Germinal center architecture disturbance during Plasmodium berghei ANKA infection in CBA mice

Abstract

Background: Immune responses to malaria blood stage infection are in general defective, with the need for long-term exposure to the parasite to achieve immunity, and with the development of immunopathology states such as cerebral malaria in many cases. One of the potential reasons for the difficulty in developing protective immunity is the poor development of memory responses. In this paper, the potential association of cellular reactivity in lymphoid organs (spleen, lymph nodes and Peyer's patches) with immunity and pathology was evaluated during Plasmodium berghei ANKA infection in CBA mice.

Methods: CBA mice were infected with 1 x 10(6) P. berghei ANKA-parasitized erythrocytes and killed on days 3, 6-8 and 10 of infection. The spleen, lymph nodes and Peyer's patches were collected, fixed in Carson's formalin, cut in 5 mum sections, mounted in glass slides, stained with Lennert's Giemsa and haematoxylin-eosin and analysed with bright-field microscopy.

Results: Early (day 3) strong activation of T cells in secondary lymphoid organs was observed and, on days 6-8 of infection, there was overwhelming activation of B cells, with loss of conventional germinal center architecture, intense centroblast activation, proliferation and apoptosis but little differentiation to centrocytes. In the spleen, the marginal zone disappeared and the limits between the disorganized germinal center and the red pulp were blurred. Intense plasmacytogenesis was observed in the T cell zone.

Conclusion: The observed alterations, especially the germinal center architecture disturbance (GCAD) with poor centrocyte differentiation, suggest that B cell responses during P. berghei ANKA infection in mice are defective, with potential impact on B cell memory responses.

Figure 1

Course of parasitaemia (mean ± 2 standard deviations of 3 separate experiments) in CBA mice inoculated with 1 × 10⁶ P. berghei ANKA-parasitized erythrocytes.

Figure 2

Spleen. A) Panoramic view of non-infected control mouse spleen. White and red pulps (Rp) with well-defined limits. Two B cell resting follicles (Flc) with surrounding thick marginal zones (Mz). Giemsa. B) T cell area (periarteriolar lymphoid sheath) of infected mouse (day 3). Immunoblasts (arrows). Giemsa. C) Infected mouse (day 3). Deposit of fibrinoid material (Fb) between T zone (Tz) and red pulp (Rp). Haematoxylin-eosin. D) Infected mouse (day 6). Disorganized germinal center with intense centroblast (Ct) activation and proliferation and apoptosis (white arrows), without centrocyte differentiation. Small lymphocytes (black arrows) permeating the disorganized germinal center. Giemsa. E) Panoramic view of infected mouse spleen (day 6). Disorganized germinal center (Gc), without definition of light and dark areas, absent marginal zone and blurred limits between white and red pulps (Rp). Haematoxylin-eosin. F) Infected mouse (day 6). Blurred limits in the border between disorganized germinal center (Gc) and red pulp (Rp), absent marginal zone, and malarial pigment in red pulp macrophages. Giemsa.

Figure 3

Spleen/Lymph nodes. A) Spleen T cell zone of infected mouse (day 6). Intense plasmacytogenesis with mitotic figures (arrows). Giemsa. B) Spleen T cell zone of infected mouse (day 10). Intense plasmacytogenesis at a more advanced stage, with plasma cell (arrow) differentiation. Giemsa. C) Lymph node of non-infected control mouse. Panoramic view of the paracortical (Pc) and follicular (Flc) areas. Giemsa. Arrows: high endothelial venules with trafficking lymphocytes. D) Detail of lymph node paracortical area of non-infected control mouse, showing interdigitating cells (arrows). Giemsa. E) Detail of lymph node paracortical area of infected mouse (day 3). Many immunoblasts (arrows). Giemsa. F) Lymph node of infected mouse (day 3). Immunoblasts (white arrows) and high endothelial venules (black arrows) showing trafficking lymphocytes. Giemsa.

Figure 4

Lymph nodes/Peyer's patches. A) lymph node of infected mouse (day 7). Disorganized germinal center (Gc), with small lymphocytes permeating an area of centroblastic proliferation, with several apoptotic bodies (white arrows). Paracortical area (Pc) with high endothelial venules (black arrows). Giemsa. B) lymph node of infected mouse (day 7). Medullary cords showing increased numbers of trafficking small lymphocytes and some immunoblasts (arrows). Giemsa. C) Panoramic view of Peyer patches of non-infected control mouse. Giemsa. D) Peyer patch of non-infected control mouse. Macrophages (arrows) permeating small lymphocytes. Haematoxylin-eosin. E) Panoramic view of Peyer patches of infected mouse (day 7), showing shrinkage of the patch. Giemsa. F) Peyer patch of infected mouse (day 7). Lymphocyte depletion and apoptosis foci (arrows). Haematoxylin-eosin.