In vivo ultrastructural analysis of the intimate relationship between polymorphonuclear leukocytes and the chlamydial developmental cycle

Abstract

We utilized a recently developed model of intracervical infection with Chlamydia muridarum in the mouse to elicit a relatively synchronous infection during the initial developmental cycle in order to examine at the ultrastructural level the development of both the chlamydial inclusion and the onset of the inflammatory response. At 18 h after infection, only a few elementary bodies attached to cells were visible, as were an occasional intracellular intermediate body and reticulate body. By 24 h, inclusions had 2 to 5 reticulate bodies and were beginning to fuse. A few polymorphonuclear leukocytes (PMNs) were already present in the epithelium in the vicinity of and directly adjacent to infected cells. By 30 h, the inclusions were larger and consisted solely of reticulate bodies, but by 36 to 42 h, they contained intermediate bodies and elementary bodies as well. Many PMNs were adjacent to or actually inside infected cells. Chlamydiae appeared to exit the cell either (i) through disintegration of the inclusion membrane and rupture of the cell, (ii) by dislodgement of the cell from the epithelium by PMNs, or (iii) by direct invasion of the infected cell by the PMNs. When PMNs were depleted, the number of released elementary bodies was significantly greater as determined both visually and by culture. Interestingly, depletion of PMNs revealed the presence of inclusions containing aberrant reticulate bodies, reminiscent of effects seen in vitro when chlamydiae are incubated with gamma interferon. In vivo evidence for the contact-dependent development hypothesis, a potential mechanism for triggering the conversion of reticulate bodies to elementary bodies, and for translocation of lipid droplets into the inclusion is also presented.

Fig. 1.

Initial stages of chlamydial infection at 18 h after mouse endocervical inoculation. (A) Note an intermediate body (arrow) within an endosomal vacuole. (B) Inclusion with a single RB (arrow). Scale bars: 500 nm.

Fig. 2.

Events occurring 24 h after infection. (A) Multiple early inclusions containing one to a few RBs. Some epithelial cells contain multiple inclusions. RBs are clearly dividing, and the proximity of inclusions suggests that the fusion process is under way (arrows). (B) Enlarged view of early, 24-h inclusions clearly showing multiple inclusions in a single cell. The RBs are generally larger at this time of the cycle in preparation for division than at later times. (C) PMNs (P) are seen directly in contact with an infected cell even though only a single RB (arrow) is present. Note the obvious pseudopodia of the PMNs extending toward and actually forming pockets in the infected cell. (D) Another view showing PMNs (P) in contact with an infected cell containing a single RB (arrow). Scale bars: 2 μm.

Fig. 3.

Events at 30 and 36 h after infection. (A) Inclusion with multiple RBs at 30 h after infection and PMNs (P) in contact with the infected epithelial host cell. (B) By 36 h after infection, the inclusions have increased dramatically in size and contain plentiful EBs, indicating that the inclusion is in its late stages. PMNs (P), again, abut the infected epithelia. (C) An infected cell, with PMNs (P) on either side (arrow), is becoming detached from the epithelium. Note that the inclusion membrane is no longer apparent and the chlamydiae appear to be free in the cytoplasm. The highly vacuolated nature and lack of microvilli of the infected cell suggest that the epithelial cell is dying. (D) Multiple large mature inclusions are seen. In particular, one infected cell (arrow) is being detached from the epithelium with a PMN (P) directly in the space beneath the cell, giving the appearance of “pushing” the cell off the epithelial surface; the PMN appears to have phagocytized numerous RBs. Nuclei (N) of the infected cells are visible. Scale bars: 2 μm (A and C), 10 μm (B and D).

Fig. 4.

PMNs enter into the infected host epithelial cell to make direct contact with chlamydiae. (A) At 42 h after infection, a PMN has gained access to and is completely inside the infected target cell. Note the extended pseudopodia, one directly in contact with the epithelial cell cytoplasm close to the inclusion. (B) Another view showing an invading intracellular PMN directly in contact with the chlamydial inclusion membrane. (C) The inclusion membrane is no longer apparent, and the intracellular PMN is in direct contact with chlamydiae. Note the projections from the PMNs touching the IBs or EBs (arrows). (D) A PMN (P) within a large space containing chlamydiae and cellular debris, including a cell nucleus (N), suggesting either that this epithelial cell was destroyed by the action of the PMN or that the PMN has entered the site following lysis of the cell. Scale bars: 2 μm (A and C), 10 μm (B and D).

Fig. 5.

Termination of the developmental cycle by breakdown of the inclusion membrane. (A) Host epithelial cell with multiple RBs and EBs distributed in the cytoplasm (arrow). The inclusion membrane is no longer obvious. (B) Similar micrographs showing a terminal infected cell with chlamydiae freely distributed in the cytoplasm. A PMN (P) is adjacent to the infected cell. (C) Terminal infected cell with chlamydiae distributed in the cytoplasm. The cell is in the process of being dislodged from the epithelial layer. Scale bars: 2 μm (A and C), 10 μm (B).

Fig. 6.

Inclusions at 42 h after infection. (A) Note that the majority of the RBs in this inclusion (arrow) are along the periphery of the inclusion adjacent to the inclusion membrane while the EBs are in the internal portion. (B) Again, the majority of the RBs are attached to the inclusion membrane. Note the multiple lipid droplets (arrows) in the infected cell. On the left side of the inclusion, lipid droplets are actually entering the inclusion, suggesting an active process of droplet translocation. Scale bars: 10 μm (A), 500 nm (B).

Fig. 7.

RBs attached to the inclusion membrane reflecting the contact-dependent hypothesis. (A) Enlarged replicative RB with extensive inclusion membrane contact, likely via the type 3 secretion injectisomes; the contact areas are almost always associated with closely apposed endoplasmic reticulum (arrow), from which the chlamydiae are likely scavenging nutrients. (B) Smaller RB with less extensive area of contact with the inclusion membrane. Scale bars: 500 nm.

Fig. 8.

In vivo depletion of PMNs by anti-PMN antibody results in increased number of IFU between 42 and 48 h after infection when quantified from cervicovaginal swabs or directly from cervical tissue. Asterisks indicate that the difference in IFU between 42 and 48 h was significantly different according to a one-tailed t test.

Fig. 9.

Inclusions containing aberrant RBs were observed in the cervix of a mouse depleted of PMNs by antibody treatment. (A) Note the large aberrant RBs (AB) as well as packets of miniature RBs (black arrow). Also, this inclusion is devoid of granular material representing glycogen accumulation (white arrow). (B) A large mature inclusion is in the lower left of the photomicrograph, while the upper right inclusion has both aberrant RBs (arrows) and normal size RBs as well as IBs and EBs. (C) Another view showing a normal inclusion (top center) and two inclusions with aberrant RBs (AB), packets of miniature reticulate bodies (arrow), and a lack of glycogen. Scale bars: 2 μm (A and C), 500 nm (B).