Phenylhydrazine-induced leukocytosis in the rat.

Rats were injected with phenylhydrazine (PHZ) for periods of up to 6 months, during which time a marked leukocytosis was induced. The highest leukocyte counts occurred within 4-5 days following injection. An initial injection of 4 mg/100 gm body weight evoked a mean total leukocyte count of 129 X 10(3) cells/microliter. Successive weekly injections of 2 mg/100 gm resulted in a mean total leukocyte count of 70 X 10(3) cells/microliter compared to a mean total leukocyte count of 12.5 X 10(3) cells/microliter in saline-injected rats. Lymphocytes and monocytes accounted for approximately 75% of the total cell counts in both the PHZ-treated and control rats. The presence of increased numbers of mononuclear cells was confirmed by Percoll gradient separation and by phase-contrast microscopy. Although a leukocytosis was evident when using the automated Coulter electronic cell counter, it was not discernible when blood samples were counted manually in a hemocytometer by light microscopy. Histological examination of the thymus, lymph nodes, and spleen of the PHZ-treated rats indicated that lymphocytes and monocytes were mobilized from these sites. Lymphocyte depletion was evident, and germinal centers were found in all these lymphoid organs, indicating that PHZ induced a lymphopoietic response. A possible autoimmune etiology for PHZ-induced red blood cell destruction is discussed.

Phenylhydrazine is a mitogen and activator of lymphoid cells.

Rats were administered a single injection of phenylhydrazine (PHZ) which induced an hemolytic anemia that reached maximal levels two to four days following injection. This was accompanied by a leukocytosis which was most pronounced four to six days after injection; lymphocytes and monocytes accounted for 75 percent to 80 percent of the leukocyte count, respectively. All peripheral blood cell values, including the red cell count and hematocrit, returned to their pre-injection levels by the 11th post-injection day. Analysis by flow cytometry of peripheral blood mononuclear cells (PBMC) isolated from PHZ-treated rats by Ficoll-Hypaque gradient separation showed a marked increase in the B cell population of the peripheral blood. This was also seen in cultures of PBMC obtained from untreated rats following incubation with PHZ. Cultures of PBMC obtained from rats four to five days after PHZ injection which were incubated with pokewood mitogen (PWM) or phytohemagglutinin (PHA) showed significant increases in blastogenesis as indicated by [3H] thymidine incorporation when compared to cultures of PBMC obtained from untreated rats incubated with these mitogens. Incubation of cultures of PBMC obtained from untreated rats with PHZ significantly increased blastogenesis in cultures of five day duration. Atypical and blastic lymphoid cells were evident in cytosmears of PBMC isolated from PHZ-treated rats and also in sections of PBMC pellets studied using the transmission electron microscope. Serum of the PHZ-treated rats contained elevated immunoglobulin titers as measured by radial immunodiffusion. The results show that PHZ stimulates lymphoid cell blastogenesis and can sensitize circulating lymphoid cells to PHA and PWM indicating that PHZ is capable of stimulating the immune system of the rat.

Dexamethasone suppresses the generation of phenylhydrazine-induced anemia in the rat.

The immunoactive steroid dexamethasone (DXM) was administered to rats injected with a dose of phenylhydrazine (PHZ) known to induce anemia. PHZ treatment alone resulted in a hemolytic anemia that was most pronounced on Days 1-7 after injection. This anemia was accompanied by a leukocytosis that was greatest on Days 2-7 following PHZ treatment. Lymphocytes accounted for greater than 75% of this incremental increase. In contrast, rats treated with PHZ as well as with DXM displayed erythrocyte counts and hematocrits within the normal range. Although the reticulocyte counts of these rats were higher than those of controls, they were significantly lower than those of animals receiving PHZ alone. In addition, DXM suppressed the leukocytosis and splenomegaly resulting from PHZ administration and inhibited the rise in plasma IgG titers induced by PHZ exposure. DXM also altered the ratio of peripheral blood T and B lymphocytes of PHZ-treated rats. DXM suppression of PHZ-induced anemia is further confirmation that this anemia is associated with immune activation.

Hemostatic alterations associated with phenylhydrazine-induced anemia in the rat.

Phenylhydrazine (PHZ) is a hemolytic agent which has been used in the treatment of polycythemia vera. Recent studies performed in our laboratory have indicated that the PHZ-induced anemia is immuno-hemolytic in etiology, and a prolonged bleeding time was present in some of the rats chronically treated with PHZ. The nature of this bleeding tendency was explored in the present experiment. PHZ was administered to rats once a week for a six week period. During this time, the animals were monitored for prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen concentration, and individual coagulation factor levels as well as routine plasma chemistries and blood cell counts. In addition, radioimmunoassays (RIA) for prostacyclin, a platelet aggregation inhibitor, and prostaglandin (PG) E2 were performed. PHZ-treated animals displayed a significant elevation in both PT and APTT when compared with saline injected controls, although plasma fibrinogen levels were not appreciably altered. Further tests revealed a PHZ-induced decrease in prothrombin and factor V levels. In addition, a significant increase in plasma serum glutamate oxaloacetate transaminase (SGOT), lactate dehydrogenase (LDH), and alkaline phosphatase levels was observed as well as a diminution in cholesterol and triglycerides following PHZ administration. PHZ treatment also induced an elevation in prostacyclin levels and transient thrombocytopenia. These findings indicate that several factors may contribute to the prolonged bleeding time in PHZ-treated rats including a drug induced thrombocytopenia possibly associated with enhanced synthesis of autologous immunoglobulin G (IgG) against the senescent red cell antigen, and diminished synthesis of vitamin K-dependent coagulation factors which may be mediated by reduced vitamin K uptake by the hypo-cholesterolemic subjects.

Evidence for a hepatic-renal antagonism in the production of hepatic erythropoietin in a phenylhydrazine-induced compensated hemolytic state in the rat.

A compensated hemolytic state was induced in rats by injection of phenylhydrazine (PHZ) over a 6-week period. The liver and kidney were perfused to determine the levels and time of appearance of a hepatic erythropoietic factor (HEF) which induces the production of hepatic erythropoietin (Ep) and its antagonist, a renal inhibitory factor (RIF). Erythropoietin assays on the perfusates have been previously reported. The amount of HEF in perfusates recovered from the livers of the PHZ-treated rats was significantly higher during the 4th to 5th weeks of treatment, coinciding with the time of increased liver production of Ep. During the 6th week of PHZ treatment, the titer of RIF in perfusates recovered from the kidney was markedly increased and the HEF titer was decreased to near the control level, suggesting inhibitory action of RIF on HEF synthesis and/or effect. These findings indicate that an HEF/RIF control mechanism regulates hepatic Ep production in this compensated hemolytic condition.

Immune activation is associated with phenylhydrazine-induced anemia in the rat.

Long-term phenylhydrazine (PHZ) treatment caused pronounced anemia and a concomitant increase in the numbers of circulating leukocytes in Long-Evans rats. The leukocytosis was caused mainly by an elevation in mononuclear cells, most notably in the lymphocyte population. PHZ has been reported to cause the direct lysis of erythrocytes by nonimmune mechanisms. However, recent reports indicate that PHZ can cross-link red cell band 3 protein (senescent antigen), resulting in the binding of autologous immunoglobulin G (IgG). Recognition of this complex by macrophage Fc receptor mechanisms triggers rapid erythrophagocytosis-in the spleen and possibly the liver as well. In our study, analysis of the blood, bone marrow, and spleen cells of long-term (1 to 6 weeks) PHZ-treated rats was performed by using flow cytometry. Total serum IgG levels were determined by radial immunodiffusion, and antibodies reactive with red cells that were sensitized to PHZ either in vivo or in vitro were titered by using the indirect Coombs' method. Serum prostaglandin E2 titers also were determined at different time intervals after PHZ administration. The results indicate that PHZ induces an increase in circulating antibody and prostaglandin E2 titers that correlates with the onset of anemia and that the serum of PHZ-treated rats can induce anemia in normal recipients after passive transfer. Cytofluorographic studies revealed a marked increase in the B-cell population of the peripheral blood and spleen and an altered ratio T-helper to T-suppressor cells at certain time intervals after PHZ injection. The results indicate that in addition to inducing senescence-like alterations in erythrocyte membrane proteins, PHZ stimulates the production of the autologous IgG that recognizes these sites and promotes lymphoid blastogenesis, most notably in the B-cell lineage.