Dr Keith Zucker, Children's Hospital Central California, Department of Laboratory Medicine (SC-12), 9300 Valley Children's Place, Madera, CA 93638-8762, USA E-mail: firstname.lastname@example.org
Strokes due to transmural vasculitis associated with coccidioidal meningitis result in significant morbidity and mortality. The immunological and inflammatory processes responsible are poorly understood. To determine the inflammatory mediators, i.e. cytokines, chemokines, iNOS, matrix metalloproteinase-9 (MMP-9), that possibly contribute to vasculitis, temporal mRNA expression in brain basilar artery samples and MMP-9 protein in the CSF of male NZW rabbits infected intracisternally with 6·5 × 104 arthroconidia of Coccidioides immitis were assessed. Five infected and 3 sham-injected rabbits at each time point were euthanized 4, 9, 14 and 20 days post infection. All infected rabbits had neurological abnormalities and severe vasculitis in the basilar arteries on days 9–20. In basilar arteries of infected animals versus controls, mRNAs encoding for IL-6, iNOS, IFN-γ, IL-2, MCP-1, IL-1β, IL-10, TNF-α, CCR-1, MMP-9, TGF-β, as well as MMP-9 protein in CSF, were found to be significantly up-regulated. Thus, this study identified inflammatory mediators associated with CNS vasculitis and meningitis due to C. immitis infection. Assessment of the individual contribution of each mediator to vasculitis may offer novel approaches to the treatment of coccidioidal CNS infection. This study also provides unique methodology for immunology studies in a rabbit model.
Infarcts in the central nervous system (CNS) parenchyma are common complications of coccidioidal meningitis in humans [1–3] and in animal models [4–6] and can result in significant morbidity and mortality. The underlying immunological mechanisms responsible are poorly understood but molecules known to be associated with immune response in systemic mycoses, especially those associated with Th-1 responses, are probably involved [1,7]. Several pro-inflammatory cytokines, chemokines, matrix metalloproteinases (MMPs) and molecules associated with nitric oxide production are up-regulated in models of viral and bacterial meningitis as well as other pathological processes affecting the CNS [8–12]. Furthermore, activated MMPs, particularly MMP-9, have been implicated in the pathogenesis of ischaemic brain injury associated with vasculitis in bacterial meningitis [13–16]. The pathogenic mechanisms leading to vasculitis, vasospasm and thrombosis of vessel lumens are not fully understood. In humans with CNS vasculitis associated with bacterial meningitis, compression of vessels, subintimal infiltration of inflammatory cells and loss of autoregulation appear to lead to neuronal injury and infarction are observed [17,18].
To identify pro-inflammatory cytokines, chemokines, nitric oxide derived from inducible nitric oxide synthetase (iNOS) and MMP-9 (termed inflammation-mediating molecules, IMMs) that might contribute to the development of vasculitis in a rabbit model of coccidioidal meningitis, we evaluated the temporal production of the mRNA of these IMMs in brain basilar artery (BA) samples of Coccidioides immitis (CI)-infected male New Zealand White rabbits. The use of the rabbit model allowed the isolation of these relatively large vessels for this analysis. It was previously demonstrated that the brain BA develops arteritis following intracisternal administration of 5·0–5·4 × 104 arthroconidia [4,6]. To isolate the brain BAs, we removed as much of the leptomeningeal inflammatory exudate on their outer surfaces as possible using careful dissection and irrigation. We then used reverse-transcription PCR-amplification to test for IMM mRNA production in BA samples at defined time points after infection. We also evaluated the temporal production of MMP-9 protein in the CSF over the same 20-day period using zymography. The findings indicate potential involvement of numerous IMMs in the pathogenesis of BA vasculitis in CI meningitis.
Materials and methods
Use of research animals
The animal studies presented in this manuscript were conducted according to the guidelines of the Institutional Animal Care and Use Committee of the California Institute for Medical Research.
Non-immunosuppressed New Zealand White rabbits (Myrtle's Rabbitry, Thompson Station, TN, USA) were inoculated with 6·5 × 104 arthroconidia (infected) or saline (sham infection control) administered intracisternally on day 0 as described previously . Five groups of animals were established according to day of euthanasia after inoculation. Group 1 consisted of 5 control animals euthanized on day 0. Groups 2, 3, 4 and 5 consisted of 3 control animals and 5 infected animals euthanized on days 4, 9, 14 and 20, respectively. Rabbits were assessed daily for fever, weight and neurological abnormalities, including paresis, gait disturbance and abnormal mobility and posture .
At the experimental completion day for each group, rabbits were euthanized via intravenous injection of a concentrated pentobarbital solution as described previously . Prior to euthanasia, and while under a surgical plane of inhalation anaesthesia, CSF was collected from each animal and evaluated for WBC count, protein, lactic acid and MMP-9 concentration by zymography, as previously described [6,19]. After euthanasia, brain and proximal spinal cord were harvested and processed for histological assessment and quantitative fungal cultures, as previously described .
Brain basilar arteries were removed and washed immediately after necropsy. Tissues were divided and one half of each sample was placed into 10% formalin for histopathological evaluation. The other half was quick-frozen on dry ice and stored at −80 °C until it was processed for RNA extraction and purification as described below.
Due to the lack of reagents or published protocols available to perform immunology studies in the rabbit, all RT-PCR tests in this study were developed in-house using published rabbit gene sequences and Oligo 5·0 primer design software (Molecular Biology Insights, Inc., Cascade, CO, USA). Table 1 shows the primer sequences, annealing temperatures and PCR product sizes for each IMM mRNA tested. The specificity of each amplicon was verified by direct sequencing (data not shown).
Table 1. RT-PCR Primers used for IMM mRNA Assessment.
Sequence (5′– 3′)
Annealing temp (°C)
PCR product size (base pairs)
TCA CCA TCT TCC AGG AGC GA
CAC AAT GCC GAA GTG GTC GT
CTG CTT TGT GCG GAG TG
CAC CCG AAC ACC AGG AT
GCA GGG TAG GGG GTA TGG A
ACA GGG CTT GGC TTT GGA
TGC TGG ATT TAC AGG TGC TTT TGA
GGT ATT TCC CCC ATG AGA GTT TTT G
ATC TTG GGT TCT TAC GGC TGT T
TTC ACT TAC TGC TTT ACG CTG GAC
GGC TCA GAA TCA GAC CTC AG
GCT CCA CAT TGC AGA GAA GA
TCC AGC TGC GCA TCT CCT GC
CTT CTC CTT GCA CAA AAC TC
GCT CCT GGT GGT GGC TAC
GGG TGG CTT CTT CAT TCA AA
CTA TGT TGC CTG GTC TTC CTG
CTG CCT TGC TCT TGT TTT CAC
ATC GTC ACC AGC ATC GTC A
CAG CAG GGG CAG AAC AAG
CCA GCC AGA TGC CGT GAA T
AGA TCC CCT TGG CCA GTT TG
CGG CAG CTG TAC ATT GAC TTC C
GCG CAC GAT CAT GTT GGA C
GAC TAC CTG CAC CGC AAC A
CAT GTA GCC GCC GTC ACT
Total RNA was extracted from BA samples using mechanical homogenization of thawed tissue and purified with the mini-RNA extraction kit of Qiagen, Inc (Valencia, CA, USA). Purified RNA was stored as single use (2 µl) aliquots at −80 °C until RT-PCR assays were performed.
RT-PCR reactions were performed on all rabbits’ mRNA at the same time for each individual IMM tested. Assays were in a total volume of 25 µl using the One-Step RT-PCR kit of Qiagen and 0·10 µM of each IMM-specific primer set (forward and reverse primers). Thirty cycles of amplification were performed on all primer sets, except the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control set (22 cycles) in a PE Biosystems GeneAmp 9700 thermocycler. Cycle parameters for all assays were 50 °C for 30 min (RT step), 95 °C for 15 min, 30 (or 22) cycles of 95 °C for 30 s, specific annealing temperature for 30 s and 72 °C for 10 s and the last cycle was followed by 10 min at 72 °C. Under these conditions, we were able to reproducibly demonstrate a linear correlation between amplicons produced and cycle number as well as with increasing amounts of RNA added (data not shown).
Assessment of MMP-9 in CSF
MMP-9 in the CSF was quantified by electrophoretic gelatin zymography as described previously .
The overall normality of the RT-PCR data for each group was assessed using the Shapiro-Wilk test . For those differences between control and infected groups for each day postinfection, the Kruskal–Wallis nonparametric test was utilized for continuous factors and the χ2 test for categorical data (i.e. paresis and gait performance). The Spearman rank-correlation coefficient was used for the correlations. P-values ≤ 0·05 were considered significant.
Clinical and neurological abnormalities
All infected rabbits showed intermittent fever and weight loss starting on day 1 postinfection. In addition, starting on day 9 postinfection, infected rabbits showed increased paresis, gait disturbance, posturing and decreased mobility as compared to control animals.
Representative histopathological findings for normal rabbit basilar arteries and arteries with mild and severe meningitis are shown in Fig. 1. Brains and spinal cords were histologically normal in six controls. Mild nonspecific findings, i.e. parenchymal ischaemia or minimal meningitis without granulomas or evidence of fungal infection and attributable to the normal saline injections were present in brain or spinal cord sections of five of the controls (two from day 14 postinfection and one each from the other time points). Eight of nine basilar artery samples from controls that were analysed separately were entirely normal (Fig. 1a). Minimal meningitis was present in the adventitia of one of the control samples from Day 14. Mild or moderate meningitis was present in the brain, spinal cord and separate basilar artery samples of five infected rabbits on Day 4 postinfection (Fig. 1b). All 15 of the infected rabbits sampled on days 9, 14 and 20 had severe granulomatous meningitis and arteritis in brain and spinal cord samples and arteritis in separately analysed basilar artery segments. In these segments, there were variable patterns, i.e. either focal fibrinoid necrosis associated with adherent adventitial inflammation or complete circumferential fibrinoid necrosis associated with more marked adventitial mononuclear cell accumulations. Neutrophils and karyorrhectic cells were also seen in some of the vessel walls (Fig. 1c). None of these samples included adjacent coccidioidal organisms. Because of the variability and the small sizes of the samples, no temporal progression of inflammatory cell subset infiltration patterns could be identified.
Spinal fluid characteristics of control and infected rabbits
Figure 2 shows the results of assays performed on CSF collected at necropsy. The total WBC count (Fig. 2a) shows a significant increase (P < 0·05) in the infected rabbits starting at day 4 postinjection and continuing to day 20, with the highest numbers of WBC noted on day 14. None of the control animals showed an elevated WBC count in the CSF. The levels of both protein (Fig. 2b) and lactate (Fig. 2c) were significantly elevated (P < 0·05 and P < 0·03 for protein and lactate, respectively) in the infected animals as compared to controls on days 9 and 20 postinfection. Protein levels on day 20 remained significantly (P < 0·03) higher in the infected group, although at a much lower overall level as compared to days 9 and 14. Both protein and lactate analyses revealed peak levels at day 14 postinfection.
Fungal cultures in brain and spinal cord tissues
The results of quantitative tissue fungal cultures for C. immitis are shown in Fig. 2d. Both brain and spinal cord tissues from infected animals grew C. immitis on all days postinfection, with peak numbers of CFU noted on day 14 for both tissue sources. No C. immitis was recovered from the brains or spinal cords of control rabbits at any day postinfection.
Temporal assessment of mRNA for IMMs in brain basilar arteries
Figure 3 shows an example of an ethidium bromide-stained agarose gel after separation of the RT-PCR products for interferon-γ and the GAPDH internal control. The relative level of each IMM RT-PCR product was calculated as a fraction of the constitutively expressed GAPDH control RT-PCR product after densitometric analysis. The reagent blank (water) RT-PCR displayed no PCR products, as expected.
Temporal analysis of the mRNA levels for 12 IMMs in brain BA samples of all rabbit groups is shown in Fig. 4. Many of the IMM mRNAs show an up-regulation in the control groups, likely as a result of the sham infection protocol alone as observed in a previous study in the same model . With the exception of PDGF-β, which shows no difference in mRNA levels between control and infected animals at any time, all IMM mRNAs from infected rabbits display significantly higher levels than the corresponding control animals on at least one time point postinfection. The most significantly up-regulated IMM mRNA was IL-6, which displayed higher mRNA levels in infected rabbits at all days postinfection. Levels of iNOS and IFN-γ mRNAs were significantly elevated in infected animals on days 9, 14 and 20 postinfection. All remaining IMMs display elevated mRNA levels at only a single day postinfection as compared to controls. Significant IMM up-regulation in brain BA samples (mRNA) or CSF (MMP-9) is summarized in Table 2.
Table 2. Summary of IMM mRNA or protein up-regulation in the brain basilar artery or CSF.
Time post-infection (days)
+ P < 0·05/day compared to controls using Kruskal–Wallis nonparametric test.
iNOS, IFN-γ mRNA
IL-2, MCP-1 mRNA
IL-1β, IL-10 mRNA
TNF-α, CCR-1, MMP-9 mRNA
CSF MMP-9 Protein
MMP-9 mRNA in brain basilar arteries and MMP-9 concentrations in CSF
Brain BA MMP-9 mRNA levels and CSF concentrations of MMP-9 as measured by zymography are shown in Fig. 5. Control animals displayed relatively low levels of both MMP-9 mRNA and MMP-9 protein up to day 14. In contrast, infected rabbits showed a marked increase in MMP-9 mRNA starting at day 4 postinfection and continuing to day 14, and in MMP-9 protein starting at day 4 postinfection and continuing to day 20. MMP-9 protein levels in infected animals were significantly (P < 0·05) higher than controls on days 4, 9 and 14. Although day 20 MMP-9 protein levels remained higher in infected animals than in controls, they failed to reach statistical significance due to an increase in MMP-9 protein levels in the control group on this day. MMP-9 mRNA levels in brain BA generally paralleled MMP-9 protein levels in the CSF, but significantly (P < 0·05) higher levels of MMP-9 mRNA in infected animals versus controls was only observed on day 9. The kinetics of MMP-9 protein shown in Fig. 5 is distinct from that of the CSF total protein levels shown in Fig. 2b. It is therefore unlikely that the observed increase in MMP-9 protein in this study is simply related to the increase in total protein levels.
Comparison of the MMP-9 mRNA data in the BA samples and CSF MMP-9 in individual rabbits demonstrated a high degree of correlation (r = 0·78, P < 0·001) between the two assays (data not shown).
Several IMM mRNAs were up-regulated in BA samples from rabbits with CNS coccidioidomycosis. This study was designed to assess the temporal profile of IMM mRNA expression over three weeks of infection. Such studies  demonstrate the utility of RT-PCR in mapping the immune response in fungal CNS infections, and enable comparisons with other pathogens. Because of the relatively low numbers of animals examined at individual time points, the power of the statistical analysis at each time point was lessened and it is possible that biologically significant up-regulation may have been underestimated. Furthermore, due to the inclusion of adherent fibrinous inflammatory exudate on the surface of the BA in the mRNA expression analysis, the data generated may represent a composite of signals from both the wall of the artery and from the fibrinous exudate on the surface. Thus, both pathomorphologic correlates of vasculitis, the vessel and the adjacent inflammation, were included in the mRNA analysis. It should also be noted that although several IMM mRNAs (notably IL-6, iNOS and interferon-γ) were increased on more days than other IMM mRNAs (IL-1β, IL-2, TNF- α), it does not necessarily follow that they contribute more to vasculitis development. Nevertheless, the present results indicate the likely involvement of specific IMMs in the pathogenesis of vasculitis in this model over the course of the infection.
We found that iNOS mRNA was dramatically up-regulated over the course of the infection. Nitric oxide is a free radical that is constitutively expressed throughout the CSF under normal physiological conditions. When produced in high quantity via stimulation of iNOS in inflammatory states, it exhibits both potentially deleterious effects due to direct cytotoxicity and proinflammatory action and potential beneficial effects due to prevention of ischaemia by its vasodilatatory effect [22–25]. The nitric oxide metabolite peroxynitrate can directly damage DNA within the CNS via tyrosine nitration, resulting in lipid peroxidation associated with increased intracerebral pressure, BBB disruption and increased CSF leucocyte extravasation .
Evidence for a beneficial role of NO at the level of the cerebral vasculature during meningitis is provided by studies in rodent models of bacterial meningitis, where both the lack of endothelial nitric oxide synthetase and inhibition of the iNOS significantly aggravated the disease and increased the extent of neuronal injury in the brain, respectively [26,27]. These results suggest that NO produced in the vasculature has a protective effect against ischaemia in vasculitis by counteracting the decline in cerebral blood flow under the influence of vasoconstrictive factors. Thus, attempts to down-regulate NO production during meningitis are potentially dangerous, despite evidence that NO contributes to some of the potentially harmful changes such as CSF inflammation, brain oedema and intracranial pressure.
Messenger RNA encoding the pro-inflammatory interleukins IL-1β, IL-2 and IL-6 were up-regulated on different days of infection. These interleukins are also up-regulated in CNS trauma, ischaemia, atherosclerosis, lupus and infections in both humans and animals [28–32]. Their effects include enhancement of endothelial cell adhesion molecule expression, thereby facilitating WBC trafficking into the CNS  and induction of other pro-inflammatory IMMs, including iNOS, IL-8, IL-6, MCP-1, TNF-α and IFN-γ[28–30]. In particular, IL-2 appears to be important in the development of Th-1 immune responses that are critical to controlling fungal infections [33,34]. Transgenic mice that over-express IL-6 have brain perivasculitis . CSF IL-1β levels are closely correlated with clinical symptoms and WBC counts in patients with C. immitis meningitis .
TNF-α, the mRNA of which was significantly up-regulated on day 5 postinfection, is another proinflammatory cytokine produced in response to ischaemic injury and infection. It also up-regulates endothelial cell adhesion molecules  and when directly administered into the CSF, elicits perivascular oedema, increased leucocyte adhesion and extravasation and vascular damage . It has previously been shown in a rat model of pneumococcal meningitis that TNF-α modulates the expression and regulation of MMPs. In return, MMPs and related metalloproteinases can act as sheddases or convertases as they transform membrane-bound cytokines, cytokine receptors and adhesion molecules to their soluble forms .
IFN-γ mRNA progressively increased over the course of the infection. This Th1-associated cytokine also enhances adhesion molecule expression  and is crucial in immune responses to C. immitis infection  and other fungal infections . IFN-γ also stimulates other IMMs including IL-12, MCP-1, RANTES and TNF-α[28,38] and may contribute to infiltration of inflammatory cells into the brain . Interferon-γ is a key cytokine in giant cell arteritis  but its specific role in C. immitis-associated vasculitis is presently unclear.
The mRNA encoding TGF-β was also up-regulated in our study on day 4 postinfection and is a multifunctional cytokine shown to be both stimulatory and anti-inflammatory under various conditions [28,41,42]. Weyand et al.  have shown TGF-β to be a proinflammatory effector molecule related to its ability to induce expression of adhesion molecules and affect chemotaxis of mononuclear cells in temporal arteritis.
Previously, elevated levels of MMP-9 protein were demonstrated over the course of experimental C. immitis meningitis . We studied MMP-9 protein in CSF to correlate with our mRNA studies, and confirm the earlier results in the CSF and demonstrate up-regulation of MMP-9 mRNA in the BAs. MMPs appear to play a central role in the development of brain injury in meningitis. MMP-9 protein is up-regulated in experimental pneumococcal meningitis and it has also been implicated in BBB disruption leading to brain oedema and facilitation of inflammatory cell extravasion and is a potential risk factor for the development of CNS parenchymal damage [13–15]. We observed a significant correlation between the temporal expression of MMP-9 protein in the CSF and MMP-9 mRNA in the brain BA (Fig. 5). It may be possible to monitor CSF levels in patients and be forewarned of an impending vasculitis-induced stroke when a significant elevation from baseline is observed. This would allow for pre-emptive preventative measures when these treatments become available. We also observed slight elevations at different time points of mRNA for the chemokine MCP-1 and the chemokine receptor CCR-1, suggesting possible roles for these molecules in the vasculitis.
With the possible exception of the Th-2 cytokine IL-10 (the mRNA of which was up-regulated on day 14 only) and TGF-β (the mRNA of which was slightly up-regulated on day 4), all of the IMM mRNAs discussed above could contribute to the complex molecular cascades that result in the development of vasculitis associated with C. immitis meningitis. We do not observe direct invasion of the BA walls by the fungus and we have not observed deposition of complement or immune complexes in the vessel walls (unpublished data). Therefore, the vasculitis appears to be secondary to the proximity of organisms and a florid inflammatory response in the subarachnoid space, rather than requiring direct invasion of the organisms into the wall. The IMM mRNAs within the BA walls and immediately surrounding leptomeninges likely promote the transmural inflammatory cell infiltration and fibrinoid necrosis of arterial walls characteristic of vasculitis.
In summary, this study represents a significant advance in knowledge with regards to inflammatory markers in the lagomorph. To our knowledge, this is the only model of a consistently reproducible CNS vasculitis induced by infection. The present findings represent an insight into which IMM mRNAs may contribute to CNS vasculitis associated with experimental C. immitis infection. Moreover, the methodology and reagent specifications used in this study importantly enhance the capabilities of investigators to perform any future immunologic studies in rabbits, as there has been a paucity of resources for this species. The results could facilitate future research efforts using gene knock-out mice , as transgenic animals are available in this model, allowing determination of which IMM may be critical for vasculitis development. Although multiple IMMs likely participate in the pathogenesis of vasculitis, it may be possible to inhibit specific vasculitis-associated IMMs, e.g. using blocking agents or antibodies, without inducing broader immunosuppression. This targeted approach to specific immune inhibition may lead to new treatments of vasculitis associated with C. immitis meningitis.
This work was supported by Community Outreach Funds; granting agency Kaweah Delta Health Care District, Visalia, California 93291. Additional funding was granted by the Swiss National Science Foundation (632–066057); the Bank of Stockton, Stockton, California; The Valley Foundation, Los Gatos, California; and Children's Hospital Central California, Madera, California.