Acute Bacterial Rhinosinusitis Causes Hyperresponsiveness to Histamine Challenge in Mice

ORIGINAL ARTICLE Acute Bacterial Rhinosinusitis Causes Hyperresponsiveness to Histamine Challenge in Mice

James J. Klemens, MD; Virat Kirtsreesakul, MD; Thongchai Luxameechanporn, MD; Robert M. Naclerio, MD

Objectives: To develop a physiologic test of nasal re- termine whether acute sinusitis causes nasal hyperresponsiveness in mice and to evaluate whether mice with sponsiveness to histamine exposure. acute bacterial sinusitis develop nasal hyperresponsiveness. Results: Nasal histamine challenge led to a reproducible, dose-dependent increase in sneezing and nose rubs. Design: Several experimental studies will be described. The response to histamine exposure was blocked by des- The first was a titration pilot study. The second was a loratadine (PՅ.05). Allergic mice had a significant in- randomized, placebo-controlled study. The remainder crease in responsiveness (PՅ.05) over baseline after ex- were before-and-after trials. posure to antigen. Mice with acute sinusitis had a sustained increase in responsiveness, although less se- Species: BALB/c or C57BL/6 mice. vere than after allergy, compared with baseline values that lasted 12 days after infection (PՅ.05). Interventions: For these experiments, we exposed mice to histamine intranasally, then counted the number of Conclusions: Nasal challenge with histamine is a physi- sneezes and nose rubs as the primary outcome measure ologic test of nasal responsiveness. The hyperrespon- of nasal responsiveness. First, we constructed a dose- siveness of allergic mice to histamine exposure parallels response curve. Second, we treated the mice with des- the response to nonspecific stimuli during the human al- loratadine, a histamine 1 receptor antagonist, prior to his- lergic reaction. In addition, we showed that acute bac- tamine exposure. Third, we challenged, with intranasal terial sinusitis causes nasal hyperresponsiveness in mice. histamine, mice made allergic using 2 techniques. Fourth, we infected mice with Streptococcus pneumoniae to de- Arch Otolaryngol Head Neck Surg. 2005;131:905-910

ASAL HYPERRESPONSIVE- ity. In allergic patients, an increase in the ness to irritants and to re- number of muscarinic receptors has been petitive allergen expo- described by some investigators,5,6 whereas sure is a well-established van Megen et al7 described not only a phenomenon associated decrease in the number of muscarinic re- with allergic rhinitis.1 In humans, hyper- ceptors, but also an increase in their N 6 responsiveness to allergens increases af- sensitivity. Ishibe et al described down- ter an antigen challenge (priming).2 Non- regulation of adrenergic receptors in al- specific hyperresponsiveness to irritants lergic patients. Local changes related to an Author Affiliations: such as histamine, methacholine, brady- increase in the numbers of eosinophils, Department of Surgery, Section kinin, and cold, dry air also increases af- neutrophils, basophils, and lymphocytes of Otolaryngology–Head and 1 Neck Surgery, The University of ter allergen challenge. These laboratory and their released mediators after anti- Chicago Hospitals, Chicago, Ill events correlate with the response of pa- gen exposure have been implicated in the (Drs Klemens, Kirtsreesakul, tients to cold weather and strong odors, development of nasal hyperresponsive- Luxameechanporn, and like that of gasoline. ness.8 Naclerio); Department of How allergic exposure causes nasal hy- When histamine contacts the nasal Otolaryngology, Prince of perresponsiveness is unknown.3 There are mucosa, it causes an increase in vascular Songkla University, Hat Yai, several proposed hypotheses for the un- permeability, dilation of the cavernous Songkla, Thailand derlying mechanism. Increased epithe- sinusoids, and neuronal stimulation.9 (Dr Kirtsreesakul); and lial permeability may allow easier access Increased nasal secretion is caused Department of 4 Otolaryngology–Head and Neck of stimuli to nerve endings. An increase primarily by nerve-mediated parasym- Surgery, Ramathibodi Hospital, in sensitivity or in the number of irritant pathetic glandular secretion and an Mahidol University, Bangkok, receptors, or a decrease in the number or increase in vascular leakage. Nasal con- Thailand responsiveness of sympathetic receptors, gestion is mediated primarily by pooling (Dr Luxameechanporn). may result in increased mucosal sensitiv- of blood in the vascular sinusoids. Nasal

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 itchiness and sneezing are thought to be secondary to NASAL CHALLENGE WITH HISTAMINE histamine’s actions on the trigeminal nerve.9-11 Clement et al12 showed that allergic patients have increased nasal Because mice are obligate nose breathers, small droplets of so- resistance after topical application of histamine, sug- lution were placed on the external nares of awake mice to be gesting that there is pooling of blood in the cavernous drawn into the nasal passages during inhalation. The nasal chal- sinusoids. However, nasal airway resistance is variable lenge with histamine consisted of intranasal application of 50 µL of various concentrations of histamine (Sigma-Aldrich, St in humans. There is considerable overlap between Louis, Mo) applied gradually over 2 minutes. In our initial ef- healthy and allergic patients in their response to hista- forts, we used 5 different concentrations: a isotonic sodium chlo- mine exposure; thus, it is not a reliable test of hyperre- ride (vehicle for dissolving histamine); 1-, 10-, 30-, and 100mM sponsiveness.3 Numata et al10 found that, although histamine; but, because of the stress on the mice, the chal- patients reported a lesser degree of congestive symp- lenge was changed to 3 exposures of 0.3-, 3.0-, and 30mM his- toms after antihistamine treatment, this did not corre- tamine. late with objective rhinomanometry. Gerth van Wijk et After each exposure, we observed the mice for 10 minutes al13 suggested that measurements of sneezing and secre- and counted the number of sneezes and nose-rubbing epi- tions were more accurate assessments of hyperrespon- sodes. Each count was performed by the same investigator siveness to histamine exposure. (J.J.K.) who was blinded to the treatment groups. From these data, we constructed dose-response curves. We investigated whether acute bacterial rhinosinusitis causes nasal hyperresponsiveness to histamine exposure in mice. Research on sinusitis in humans is lim- DESLORATADINE GAVAGE ited by access to the sinuses and the types of studies that ethically can be done. Therefore, we developed a mouse Mice were treated with 10 mg/kg of desloratadine by gavage in a methylcellulose vehicle 4 hours prior to nasal challenge with model of acute bacterial rhinosinusitis. histamine. The controls for this experiment were given an Little is known about sinusitis-induced hypersensi- equivalent volume of methylcellulose (Dow Chemical Co, Mid- 14 tivity in humans. Sampaio et al, in an abstract, re- land, Mich), the vehicle for desloratadine, by gavage. ported decreased ipsilateral nasal secretions after intranasal histamine challenge in a few patients with chronic ALLERGIC SENSITIZATION sinusitis compared with healthy control subjects and no difference in the contralateral secretory or the sneezing Two techniques were used. For the first technique, mice were response. Whether acute bacterial sinusitis causes dif- sensitized by intraperitoneal (IP) injection of 20 µg of ovalbu- ferences in nasal mucosal hyperresponsiveness has not min (Sigma-Aldrich) together with an aluminum hydroxide been studied. (Pierce Biotechnology, Inc, Rockford, Ill) adjuvant. Eight days To test the hypothesis that acute bacterial sinusitis after the first IP exposure, the mice received a second injec- causes hyperresponsiveness, we needed to develop a non- tion. Two days after the second injection, the animals were ex- specific test of nasal hyperresponsiveness in mice. posed to a 6% ovalbumin solution in 0.1M phosphate- Imamura and Kambara15 used substance P and hista- buffered saline (PBS) (Roche Diagnostics Corporation, mine applied intranasally to measure the sneeze re- Indianapolis, Ind) by intranasal inoculation daily for 5 days. 16 For the second technique, mice were injected with aluminum sponses in guinea pigs. Saito et al applied intranasal his- hydroxide and PBS, but no ovalbumin. These mice were then tamine to study the kinetics of hyperresponsiveness in exposed to either 50 µL of 6% ovalbumin or an equivalent vol- allergic mice. Thus, we chose histamine as a stimulus. ume of PBS daily for the duration of the experiment. We first established a dose-response curve to intranasal histamine exposure in healthy mice. We then dem- INDUCTION OF SINUSITIS onstrated that the response to nasal histamine challenge could be blocked by treatment with a histamine Soy broth was used for control animals and soy broth contain- 1–antihistamine, desloratadine (Schering=Plough, Ke- ing Streptococcus pneumoniae for experimental animals. The nilworth, NJ). Next, we histamine-challenged mice that American Tissue Culture Collection, Rockville, Md, strain of had been sensitized to ovalbumin by 2 different meth- S pneumoniae was obtained from the Clinical Microbiology Labo- ods to determine whether mice, like humans, became hy- ratories of the University of Chicago Hospitals. Fifty microli- perresponsive after allergen stimulation. Finally, we ap- ters of 109 colony-forming units/mL solution was adminis- plied the histamine challenge to our model of acute tered to each mouse over 5 minutes. bacterial sinusitis. NASAL CULTURE METHODS Animals were killed with 120 mg/kg of phenobarbital. As respiratory depression occurred, we disinfected the animal’s head, external nose, and oropharynx with a swab of 70% isopropyl MICE alcohol. We then lavaged the nasal and sinus cavities with 200 µL of sterile sodium chloride. We quantified the bacteria ob- We purchased 6-week-old, pathogen-free, BALB/c or C57BL/6 tained by nasal lavage by using serial 10-fold dilutions and spread mice from Jackson Laboratories (Bar Harbor, Me). They were the lavage fluid on blood agar plates. The plates were incu- maintained in the biohazard suite of the Carlson Animal Fa- bated for 24 hours, and then colony-forming units of S pneu- cility, Chicago, Ill, in cages with microisolator tops. The stud- moniae were counted. In prior experiments, there was an ex- ies were approved by the Animal Care and Use Committee of cellent correlation between nasal lavage correlations and cultures the University of Chicago. of sinus tissues.17

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10 4 8 3 6 2 4 No. of Sneezes per 10 min No. of Nose Rubs per 10 min 2 1 (n = 4) (n = 4) 0 0 0 1 10 30 100 0 1 10 30 100 Concentration, mM Concentration, mM

Figure 1. Mean±SEM of nose rubs after various concentrations of intranasal Figure 2. Mean±SEM of sneezes after various concentrations of intranasal histamine in mice. The number of nose rubs was significantly higher than histamine in healthy mice. The number of sneezes was significantly higher control vehicle for 10-, 30-, and 100mM histamine. than with control vehicle for 30 and 100mM-histamine.

STATISTICAL ANALYSIS Control Mice All calculations were done by use of Minitab software (Sigma Desloratadine-Treated Mice Breakthrough Technologies, San Marcos, Tex). We looked for A B 20 10 differences between groups and between treatments with analy- ∗P ≤.05 vs ∗ ∗P ≤.05 vs sis of variance; PՅ.05 was statistically significant. If statisti- Desloratadine Desloratadine ∗ Treatment Treatment cally significant, the Tukey test was applied. 8 15

6 RESULTS 10 4 Histamine causes a dose-dependent increase in sneezes 5 2

and nose rubs in nonallergic, uninfected mice. Five No. of Sneezes per 10 min No. of Nose Rubs per 10 min C57BL/6 mice were given intranasal histamine in 5 doses: (n = 4) (n = 4) 0 0 0 (isotonic sodium chloride), 1, 10, 30, and 100 mM. We 0.3 3.0 30 0.3 3.0 30 found a dose-dependent increase in the response to his- Concentration, mM Concentration, mM tamine exposure. There was a significant increase in nose rubs from the control vehicle for the 3 highest concen- Figure 3. Treatment with desloratadine 4 hours prior to intranasal histamine trations of nasal histamine challenge (PՅ.05), and the exposure significantly decreased the mean number of nose rubs (A) and sneezes (B) at 30mM histamine. number of nose rubs reached a plateau at 10mM (Figure 1). There was a significant increase in sneezes at the 2 highest doses of histamine (PՅ.05), which reached between the lowest doses of histamine (0.3- and 3.0mM) a plateau at 30mM (Figure 2). and the highest dose of histamine (30mM) for both sneez- We found that the mice did not tolerate more than 3 ex- ing and nose rubbing (PՅ.05). In the desloratadine treat- posures. One mouse died after the fourth dose challenge, ment group, there was no difference between the lowest and several of them seemed dazed after the fourth challenge, and the highest dose of histamine. The difference at the with rapid respiratory rates and absence of nose rubbing 30mM concentration between the desloratadine-treated and sneezes. When given a 30-minute rest period, these mice group and the control group was significant for both responded again with nose rubs and sneezing in the dose- sneezing and nose rubs (PՅ.05) (Figure 3). dependent manner presented in Figure 1 and Figure 2. Be- Allergic inflammation increases sensitivity to the na- cause the mice did not tolerate more than 3 exposures, we sal challenge with histamine. We hypothesized that mice decreased the number of nasal histamine challenges to 3, made allergic to ovalbumin would show hyperresponsive- with the highest concentration set at 30mM, where the pla- ness to histamine. For this experiment, we used 5 BALB/c teau of the sneezing dose-response curve began. mice, because these mice have a greater tendency than do

The response to histamine exposure is blocked by treat- C57BL/6 mice to develop TH2 or allergic responses. Base- ment with desloratadine. To demonstrate the specificity line histamine challenges were compared with challenges of the response, we hypothesized that the dose- performed after allergic sensitization. Nasal histamine chal- response curve generated by nasal histamine challenge lenge was performed prior to the sensitization process and would be reduced by treatment with a histamine 1 re- after IP sensitization, but prior to intranasal exposure to ceptor blocker. Five C57BL/6 mice were treated 4 hours antigen. A third histamine challenge was performed 6 days prior to histamine challenge with desloratadine (10 mg/ after intranasal antigen exposure, and the last nasal his- kg) by gavage, and 5 were treated with methylcellulose. tamine challenge was performed 1 week after the last in- For the control group, there was a significant difference tranasal antigen exposure.

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 A Baseline 1 Postexposure 45 ∗ P ≤.05 vs Baseline Values Baseline 2 1 wk After Postexposure 40 (n = 5) A B 35 ∗ ∗ 20 ∗P ≤.05 vs 40 ∗P ≤.05 vs ∗ 30 Baseline Value 35 Baseline Value ∗ ∗ 25 15 30 ∗ 20 ∗ 25 ∗ 15 10 20 10 15 No. of Nose Rubs per 30 min 5 5 10

0 No. of Sneezes per 10 min

No. of Nose Rubs per 10 min 5 B (n = 5) (n = 5) 0 0 120 ∗ P ≤.05 vs Baseline Values 0.3 3.0 30 0.3 3.0 30 (n = 5) Concentration, mM Concentration, mM 100 ∗

80 Figure 5. Nose rubbing and histamine concentration. A, There was a significant increase in nose rubbing at 3 and 30mM-histamine 60 concentrations from baseline. In addition to a total increase in nose rubs, a lower concentration of histamine caused more nose rubs after allergen 40 exposure. B, There was a significant increase from baseline in the number of sneezes seen at each concentration. In addition to a total increase in sneezes,

No. of Sneezes per 30 min 20 allergen exposure significantly increased the number of sneezes seen after exposure to the 0.3mM-histamine concentration. 0 Baseline 1 Baseline 2 Postexposure 1 wk After Postexposure Time Point of intranasal ovalbumin without systemic sensitization to ovalbumin developed increased ovalbumin-specific IgE and Figure 4. Mean±SEM of the sum of the nose rubs (A) and sneezes (B) for IgG after 28 days of exposure. These mice also had goblet each histamine concentration. There was a significant difference between cell hyperplasia, recruitment of eosinophils and lympho- baseline values and postallergen exposure. A, For nose rubs there was a difference between baseline values and 1 week after the last allergen cytes, and increased interleukin 5 in bronchoalveolar la- exposure. B, For sneezes this difference was no longer present by 1 week vage fluid. We hypothesized that mice exposed to intra- after the last exposure to allergen. nasal ovalbumin without IP sensitization would develop nasal hyperresponsiveness. Six BALB/c mice were ex- There were no significant differences between the 2 posed daily to intranasal ovalbumin. The timeline of the baseline histamine challenges for either outcome, dem- sensitization protocol described earlier was used; how- onstrating the reproducibility of this test of hyperrespon- ever, instead of IP injection of ovalbumin and aluminum siveness (Figure 4). After sensitization and 6 days of hydroxide, the mice were injected IP with PBS and alumi- intranasal allergen exposure, there was a statistically sig- num hydroxide adjuvant and given intranasal ovalbumin nificant increase in the number of sneezes seen at each 5 days a week for 4 weeks. A baseline histamine challenge concentration of histamine (PՅ.05), and a significant in- was performed prior to intranasal ovalbumin exposure. His- crease in nose rubbing at 3mM and 30mM histamine con- tamine challenges were performed 4, 12, 19, and 26 days centrations from baseline (PՅ.05) (Figure 5). after the first ovalbumin exposure. Both sneezing and nose In addition to a total increase in sneezes and nose rubs, rubbing end points increased significantly in mice ex- the first concentration of histamine to be statistically dif- posed daily for 19 days to ovalbumin. Responses to hista- ferent from baseline was lower after allergen exposure mine challenge on day 26 were significantly higher for both (Figure 5). Significant sneezing and nose-rubbing response parameters than on day 19 (Figure 6). was reached at 0.3mM and 3.0mM histamine concentra- Acute bacterial sinusitis increases sensitivity to nasal tions, respectively, vs 30mM for both end points before challenge with histamine, but to a lesser degree than al- allergen exposure. lergy. We hypothesized that mice with acute bacterial si- One week after the last allergen exposure, the area un- nusitis would develop hyperresponsiveness to hista- der the curve for the number of nose rubs was un- mine exposure. For this experiment, we infected 6 BALB/c changed from that for the third histamine challenge, still mice with S pneumoniae. This group received daily in- exhibiting the same increase from baseline (Figure 4A). tranasal PBS after an initial IP injection with PBS and alu- By 1 week after antigen exposure, there had been a de- minum hydroxide adjuvant. crease in response for the sneezing outcome compared Culture results at day 14 showed 9ϫ105 colony-forming with the third time point (Figure 5). When we analyzed units of bacteria, and this decreased to 1ϫ105 colony- each concentration of histamine separately, only the re- forming units of bacteria by 28 days (PՅ.05) (Figure 7). sponse at the 3 mM concentration was significantly dif- These results paralleled the change in hyperresponsive- ferent from baseline (PՅ.05) (Figure 5B). ness measured by histamine challenge. By day 4, these mice The response to nasal challenge with histamine in- had a small, but significant increase in nasal hyperrespon- creases when BALB/c mice are exposed to daily nasal ov- siveness as measured by nasal rubbing, which persisted albumin without IP sensitization. McCusker et al18 ob- to day 12 (Figure 8A). At day 19, the next time point, served that BALB/c mice exposed to high concentrations the number of recorded nose rubs was no longer signifi-

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 A A 50 ∗ ≤ P .05 vs Baseline Value 50 ∗P ≤.05 vs Baseline Value †P ≤.05 vs Day 19 45 (n = 3) (n = 3) † 40 40 35 30 ∗ 30 25 ∗ ∗ 20 20 15 10

Total Nose Rubs per 30 min Total 10 Total Nose Rubs per 30 min Total 5 0 0

B B † 50 50 ∗P ≤.05 vs Baseline Value (n = 3) †P ≤.05 vs Day 19 45 (n = 3) 40 40 ∗ 35 30 30 25 20 20 15 Total Sneezes per 30 min Total Total Sneezes per 30 min Total 10 10 5 0 0 Baseline 4 12 19 26 Baseline 4 12 19 26 Time, d Time, d

Figure 6. Mean±SEM of the summation of nose rub (A) and sneeze (B) Figure 8. A, There was a significant increase in nose rub counts above counts for each histamine concentration. There was a slow increase in nasal baseline values 4 days and 12 days after infection with Streptococcus responsiveness with daily intranasal allergen application, which became pneumoniae (the decrease in responsiveness corresponds temporally to significant by day 19. Responsiveness also increased from day 19 to day 26. decreasing bacterial counts); B, there was no difference in sneeze counts in response to histamine challenge after infection with S pneumoniae.

inflammatory nasal disease and should allow us to de- 8 10 ∗P ≤.05 vs 0mM termine the underlying mechanism of increases in hy-

107 perresponsiveness to different inflammatory stimuli. The 2 end points, nose rubbing and sneezing, have 106 ∗

, CFU/mL different thresholds of responses. In the first experi- 105 ment, the nose rubbing measures were increased signifi-

4 cantly from baseline at the 10mM concentration of his- 10 tamine, whereas the first significant increase for sneezing 103 was at 30mM. Mice begin to rub their noses with less ir-

102 ritation than it takes to generate a sneeze. In IP- sensitized allergic mice, there were maximal increases in Streptococcus pneumoniae 101 hyperresponsiveness 5 days after the first exposure to al- (n = 5) 100 lergen, followed by a decline. However, with the nose- 14 28 rub end point, we noted that nasal irritability remained Time, d maximally elevated over baseline even 1 week after the last ovalbumin exposure. The lower threshold for nasal Figure 7. In infected mice there was a significant decrease in colony-forming rubbing is analogous to the human condition where symp- units (CFUs) of Streptococcus pneumoniae from 14 days to 28 days after 9 infection. toms of nasal itching precede sneezing. The lower threshold of irritability for nose rubbing was also important for measuring the nasal hyperresponsiveness associated with acute sinusitis. Although nose rub- cantly increased over baseline. There was no significant bing was significantly increased from baseline, showing increase in the number of sneezes throughout the time nasal irritability, the mucosal hyperresponsiveness as- course of the bacterial sinusitis infection (Figure 8B). sociated with sinusitis did not stimulate sneezing above baseline in our model. This could also represent the ef- COMMENT fects of different inflammatory stimuli on the manifes- tations of hyperresponsiveness. Nasal challenge with histamine is a reproducible and easy- Nasal hyperreactivity testing in humans derives from to-use physiologic test of nasal hyperresponsiveness. It the concept of bronchial hyperresponsiveness testing in provides a useful outcome measure in murine models of asthma. In fact, sensitivity to methacholine is often used

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 in the diagnosis of asthma. Its use in the nose as a test for Chicago, 5841 S Maryland Ave, MC 1035, Chicago, IL distinguishing disease states is limited because of large 60637-1035 ([email protected]). variability in responses.13 We found little variability in our Financial Disclosure: None. histamine test, as even small changes were statistically significant, making this an easy-to-use, sensitive model of nasal hyperreactivity in mice. REFERENCES It has been reported that changes in the nasal mucosa associated with sinusitis in humans include in- 1. Walden SM, Proud D, Lichtenstein LM, Kagey-Sobotka A, Naclerio RM. Antigen- 19 provoked increase in histamine reactivity: observations on mechanisms. Am Rev creased basal secretion of lysozyme and lactoferrin. In Respir Dis. 1991;144:642-648. the same study, the quantity of enzymes in the secre- 2. Connell JT. 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