Cotton bracts

Aqueous extracts of cotton bract and mill dust are leuco-tactic (15,40). In experimental animals, aqueous extracts of cotton dust (AECD) attract PMNs to airways in a dose dependent fashion beginning at 3 hours, and reaching a peak at 18-24 hours. Macrophage levels Immediately drop and remain low until 18 hours post-exposure when a modest increase occurs. Cell infiltration, with subsequent release of intracellular enzymes and mediators, is thought to be important in the pathology of byssinosis (15). 

Small airway constriction and recruitment of leukocytes on pulmonary surfaces are prominent, documented responses to the inhalation of cotton dust. Currently, one or both of these effects are generally ascribed to endotoxin (8-10), to antigen-antibody reactions (11), to lacinilene C-7 methyl ether (1, 13), to a low molecular weight ( 1000 daltons), neutral, highly water soluble substance that is stable in boiling water and found in cotton bracts (14), to chemotaxins present in cotton mill dust extracts (15, 16) or to histamine releasing substances (17). 

Histamine Releasing Agents in Extracts of Cotton Mill Dust and Cotton Bract... 

The platelet hist UIline release assay demonstrated that cotton mill dust extract, cotton bract extract, cotton leaf extract, dialyzed CMD extract, polyphenols, compound 48/80, rutin, trimethylamine HCl, quercetin, catechin, tannic acid, ellagic acid and sodium metasilicate all release histamine directly (48). Thus not only do tannin compounds induce histamine release, but they may also form higher molecular weight polymers and contain components that survive acid hydrolytic conditions (48). Tannins are widely distributed in the plant kingdom. 

Silicate is another type of widely distributed substance that induces the release of histamine from platelets. It is a common constituent of plant tissues in the form of dissolved hydrous silica or silicic acid (24), which may contribute to the byssinotic syndrome. Cotton bracts contain 0.4-0.8% silica (24). For many of the byssinogenic substances, nanogram concentrations are sufficient to release histamine (48). 

Nicholls and Skidmore (65) demonstrated that dust collected from mills with a higher prevalence of byssinosis caused greater smooth muscle contractile activity than dust from mills with a lower prevalence. Recently, Russell e al. (66) used an isolated tissue bath to measure canine trachealis muscle contraction caused by cotton bract extracts (CBE). Morey et al. (67) showed that cotton bract represents 20-43% of the cotton dust total thus, the findings of Paton and Davenport using cotton dust agree in principle with Russell e al., who used cotton bract. Davenport and Paton (62) found a percentage of the activity of CDE represented by a 5HT-like component, but they also found at least one more active substance in the CDE than Russell et al. (66). 

Cotton dust and cotton bract extract contracted smooth muscle with forces equivalent to a 0.32jj0.05 jug/ml concentration of 5HT and 0.7+0.1 ug/ml concentration of 5-HT, respectively ( ). However, cotton bract extracts were blocked by methysergide, a 5-HT antagonist, while cotton dust extracts were not. Diphenhydramine, a histamine antagonist, failed to significantly affect either of the extracts ( ). 

Though the contractions caused by cotton dust and cotton bract have important differences, there are also similarities between their chemical properties. It is doubtful that either of the active agents are proteins because pronase has no affect on the extracts and they are labile even at 4 C. In addition, both active substances were extracted with butanol (69). 

Cotton Bract and Acute Airway Constriction in Humans... 

Preparation of cotton bract extracts. Figure 1 is a flow chart showing our procedures for preparing the various bract extracts. Dried bracts (frost killed) were hand picked just prior to harvest from cotton fields in the Lubbock, Texas area. These were stored at room temperature. Extracts were freeze-dried and stored at -4°C. For inhalation challenge by our subjects each extract was reconstituted with water or saline, as indicated, at a concentration equivalent to the standard crude extract. This Insured that for challenge purposes components were not concentrated as purification progressed. 

Figure 1. Procedures for preparing cotton bract extracts.

Table I is an attempt to compare the responses in lung function we observe with our naive subjects on exposure to cotton bract extracts with the responses reported in literature of both naive subjects and workers exposed to cotton dust. The comparison suffers from the fact that neither the exposure time or concentration nor the post-exposure time of FEV. q readings are standardized for the different investigating laboratories. The various cotton dusts or extracts are not standardized either and the airway constrictor potency varies with the dusts. Bracts also vary in their potency. We have observed variations in potency with harvest year from the same location (Lubbock, Texas).

We thank R.V. Baker and C. Brown (Lubbock, Texas) who supervised the collection of cotton bracts by members of Boy Scout Troop 103. This work was In part supported by the Physiology Institute, University of Utrecht, Utrecht, The Netherlands, and Cotton, Inc. 

A close relation between the extent of byssinosis and the contamination of cotton by GNB has been demonstrated in several studies ( > j )- It has been shown that FEV. decrements in textile mills correlated best with the square root or the product of dust concentration and numbers of GNB in raw cotton ( ). It has also been demonstrated that the cotton bract contains an agent or agents capable of eliciting acute respiratory responses during human challenge tests (, 7.) ... 

Aqueous extracts of cotton dust and cotton bract induced the formation of specific precipitating antibodies in rabbits. The antisera cross-reacted with both extracts as well as with extracts of cotton stem, leaf, and burr, baled cotton and gin trash. Cross-reactivity was also demonstrated with extracts of flax, soft hemp, sisal, and jute. No antigen-antibody reaction was obtained with extracts of cottonseed hulls, cottonseed proteins, noncontami-nated cotton lint, or house dust. No reaction was obtained between the antisera to dust and several connmercial preparations of bacterial lipopolysac-charides believed to be present in cotton dust. 

Cotton dust IS a heterogeneous mixture of varied composition (13), but cotton bract has been identified as one of the major contaminants (32-52%) of the plant trash (14). 

Recently, other reports have appeared (31). as well as our own studies (32). showing that cotton dust activates the alternate pathway of complement. Previous studies in this laboratory (33.34). showed that antibodies to water extracts of cotton dust, cotton bract, and a highly purified fraction of dust elicited positive immunological responses in rabbits. 

Preparation of Aqueous Extract of Cotton Bract. Field-dried cotton bract, 10 g, picked directly from plants in a field near Lake Providence, Louisiana, was ground with mortar and pestle, extracted twice with 100 ml of deionized water at pH 7 for 30 min each time at 250c with stirring. The extracts were combined and the solution was filtered the filtrate was freeze-dried. This bract extract (bAg) was also used to immunize rabbits. Additional bAg was further purified by treatment with 85% methanol in a similar manner as with cotton dust. 

These reactions differ from the pseudoimmune reactions reported by Edwards and Jones (30). because the cotton dust anti gen(s) does not react nonspecifically with both normal and immunized rabbit sera. Precipitin arcs in a reaction of identity between antigens in bracts and dust in Figure 2 confirm that antigens present in dust are derived at least partly from cotton bract. 

Figure 2. Double diffusion of cotton dust fractions and cotton bract extract against rabbit antiserum to dust (AD) and normal rabbit serum (NS). Conditions described in Material and Methods.

---