had on admission.
Phagocytosis on the day of admission was found to be half of the control values, independent of the severity of the disease; and it continued to remain low throughout the period of hospitalisation.
Decreased phagocyte function of PMN is reported by McCall et al during infection. Alexander and Meakins reported abnormality of neutrophil function in patients with septic burn wounds. They also reported that burn patients with normal neutrophil activity did not develop sepsis.
We observed that though the patients in severe group (14 out of 40) developed secondary infection, there was no difference in their phagocyte function as compared to that in mild or moderate group. These patients, however, were effectively treated by suitable antibiotics. In vitro study showed that benzyl penicillin, diazepam and tetanus antitoxin did not have any effect in phagocytosis, while ampicillin at concentration of 3 ug/ml significantly increased percentage phagocytosis. This effect was not seen in those patients who received ampicillin. One of the reasons could be failure of correlation of drug effects on phagocytosis, in vivo and in vitro. Many workers have reported such failure and our previous work on gentamicin also failed to correlate in vivo findings with in vitro observation., ,  Thus reduced phagocyte function could be attributed to either effect of treatment received or secondary infection.
Absolute count of patients in severe group was high as compared to the mild group, which indicates the higher rate of secondary infection and the severity of the disease in the former group.
Chemotaxis like phagocytosis, in the tetanus patients was significantly low on admission, and could be due to the effect on tetanus toxin. An increase in chemotaxis was seen on day 7 [Table - 3], whereas on days 3 and 5 it remained low, i.e. comparable to the basal value
An increase on day 7 could be related to the developing secondary infection. McCall et al have observed that the migration of toxic neutrophils after onset of infection is decreased. But contrary to this, Hill et al observed that toxic neutrophil during bacterial infections were hyperactive in unidirectional movement towards the chemotactic stimulus. Similar results were obtained by Hill et al in patients with pulmonary infection. The chemotactic function of neutrophils in this group, again decreased on days 10 and 15, and could be the result of drugs administered during the second week. Effect of drugs on chemotaxis in vitro was not studied, and thus the possibility of their effect on chemotaxis cannot be excluded.